Posters (in order of presentation)

P1 a/1 Accessory LYR subunit LYRM6/NDUFA6 has a critical function for complex I activity Heike Angerer1, Etienne Galemou Yoga1, Karin Siegmund1, Ilka Wittig2, Juliane Heidler2, Klaus Zwicker3, Volker Zickermann1,4 1Goethe University Frankfurt, Medical School, Institute of Biochemistry II, Structural Group, Frankfurt, Germany 2Functional Proteomics, SFB 815 core unit, Goethe University Frankfurt, Medical School, Frankfurt, Germany 3Goethe University Frankfurt, Medical School, Institute of Biochemistry I, Frankfurt, Germany 4Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Germany

Mitochondrial complex I consists of 14 centrals and more than 30 accessory subunits. The interface of the peripheral and membrane arm is formed by the hydrophilic 49-kDa and PSST subunits and the hydrophobic ND1 and ND3 subunits. The functional significance of selected contact points was studied here by site-directed mutagenesis. Moreover, deletion of accessory LYR protein subunit LYRM6, that tightly interacts with the interface region, caused complete loss of ubiquinone reductase activity and prevented binding of the acyl carrier protein subunit ACPM1 to complex I [1]. Three single point mutations in subunit LYRM6, which changed interaction with the matrix loop of subunit ND3, showed decreased complex I activity below <25 % demonstrating the crucial role of an accessory subunit for complex I function. The physiological role of further LYR proteins is discussed in the context of mitochondrial metabolism.

1. H. Angerer, M. Radermacher, M. Mankowska, M. Steger, K. Zwicker, H. Heide, I. Wittig, U. Brandt, V. Zickermann, The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity, Proc. Natl. Acad. Sci. U. S. A 111 (2014) 5207-5212.

P1 a/2 Molecular simulations of quinone binding into respiratory complex I and of substrate reactivity in complex II Guilherme Menegon Arantes, Murilo Teixeira, Felipe Curtolo Department of Biochemistry, Universidade de Sao Paulo, São Paulo, Brazil

It is now possible to use computer simulation to study in detail the molecular mechanisms involved in the function of respiratory complexes. Here we present simulation results obtained with both classical molecular dynamics (MD) and hybrid quantum chemical/molecular mechanical (QC/MM) methods with careful calibration of simulation parameters and approximations. For the respiratory complex I, we estimate the free energy and mechanism of ubiquinone binding into the reactive chamber. The results show an important intermediate state that may explain several experimental observables as well as the possible binding modes of inhibitors and the molecular basis of diseases (LHON) caused by mutations in complex I. For the respiratory complex II, we study the first reaction step of succinate oxidation in order to determine the species involved in proton and electron transfer. We also compare with the reverse reaction as found in fumarate reductases. The results show a strong electron correlation effect between the succinate/fumarate substrate and the flavine acceptor which should be carefully accounted for during calculations. Several mechanistic proposals, including a carbanion intermediate and a concerted hydride-proton transfer are compared, and the participation of an arginine (Arg-A287, in E. Coli numbering) side-chain in proton transfer from the substrate is shown to be water-dependent.

P1 a/3

Functional characterisation of E. coli cytochrome b561 proteins: A membrane-associated superoxide scavenger? Olivier Biner1, Camilla Lundgren2, Dan Sjöstrand2, Martin Högbom2, Christoph von Ballmoos1 1Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland 2Department of Biochemistry and , , Stockholm, Sweden

The aerobic respiratory chain of E. coli contains six different cytochromes: cytochrome b556 that is part of the succinate dehydrogenase, the cytochromes b562, o, b558, b595 and d, which belong to terminal quinol oxidases, and cytochrome b561 with no assigned function yet.

Cytochrome b561 (CybB) was identified in a cytochrome b556 deficient E. coli strain in 1984 in the laboratory of Yasuhiro Anraku [1]. The protein was found to reside in the cytoplasmic membrane and to contain two b-type hemes [2, 3]. Experiments with cytoplasmic membrane vesicles showed that CybB is reduced by the respiratory chain with D-lactate or NADH as substrate [3]. Therefore, it was speculated that CybB may act as electron carrier between dehydrogenases and ubiquinone. In this project, we present the crystal structure of the protein at 2.0 resolution and its functional characterisation. The structure shows that CybB is an integral membrane protein poised for electron transferÅ between its two b-type hemes. Molecular docking experiments predict a ubiquinone binding pocket. We investigated the reduction of detergent-solubilised CybB by a variety of biological reductants and found that the protein is reducible by superoxide and quinols. Further experiments showed that it can catalyse electron transfer between its two substrates in either direction in a nearly diffusion-limited reaction. Investigations in proteoliposomes and inverted membrane vesicles show that the protein preferentially quenches superoxide generated at the membrane. This is the first description of a membrane-bound enzyme that oxidizes superoxide to reduce ubiquinone. The results are discussed in light of a potential role in scavenging superoxide radicals that are produced during oxidative phosphorylation.

References [1] K. Kita, H. Murakami, H. Oya, Y. Anraku, Quantitative determination of cytochromes in the aerobic respiratory chain of Escherichia coli by high-performance liquid chromatography and its application to analysis of mitochondrial cytochromes, Biochemistry international, 10 (1985) 319-326. [2] H. Murakami, K. Kita, Y. Anraku, Purification and properties of a diheme cytochrome b561 of the Escherichia coli respiratory chain, Journal of Biological Chemistry, 261 (1986) 548-551. [3] H. Murakami, K. Kita, Y. Anraku, Cloning of cybB, the gene for cytochrome b561 of Escherichia coli K12, Molecular & general genetics : MGG, 198 (1984) 1-6.

P1 a/4 Reversible decoupling of the proton pumps of mitochondrial complex I by fixing a loop in the ubiquinone reduction pocket Alfredo Cabrera-Orefice1,2, Etienne Galemou Yoga3,4, Christophe Wirth5, Karin Siegmund3,4, Klaus Zwicker6, Sergio Guerrero-Castillo1, Volker Zickermann2,3,4, Carola Hunte5, Ulrich Brandt1,2 1Radboud Institute for Molecular Life Sciences, Department of Pediatrics, Radboud University Medical Center, Nijmegen, The Netherlands 2Cluster of Excellence Macromolecular Complexes, Goethe-University, Frankfurt am Main, Germany 3Institute of Biochemistry II, Medical School and Institute of Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany 4Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany 5Institute for Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, BIOSS Centre for Biological Signalling Studies, University of Freiburg, Germany 6Institute of Biochemistry I, Medical School, Goethe University Frankfurt am Main, Germany

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) couples the transfer of 2e- from NADH to ubiquinone to the translocation of 4H+ across the inner membrane. We have proposed that the reduction of ubiquinone may trigger a coordinated movement of three protein loops located in subunits ND1, ND3 and 49-kDa resulting in an electrostatic power stroke into the membrane arm to drive proton pumping [1, 2]. Loop TMH1-2 of complex I subunit ND3 carries a cysteine shown to be involved in the active/de- active (A/D) transition of complex I [3]. In a structure-guided approach, we applied site-directed mutagenesis in Yarrowia lipolytica to generate an artificial disulfide bond locking the loop TMH1-2ND3 to the nearby subunit PSST. The cross-link did not impair ubiquinone reduction, inhibitor sensitivity or A/D transition. However, the ability of complex I to pump protons was abolished. Importantly, proton pumping was fully restored by reducing the disulfide bond. The X-ray structure of mutant complex I revealed that loop TMH1-2ND3 was indeed immobilized by the disulfide bond, but remained essentially in the same position as in the wild-type enzyme. Our results thus corroborate the mechanistic model proposed earlier [1, 2]. Moreover, the mutant allows reversible decoupling of redox reactions from the proton pumping machinery providing a unique and valuable tool for studying the still enigmatic catalytic mechanism and the regulation of mitochondrial complex I.

[1] U. Brandt, A two-state stabilization-change mechanism for proton-pumping complex I, Biochim Biophys Acta, 1807 (2011) 1364- 1369. [2] V. Zickermann, C. Wirth, H. Nasiri, K. Siegmund, H. Schwalbe, C. Hunte, U. Brandt, . Mechanistic insight from the crystal structure of mitochondrial complex I, Science, 347 (2015) 44-49. [3] S. Dröse, A. Stepanova, A. Galkin, Ischemic A/D transition of mitochondrial complex I and its role in ROS generation, Biochim Biophys Acta, 1857 (2016) 946-957.

P1 a/5 Cardiolipin affects respiratory complex I structure and dynamics Andrea Di Luca, Alexander Jussupow, Ville R. I. Kaila Department of Chemistry, Technical University of Munich, D-85747 Garching, Germany

Cardiolipin plays a key role in membrane bioenergetics by modulating the function of respiratory complexes. In recent years, considerable progress has been made in understanding cardiolipin function in many enzymes of the respiratory chain. Although presence of tightly bound cardiolipin and its requirement for optimal function have been reported for respiratory complex I (NADH:ubiquinone oxidoreductase) [1,2], details of such interactions, as well as mechanistic implications are still not known. Using large-scale molecular simulations, we highlight putative interaction sites of cardiolipin in the membrane domain of complex I, and investigate its possible roles for the enzyme function. Our results serve as a basis to understand how cardiolipin affects the structure and dynamics of respiratory complex I, and provide a general view of how lipid molecules affect enzyme function.

1. S. Dröse, K. Zwicker, U. Brandt, Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids, Biochim Biophys Acta - Bioenerg. 1556 (2002) 65–72. 2. M. S. Sharpley, R. J. Shannon, F. Draghi, J. Hirst, Interactions between phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine mitochondria, Biochemistry 45 (2006) 241–248.

P1 a/6 A highly conserved loop in the PSST subunit of complex I is essential for ubiquinone binding and reduction Etienne Galemou Yoga1, Outi Haapanen2, Vivek Sharma2,3 and Volker Zickermann1,4 1Structural Bioenergetics Group, Institute of Biochemistry II, Medical School, Goethe-University Frankfurt am Main, Germany 2Department of Physics, University of Helsinki, Helsinki, Finland 3Institute of Biotechnology, University of Helsinki, Helsinki, Finland 4Cluster of Excellence ‘Macromolecular Complexes’, Goethe-University Frankfurt am Main, Germany

Proton pumping NADH:ubiquinone oxidoreductase, also known as complex I, is the first and largest enzyme of the respiratory chain. It couples the transfer of electrons from NADH to ubiquinone (UQ) with the translocation of protons across the inner mitochondrial membrane or the membrane of . The binding site for ubiquinone is located in close proximity to its immediate electron donor cluster N2 [1]. The PSST subunit that coordinates this cluster comprises a highly conserved loop lining the ubiquinone binding site. This loop was shown to harbor a hydroxylated arginine residue in mammals and was discussed to be related to the active site loop of CO- dehydrogenase [2]. We exchanged selected residues in this loop and neighboring structural elements in complex I of Yarrowia lipolytica. We found that several mutants showed drastically compromised ubiquinone reductase activity. Fully atomistic microsecond-long molecular simulations of wild-type and mutant enzymes reveal tight coupling between the dynamic of selected protein residues and ubiquinone in the UQ- binding tunnel. Our multidisciplinary approach provides detailed mechanistic insights into the most complex enzyme of the respiratory chain that is associated with a number of mitochondrial dysfunctions.

References: 1. V. Zickermann, C. Wirth, H. Nasiri, K. Siegmund, H. Schwalbe, C. Hunte, U. Brandt, Mechanistic insight from the crystal structure of mitochondrial complex I, Science 347 (2015), 44-49. 2. V.F. Rhein, J. Carroll, S. Ding, I.M. Fearnley, J.E. Walker, NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I, J. Biol. Chem. 291 (2016), 14851-14860.

P1 a/7 The mechanism of mammalian respiratory complex I Domen Kampjut, Leonid Sazanov Institute of Science and Technology , Am Campus 1, Klosterneuburg, Austria

Mitochondrial respiratory complex I is one of the central oxidative phosphorylation enzymes that generates ~40% of the total mitochondrial proton motive force in mammals. Proton pumping is achieved by tight coupling to NADH:ubiquinone oxidoreduction but these reactions take place in different subunits more than 100 Å away in this ~1 MDa protein complex [1]. To fully understand this mechanism, we have purified and stabilised ovine complex I in the active state by LMNG or lipid nanodiscs and determined its cryo-EM structure. Lipid environment of the nanodisc preparation preserved the structural integrity of the loosely bound membrane subunit B14.7 and the majority of the particles corresponded to the active closed state of complex I. We are working on structurally characterising complex I with different substrates and inhibitors to delineate the full conformational landscape of complex I during its catalytic cycle.

References 1. Fiedorczuk, K., Letts, J.A., Degliesposti, G., Kaszuba, K., Skehel, M. and Sazanov, L.A., 2016. Atomic structure of the entire mammalian mitochondrial complex I. Nature, 538(7625), p.406.

P1 a/8 Probing the conformational heterogeneity of complex I by combination of cryo-electron microscopy, crystallography and normal mode analysis Karol Kaszuba1, Javier Gutierrez-Fernandez1, Margherita Tambalo1, David Gallagher2, Leonid Sazanov1 1IST Austria, Klosterneuburg, Austria 2The MRC Laboratory of Molecular Biology, Cambridge, UK

Complex I is the largest enzyme of the respiratory chain. It plays a central role in the process of synthesis of cellular energy, by coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane [1,2]. Detailed studies of complex I are of essential medical importance. Multiple mutations in genes, encoding structural subunits of complex I compromise its function and can also cause the increased production of the reactive oxygen species, which damage mitochondrial DNA, lipids and proteins, ultimately leading to development of several neurodegenerative disorders. Thus, understanding the action of this enzyme is of primary importance. The aim of this project is to decipher details of the proton translocation mechanism, relying on different structures of complex I, which were derived through the single-particle cryo-electron microscopy and x-ray crystallography, and subsequently analyzed, using various computational techniques. Performed normal mode analysis suggests that complex I operates via the large- scale and concerted rigid-body motions, which affect structures of both hydrophilic and membrane domain. The first two non-trivial modes can be best described as the “opening” of the structure of complex I. The mode 7 shows a large displacement of the apical part of the hydrophilic domain, and of the Nqo12 subunit of the membrane domain, which bend in opposite directions, while the mode 8 features an opposed rotation of these regions. These motions are also observed when comparing discussed cryo-EM structures to the previously published x-ray structure of a „native“ state of the enzyme [1]. Thus, the performed NMA reproduces an essential dynamics of the enzyme in a reliable manner. The remaining low-energy modes show various rotational and translational movements, which affect mainly the structure of a hydrophilic domain of the complex.

References: 1. Baradaran R, Berrisford JM, Minhas GS, Sazanov LA. Crystal structure of the entire respiratory complex I. Nature. 2013, 494(7438): 443-8. 2. Sazanov LA. A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat Rev Mol Cell Biol. 2015, (6): 375-88.

P1 a/9 Proton Pumping Mechanism of Complex I Ievgen Mazurenko1, Justin G. Fedor2, Hannah R. Bridges2, Judy Hirst2, Lars J. C. Jeuken1 1School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom 2Medical Research Council Mitochondrial Biology Unit, , Cambridge, United Kingdom

Membrane proton-pumping enzymes of electron transfer chains couple a cascade of redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP synthesis. In eukaryotes, complex I, a large multi-subunit 1MDa enzymatic complex, is responsible for about 40% of protons translocated within the mitochondrial respiratory chain. In the absence of substrates, complex I was shown to enter into a deactive state with Na+-H+ antiporter activity [1]. Complex I is also thought to be a major source of reactive oxygen species, implicated in cellular damage. Due to its complex nature, the exact mechanisms linking these processes with electron transfer and proton translocation still remain elusive for complex I. Previously we have shown that proton-pumping activity can be studied at the single-enzyme level by reconstituting complexes in the native lipid environment together with a pH-sensitive fluorescent dye. Once adsorbed on an electrode surface, electrochemical conversion of the quinone pool can be used to activate proton translocation through liposome bilayer, which can be detected by fluorescence microscopy [2]. Here, we show progress towards extending this approach to the mammalian Complex I from B. taurus.

[1] P.G. Roberts, J. Hirst, The deactive form of respiratory complex I from mammalian mitochondria is a Na+/H+ antiporter, J. Biol. Chem. 287 (2012) 34743–34751. [2] M. Li, S.K. Jørgensen, D.G.G. McMillan, Ł. Krzemiński, N.N. Daskalakis, R.H. Partanen, M. Tutkus, R. Tuma, D. Stamou, N.S. Hatzakis, L.J.C. Jeuken, Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase, J. Am. Chem. Soc. 137 (2015) 16055–16063.

P1 a/10 Characterization of the Quinone-binding Pocket of Mitochondrial Respiratory Complex I through Specific Chemical Modifications Masatoshi Murai, Takahiro Masuya, and Hideto Miyoshi Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan

The site-specific chemical modification of the quinone-binding pocket of mitochondrial respiratory complex I may provide new insights into the mechanism responsible for energy conversion. We have demonstrated that a bulky ring-strained cycloalkyne TAMRA-DIBO directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine complex I, which is located in the inner part of the pocket [1]. Although this two-step conjugation may provide technical possibility of diverse chemical modifications of complex I by various molecular probes, TAMRA-DIBO also reacted with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions. To minimize side reactions and achieve precise pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction. We found that Diels-Alder cycloaddition of a pair of cyclopropene incorporated into 49 kDa Asp160 (via ligand-directed tosyl chemistry) and externally added tetrazine (BODIPY-tetrazine) is more efficient for the pinpoint modification [2]. Side reactions were almost completely avoidable in the case of Diels-Alder cycloaddition. An excess of quinone-site inhibitors did not interfere with Diels-Alder cycloaddition between the cyclopropene and tetrazine. These results strongly suggest that in contrast to the predicted quinone-access channel modeled by X-ray crystallographic and single- particle cryo-EM studies, the channel may undergoes large structural re-arrangements to allow wide range of bulky ligands into the close proximity of 49 kDa Asp160.

1. T. Masuya, M. Murai, H. Morisaka, H. Miyoshi, Pinpoint chemical modification of Asp160 in the 49 kDa subunit of bovine mitochondrial complex I via a combination of ligand-directed tosyl chemistry and click chemistry, Biochemistry 53 (2014) 7816-7823. 2. T. Masuya, M. Murai, T. Ito, S. Aburaya, W. Aoki, H. Miyoshi, Pinpoint chemical modification of the quinone-access channel of mitochondrial complex I via a two-step conjugation reaction, Biochemistry 56 (2017) 4279-4287.

P1 a/11 Proton translocation pathways and inter-subunit coupling in the membrane domain of respiratory complex I Max E. Mühlbauer, Andrea Di Luca, Patricia Saura, Ville R. I. Kaila Department of Chemistry, Technical University Munich, Munich (Germany)

NADH:ubiquinone oxidoreductase functions as the initial entry point for electrons to the respiratory chain. The multi-subunit complex consists of a hydrophilic domain, which catalyzes the electron transfer from nicotineamide adenine dinucleotide (NADH) to ubiquinone (Q), and a transmembrane domain, which uses the energy provided by this redox chemistry to pump four protons across the membrane. The established proton motive-force (pmf) is in turn used to drive ATP synthesis and active transport across membranes. The hydrophilic and membrane domains of complex I are strongly coupled by a chain of buried charged residues that spans across the complete ca. 200 Å membrane domain. Here, we employ large-scale molecular dynamics simulations and free energy calculations to investigate how key elements transmit the redox signals across the complete membrane domain. We elucidate key elements necessary for proton pumping and show how conformational changes within ion-pairs between the subunits can form both inter-subunit or intra- subunit salt-bridges that are key for the action-at-a-distance mechanism controlling proton translocation processes in complex I [1-3].

1. A. Di Luca, A.P. Gamiz-Hernandez, V.R I. Kaila, Symmetry-related Proton Transfer Pathways in Respiratory Complex I. Proc Natl Acad Sci USA 114(31) (2017) E6314-E6321. 2. A. Di Luca, M.E. Mühlbauer, P. Saura, V.R.I. Kaila, How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics, Biochim Biophys Acta - Bioenergetics (2018), submitted 3. V.R.I. Kaila, Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I. J Roy Soc Interface (2018), in press

P1 a/12 Assembly of the Iron-Sulfur Clusters of Complex I in Escherichia coli Franziska Nuber1, Sabrina Burschel1, Doris Kreuzer Decovic1,2, Marie A. Stiller1, Thorsten Friedrich1 1Institute of Biochemistry, Albert-Ludwigs-University, Albertstr. 21, D-79104 Freiburg, Germany 2Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg

The energy-converting NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into many respiratory chains. With its sophisticated setup of 14 subunits and 10 co-factors it is likely that different chaperones are needed for the proper maturation of the complex in Eschericha coli [1,2]. Little is known about the assembly process and the enzymes involved. To contribute to the understanding of the insertion of the iron-sulphur (Fe/S)-clusters into the complex, we generated knock-out strains of the putative chaperones NfuA, BolA, YajL, Mrp, GrxD and IbaG and measured the NADH and succinate oxidase activities of the corresponding mutants. Together with activity profiles of sucrose density gradients from membrane extracts, it is suggested that NfuA, BolA, YajL and Mrp have an influence on complex I maturation to varying degrees. EPR spectroscopy of cytoplasmic membranes demonstrates that the bolA deletion results in the loss of the binuclear Fe/S-cluster N1b.

1. B. Py, F. Barras, Building Fe-S proteins: bacterial strategies, Nature Reviews 8 (2010) 436-446 2. T. Friedrich, D.K. Dekovic, S. Burschel, Assembly of the Escherichia coli NAHD:ubiquinone oxidoreductase (respiratory complex I), Biochim. Biophys. Acta. 1857 (2016) 214-223

P1 a/13 Reduction of 2-methoxy-1,4-naphtoquinone by mitochondrially-localized Nqo1 yielding NAD+ supports substrate-level phosphorylation during respiratory inhibition Dora Ravasz, Gergely Kacso, Viktoria Fodor, Kata Horvath, Vera Adam-Vizi and Christos Chinopoulos Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Provision of NAD+ for oxidative decarboxylation of alpha-ketoglutarate to succinyl-CoA by the ketoglutarate dehydrogenase complex (KGDHC) is critical for maintained operation of succinyl-CoA ligase yielding high-energy phosphates, a process known as mitochondrial substrate-level phosphorylation (mSLP). We have shown previously that when NADH oxidation by complex I is inhibited by rotenone or anoxia, mitochondrial diaphorases yield NAD+, provided that suitable quinones are present [1]. This allows for KGDHC reaction to proceed and as an extension of this, mSLP. NAD(P)H quinone oxidoreductase 1 (NQO1) is an enzyme exhibiting diaphorase activity. Here, by using Nqo1-/- and WT littermate mice we show that in rotenone-treated, isolated liver mitochondria 2-methoxy-1,4- naphtoquinone (MNQ) is preferentially reduced by matrix Nqo1 yielding NAD+ to KGDHC, supporting mSLP. This process was sensitive to inhibition by specific diaphorase inhibitors. Reduction of idebenone and its analogues MRQ-20 and MRQ-56, menadione, mitoquinone and duroquinone were unaffected by genetic disruption of the Nqo1 gene. The results allow for the conclusions that i) MNQ is a Nqo1-preferred substrate, and ii) in the presence of suitable quinones, mitochondrially-localized diaphorases other than Nqo1 support NADH oxidation when complex I is inhibited. Our work confirms that complex I bypass can occur by quinones reduced by intramitochondrial diaphorases oxidizing NADH, ultimately supporting mSLP. Finally, it may help to elucidate structure-activity relationships of redox-active quinones with diaphorase enzymes.

1. G. Kiss, C. Konrad, I. Pour-Ghaz, J.J. Mansour, B. Németh, A.A. Starkov, V. Adam-Vizi, C. Chinopoulos, Mitochondrial diaphorases as NAD donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition,⁺ FASEB J 28 (2014) 1682-97

P1 a/14 Energetics and dynamics of long-range electron transfer in respiratory complex I Michael Röpke1, Ana P. Gamiz-Hernandez1, Alexander Jussupow1, Mikael P. Johansson2, Ville R. I. Kaila1 1Department of Chemistry, Technical University of Munich, Garching, Germany 2Department of Chemistry, University of Helsinki, Finland

Many proteins involved in biological energy conversion employ iron-sulfur clusters (ISC) to shuttle electrons across large distances. One of the most intricate examples is the respiratory complex I (NADH:ubiquinone oxidoreductase), which couples electron transfer (eT) mediated by ISCs to proton pumping up to 200 Å away. [1] The ISCs are arranged in a tunneling wire, with approximately 10-14 Å spacing between individual centers, and the eT wire is located in the hydrophilic domain of this protein, reaching from the NADH oxidation site, to the quinone reduction site. [2] Here we combine quantum and classical molecular simulations to estimate electronic couplings, reorganization energies, and thermodynamic driving forces in order to derive a microscopic understanding of the eT processes in complex I. We show how the ISC spin-states have a significant influence on the transfer energetics and electronic couplings, and we also evaluate the influence of conformational changes during the electron transfer process. The predicted rates are consistent with experiments and highlight general mechanistic features of long-range biological electron transfer in biology.

1. V.R.I. Kaila, Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I, J. R. Soc. Interface. in press (2018). 2. A.P. Gamiz-Hernandez, A. Jussupow, M.P. Johansson, V.R.I. Kaila, Terminal Electron–Proton Transfer Dynamics in the Quinone Reduction of Respiratory Complex I, J. Am. Chem. Soc. 139 (2017) 16282–16288.

P1 a/15 Energetics and dynamics of proton coupled-electron transfer reactions in the NADH/FMN site of respiratory complex I Patricia Saura, Ville R. I. Kaila Department of Chemistry, Technical University of Munich, Garching, Germany

Complex I or NADH:ubiquinone oxidoreductase functions as a redox-driven proton pump in aerobic respiratory chains. It catalyzes the electron transfer (eT) from NADH to quinone (Q) through a series of iron-sulfur (FeS) centers, and employs the free energy released to pump four protons across the ca. 200 Å long membrane domain, generating a proton-motive force (pmf) [1]. The catalytic process is initiated by a hydride transfer reaction between NADH and flavin mononucleotide (FMN) cofactors of the enzyme. The two electrons are subsequently transferred to the FeS centers chain, which are one-electron acceptors. Two of the FeS clusters, N1a and N3, are located in the proximity of the NADH/FMN binding site. However, only N3 is proposed to participate in the eT to quinone, whereas the actual role of N1a remains controversial. To understand the molecular mechanism, energetics, and dynamics of the proton-coupled electron transfer (PCET) reactions in the NADH/FMN binding site of complex I, we employ here multi-scale quantum and classical molecular simulations [2]. We present a mechanistic model for the NADH/FMN hydride transfer reaction and the subsequent eT to N1a and N3, and discuss their implications for the function of complex I.

1. V.R.I. Kaila, Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I, J. R. Soc. Interface (2018) 20170916. http://dx.doi.org/10.1098/rsif.2017.091. 2. P. Saura, V.R.I. Kaila, Energetics and dynamics of proton-coupled electron transfer coupled to NADH Oxidation in Respiratory Complex I (in preparation).

P1 a/17 Two-electron reduction of ubiquinone in respiratory complex I Alexei A. Stuchebrukhov, Muhammad A. Hagras Department of Chemistry, University of California Davis, One Shields Avenue, Davis, California, USA

Respiratory Complex I performs two-electrons/two-protons reduction of a ubiquinone (Q) ligand bound at its Q-binding pocket that ultimately generates a ubiquinole (QH2) molecule. We propose a mechanism in which two electrons are transferred together with two protons in a concerted fashion. A method to evaluate the coupling matrix element that corresponds to a concerted tunneling of two electrons was developed. On one side a coupled electron/proton transfer occurs from reduced N2 cluster and protonated His38 residue respectively, while on the other side a hydrogen radical transfer occurs from neutral Tyr87 residue. Study of N2 Fe4S4 cluster spin states shows energetically favorable electron localization at the lower two iron centers which is mainly due to the extra delocalization contributed by the tandem Cys45 and Cys46 residues. In addition, His38 residue is presumed to act as a rotating proton shuttle; this is supported by our molecular dynamics simulation which shows that His38 can assume hydrogen-bonded or edged-T conformations as related to Q-benzoquinone group. Overall, our calculations indicate that the concerted reaction is feasible, in which case a transient tyrosyl radical is formed during the catalytic cycle of the enzyme.

P1 a/18 NAD+ Binding Site-Independent Energy-Linked Reverse Electron Transfer in Respiratory Complex I Andrei D. Vinogradov, Grigory V. Gladyshev, Vera G. Grivennikova Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia

The substrate (NAD+/NADH) binding site of the respiratory complex I is specifically blocked by NADH-OH, a derivative of NADH [1,2]. This tightly binding inhibitor prevents the proton motive force (pmf)-generating NADH:quinone reductase and/or reverse pmf-utilizing quinol:NAD+ (or :ferricyanide (Ferri)) reductase activities as well as non coupled FMN-mediated NADH:Ferri or :hexaammineruthenium III (HAR) reductase activities [3,4]. A simple procedure for measuring ATP-dependent reverse electron transfer (RET) from ubiquinol to HAR as catalyzed by coupled bovine heart submitochondrial particles or coupled Paracoccus denitrificans plasma membrane vesicles is introduced. ATP induces HAR-mediated oxygen consumption by the respiratory chain-inhibited particles supplemented with succinate. In contrast to pmf-dependent RET with NAD+ or Ferri, the reaction with HAR is insensitive to NADH-OH. The results suggest that a site (or mechanism) of HAR reduction in RET catalyzed by complex I is different from that for NAD+ or Ferri. Two possible explanations are discussed: (i) electron connection exists between reduced FMN and HAR different from that for NAD+ and Ferri; (ii) during RET HAR accepts electrons from iron-sulfur N-2 cluster–quinone junction site.

References 1. A.B. Kotlyar, J.S. Karliner, G. Cecchini, A novel strong competitive inhibitor of complex I, FEBS Lett. 579 (2005) 4861-4866. 2. V.G. Grivennikova, A.B. Kotlyar, J.S. Karliner, G. Cecchini, A.D. Vinogradov, Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I, Biochemistry 46 (2007) 10971-10978. 3. V.D. Sled, A.D. Vinogradov, Kinetics of the mitochondrial NADH-ubiquinone oxidoreductase interaction with hexammineruthenium(III), Biochim. Biophys. Acta 1141 (1993) 262-268. 4. E.V. Gavrikova, V.G. Grivennikova, V.D. Sled, T. Ohnishi, A.D. Vinogradov, Kinetics of the mitochondrial three-subunit NADH dehydrogenase interaction with hexammineruthenium(III), Biochim. Biophys. Acta 1230 (1995) 23-30.

P1 a/19 Using molecular approaches to understand complex I deficiency in the Ndufs4 knockout mouse model Zhan Yin, Ahmed-Noor A. Agip and Judy Hirst MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom

Complex I (NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial . Mammalian complex I contains 44 different subunits [1], encoded on both the nuclear and mitochondrial genomes, and genetic mutations in many of these proteins are associated with neuromuscular diseases [2]. The nuclear gene Ndufs4 encodes an 18 kDa protein which is a supernumerary subunit of complex I. In humans, mutations of Ndufs4 can cause complex I dysfunctions, and result in clinical presentations including Leigh syndrome [3]. Therefore, in this study, we used the Ndufs4 knockout mouse model [4] to investigate the pathogenic mechanisms of the associated complex I dysfunction on a molecular level. We have purified the enzyme from mouse heart mitochondria and characterised it by mass spectrometry and kinetic assays. By using electron cryo-microscopy and single particle analyses, we have studied the structure of the variant enzyme. The results provide insights into the pathogenic mechanisms of complex I disorders on the molecular level and may highlight strategies for developing therapeutic approaches in the future.

1. K.R. Vinothkumar, J. Zhu, J. Hirst, Architecture of mammalian respiratory complex I, Nature. 515 (2014) 80–84. doi:10.1038/nature13686. 2. E. Fassone, S. Rahman, Complex I deficiency: clinical features, biochemistry and molecular genetics., J. Med. Genet. 49 (2012) 578– 90. doi:10.1136/jmedgenet-2012-101159. 3. E. Leshinsky-Silver, A.S. Lebre, L. Minai, A. Saada, J. Steffann, S. Cohen, A. Rötig, A. Munnich, D. Lev, T. Lerman-Sagie, NDUFS4 mutations cause Leigh syndrome with predominant brainstem involvement, Mol. Genet. Metab. 97 (2009) 185–189. doi:10.1016/j.ymgme.2009.03.002. 4. S.E. Kruse, W.C. Watt, D.J. Marcinek, R.P. Kapur, K.A. Schenkman, R.D. Palmiter, Mice with Mitochondrial Complex I Deficiency Develop a Fatal Encephalomyopathy, Cell Metab. 7 (2008) 312–320. doi:10.1016/j.cmet.2008.02.004.

P1 a/20 The cryo EM structure of respiratory complex I from Yarrowia lipolytica Volker Zickermann3,5, Kristian Parey1, Ulrich Brandt2,5, Werner Kühlbrandt1,5, Deryck Mills1, Karin Siegmund3, Janet Vonck1, Hao Xie4 1Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 2Radboud Institute for Molecular Life Sciences, Department of Pediatrics, Radboud University Medical Centre, Nijmegen, The Netherlands 3Goethe University Frankfurt, Medical School, Institute of Biochemistry II, Frankfurt am Main, Germany 4Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 5Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Germany

Mitochondrial complex I is a 1 MDa membrane protein complex with a key role in aerobic energy metabolism. Redox-linked proton translocation by complex I contributes about 40% of the proton motive force that drives mitochondrial ATP synthesis. We report the cryo-EM structure of complex I from the aerobic yeast Yarrowia lipolytica in the deactive form and captured during steady state turnover. We provide evidence for an alternative binding position of ubiquinone and propose that a conformational switch of the ubiquinone reduction site is pivotal for energy conversion in the catalytic cycle of complex I.

P1 a/21 Daniel N. Grba, Polly Marino, Judy Hirst MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom

A humanised model of complex I as a tool for mitochondrial disease characterisation

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a cornerstone enzyme in metabolic energy utilisation [1]: dysfunction of the enzyme in high energy-demanding tissues can result in motor defects and neurological diseases, as well as affecting fluxes through metabolism. Complex I is a 1 MDa protein, which introduces a lot of potential mutation sites to induce such diseases. In a clinical setting, it is therefore beneficial and important to separate out benign polymorphisms and pathogenic mutations to enable the correct method of treatment, but to also provide a blueprint to map new variants to facilitate diagnosis of their pathogenic effects. Here, we have built a humanised model of complex I using structural data, via cryo-electron microscopy, from alternative mammalian species determined in house and from data on the human respirasome [2], a unit of associated mitochondrial respiratory chain enzymes, in which complex I is arguably present in a more native environment. Utilisation of both electron density maps enabled complementation in model building, both offering unique areas of structural definition due to the variation in their local resolutions. This model allows us to investigate the spatial localisation of 183 established pathogenic mutations and 595 polymorphic variants. We demonstrate that residues <15 Å away from known functional elements are highly likely to be pathogenic. Our results emphasise the role of high- resolution structural information in medicine as a tool for disease prediction and diagnosis.

1. J. Hirst, Mitochondrial Complex I, Annu. Rev. Biochem. 82 (2013) 551–575 2. R. Guo, S. Zong, M. Wu, M. Yang, Architecture of Human Mitochondrial Respiratory Megacomplex I2III2IV2, Cell. (2017) 1–11

P1 b/1 N-Acetylcysteine induced S-glutathionylation prevented oxidative stress, bioenergetic and dynamic mitochondrial alterations on kidney insufficiency generate by folic acid administration Omar Emiliano Aparicio-Trejo1; Edilia Tapia2; Laura Gabriela Sánchez-Lozada2; José Pedraza-Chaverri1 1Department of Biology, Faculty of Chemistry, National Autonomous University of Mexico (UNAM), Mexico City 04510, Mexico 2Department of Nephrology and Laboratory of Renal Pathophysiology, National Institute of Cardiology “Ignacio Chávez”, Mexico City 14080, Mexico

Kidney insufficiency induced by folic acid (FA) is widely used model for studies renal insufficiency progression[1]. Also, S- glutathionylation (reversible addition of GSH to Cys) has emerged as a mechanism able to regulate mitochondrial functions, linking energy metabolism with redox homeostasis and mitochondrial dynamics[2]. Due to renal homeostasis depends of the mitochondria functions, it has suggested that mitochondrial bioenergetic, dynamic and oxidative stress are involved in the progress of renal damage in this model[3,4]. N-acetylcysteine (NAC), a glutathione inductor, could potentially prevent mitochondrial and renal dysfunction given its ability to control the S-glutathionylation levels[4,5]. In this work, we used male Wistar rats to show that NAC preadministration prevented the FA induced acute renal insufficiency and the decrease in OXPHOS capacity, in membrane potential and in ATP synthase and complex I activities. NAC also prevented the mitochondrial oxidative stress, the increase in H2O2 production, the shift of mitochondrial dynamic to fission and the loss of mitochondrial morphology. These protective effects were associated to the conservation of mitochondrial S-glutathionylation, due to NAC preserved the activities of mitochondrial enzymes involved in S-glutathionylation and the GSH levels. Taken together our results suggested that the preservation of the mitochondrial function by NAC is a useful tool to prevent the progression of kidney damage in this model.

[1] A. Ortiz et al., Translational value…, Eur. J. Pharmacol., 759, (2015), 205–220. [2] R. J. Mailloux et al, Protein S-glutathionlyation…, Redox Biol., 8, (2016) 110–8. [3] R. Che, et al, Mitochondrial dysfunction…, Am. J. Physiol. Renal Physiol., 306, (2014), F367-78. [4] Y. Ishimoto et al, Mitochondria: a therapeutic…, Nephrol. Dial. Transplant, 31, (2015), 1062-9. [5] G. Tamma et al, Evaluating the Oxidative …, Antioxid. Redox Signal., 25, (2016),1–18.

P1 b/2 Fluorescence lifetime imaging as a tool to detect oxidative stress and associated effects of drug binding to cytochrome c oxidase Ulrike Alexiev1, Jens Balke1, Alexander Wolf1, Pierre Volz1, Robert Brodwolf1, Nan Ma2 1Physics Department, Freie Universität Berlin, Berlin, Germany 2Biomaterial Research Department, HZG Teltow, Teltow, Germany

Basal levels of reactive oxygen species (ROS) are produced by the respiratory chain and excess ROS can be reduced by antioxidant cellular defenses like catalase and superoxide dismutase. Overproduction of ROS, among others, is indicative of cellular stress that occurs under various environmental and disease conditions. Because of their highly reactive nature, elevated ROS levels damage DNA, proteins, and lipids, thereby impairing normal cellular function. We developed a highly sensitive and reproducible method for ROS detection based on fluorescence lifetime imaging microscopy (FLIM) that we named FLIM-ROX [1]. This method allows for identifying even low levels of ROS in vivo and in vitro and thus promotes the understanding of ROS-associated nanotoxicity. One example of a widely used drug that exerts adverse effects in myocardial cells via increased oxidative stress is the anti-cancer drug doxorubicin. As a mechanism of action it was proposed that doxorubicin binds to cytochrome c oxidase, which in turn leads to elevated ROS levels and may cause cardiotoxicity [2]. Using the fluorescence of DOX we show binding, as well as molecular and cellular effects on CcO function.

[1] J. Balke, P. Volz, F. Neumann, R. Brodwolf, A. Wolf, H. Pischon, M. Radbruch, L. Mundhenk, A. D. Gruber, N. Ma, U. Alexiev. Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range using Fluorescence Lifetime Imaging, Small (2018) , DOI: 10.1002/smll.201800310 [2] K. Chandran et. al.. Doxorubicin Inactivates Myocardial Cytochrome c Oxidase in Rats: Cardioprotection by Mito-Q, Biophysical Journal (2009), 96, 1388-1398

P1 b/3

Dietary supplementation with MicroActive Coenzyme Q10 increases expression of antioxidant genes in Thoroughbred skeletal muscle Caitriona E Curley1, Mary F Rooney1, Michael E Griffin2, Lisa M Katz3, Richard K Porter1 and Emmeline W Hill2 1School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, Dublin, Ireland 2Plusvital Ltd., Dun Laoghaire Industrial Estate, Dublin, Ireland 3UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland

Coenzyme Q10 (CoQ10) is a small lipophilic molecule ubiquitous in eukaryotic cells that has a principal role in aerobic respiration. The redox function of CoQ10 means that by its nature, CoQ10 is acts as an antioxidant molecule. The aim of the present study was to test the hypothesis that dietary CoQ10 supplementation effects antioxidant enzyme gene expression in equine skeletal muscle. Healthy untrained

Thoroughbred horses (N = 19, 1-3 years) were supplemented orally once per day for nine weeks with 1.5 mg/kg of Microactive CoQ10 (Maypro Industries, USA). Each individual horse acted as its own pre-supplementation control. Skeletal muscle biopsies were collected at week 0 (control) and week 9 (supplemented) for each horse. RNA was extracted from the skeletal muscle sample, cDNA synthesised and gene expression was determined by qPCR. Genes encoding the antioxidant enzymes glutathione disulphide reductase (GSR), catalase (CAT), superoxide dismutase 2, mitochondrial (SOD2) and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) were not significantly (P> 0.05) differentially expressed following nine weeks of CoQ10 supplementation. However, genes encoding the glutathione peroxidase isozymes, GPX1 and GPX8, were significantly (P≤ 0.001 and P≤ 0.05, respectively) increased post-supplementation (~ 2-

Fold and 3-Fold, respectively). These data suggest that increased availability of CoQ10 may promote an improvement in the antioxidant defence system in Thoroughbred skeletal muscle, not only by its own recognized direct antioxidant activity but also in the indirect modulation of the expression of other antioxidant enzymes.

P1 b/4 The impact of mitochondria and reactive oxygen species on the proliferation of human amniotic mesenchymal stromal cells Sergiu Dumitrescu1, Adelheid Weidinger1, Asmita Banerjee1, Susanne Wolbank1, Karlheinz Hilber2, Heinz Redl1, Andrey Kozlov1 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, , Austria 2Department of Neurophysiology and – Pharmacology, Center for Physiology and Pharmacology, Medical

The transition from a quiescent to an active stem cell is an obligatory event for the maintenance of tissue homeostasis and regeneration. Several lines of evidence suggest that mitochondria and reactive oxygen species (ROS) may play an essential role in this process. This study aims to better understand the role of mitochondria and ROS in the activation of human amniotic mesenchymal stromal cells (hAMSC). Adjusting specific components of the incubation medium and passage numbers we have established two different culture conditions, facilitating either the active (proliferating) or quiescent state of hAMSCs; both states manifested healthy cellular morphology. In both, active and quiescent cells we modulated the mitochondrial respiration, as well as generation of ROS by mitochondria and NADPH- oxidase. We have observed that FCCP, a mitochondrial uncoupler, completely inhibited cell proliferation under activating conditions and even exerted a slight cytotoxic effect, while oligomycin, an inhibitor of ATP-synthase, and antimycin A, an inhibitor of mitochondrial complex III and simultaneously an inducer of mitochondrial ROS release, did not exert any effect on proliferation rate. Similarly, the mitochondria targeted antioxidant mitoTEMPO did not change the rate of proliferation, suggesting that mitochondrial ROS do not contribute to the activation of hAMSCs. This was in line with the data obtained by laser scanning microscopy, which revealed that hAMSCs have a much lower mitochondrial ROS levels compared to parenchymal cells such as hepatocytes. In contrast to these results, inhibitors of NADPH oxidase (apocynin and DPI) as well as extracellular ROS scavengers (SOD and catalase) nearly completely inhibited proliferation. Our results indicate that the activation of hAMSCs require coupled mitochondria and ROS derived from the NADPH oxidase; NADPH- oxidase derived ROS activate hAMSCs in an autocrine and/or paracrine manner.

P1 b/5 Cytochrome c-mediated peroxidative permeabilization of cardiolipin-containing liposomes is prevented by minocycline Alexander M. Firsov, Еlena А. Kotova, and Yuri N. Antonenko Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

During the last 15 years there has been accumulated a large body of knowledge on beneficial effects of the antibiotic minocycline in various models of neurological disorders. Nevertheless, a mechanism of minocycline-induced inhibition of cytochrome c release from mitochondria, reported in 2002, is not well understood at present. It is widely accepted that peroxidase activity of cytochrome c, strongly stimulated upon its interaction with cardiolipin, plays a significant role in promoting the release of this protein from mitochondria to cytosol in the course of apoptosis initiation. Previously peroxidase activity of cytochrome c has been shown to cause permeabilization of artificial cardiolipin-containing lipid vesicles [1]. Here we will report data on the impact of minocycline on both cytochrome c peroxidase activity, as measured by luminol chemiluminescence, and liposome permeabilization induced by the combination of cytochrome c with hydrogen peroxide, as assayed by leakage of fluorescent dyes from liposomes. The substantial suppression of cytochrome c-induced liposome permeabilization by minocycline could be of relevance to the inhibiting effect of this antibiotic on cytochrome c release from mitochondria, playing a crucial role in apoptosis.

1. A.M. Firsov, E.A. Kotova, E.A. Korepanova, A.N. Osipov, Y.N. Antonenko, Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex, Biochim. Biophys. Acta - Biomembranes 1848 (2015) 767-774.

P1 b/6 Phospholipase A2γ ablation is associated with decreased expression of selected antioxidant enzymes in liver, heart and brain Martin Jaburek1, Alberto Leguina-Ruzzi1, Pavla Pruchova1, Andrey V Kozlov2, Petr Jezek1 1Department of Mitochondrial Physiology, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic 2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria

Mitochondrial phospholipase A2γ (iPLA2γ) hydrolyzes membrane phospholipids to release free fatty acids and lysophospholipids. The products of iPLA2γ and their downstream metabolites modulate numerous functions, including mitochondrial bioenergetics and cellular signaling. iPLA2γ is directly activated by H2O2, leading to consequent interactions with cellular redox regulatory mechanisms [1,2]. Glutathione peroxidases (GPxs) and peroxiredoxins (Prxs) are the major cellular antioxidant enzymes and we tested the hypothesis that the expression of GPxs and Prxs enzymes is altered following the iPLA2γ gene ablation. We have determined the relative mRNA levels of Prx1, Prx2, Prx3, GPx1 and phospholipid-specific GPx4 in liver, heart and brain of wild-type and iPLA2γ-KO mice. We found decreased levels of GPx1, GPx4, and Prdx2 in all tested tissues of iPLA2γ-KO mice compared to the WT control. In addition, subcutaneous injection of mitoparaquat (mtPQ) caused moderate decrease of GPx4 in WT mice, but nearly complete depletion of GPx4 in iPLA2γ-KO animals. The decrease in mRNA levels of selected antioxidant enzymes correlated with increased levels of lipid peroxidation products in both WT and iPLA2γ-KO animals in all tested tissues. These results support the hypothesis that iPLA2γ inhibition or ablation leads to a decreased antioxidant capacity and suggests protective role of iPLA2γ in GPx4-dependent ferroptosis. Supported by GACR 16-04788S

[1] M. Jabůrek, J. Ježek, J. Zelenka, P. Ježek, Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen, Int. J. Biochem. Cell Biol. 45 (2013) 816–825.

[2] J. Ježek, A. Dlasková, J. Zelenka, M. Jabůrek, P. Ježek, H2O2 -activated mitochondrial phospholipase iPLA2γ prevents lipotoxic oxidative stress in synergy with UCP2, amplifies signaling via G-protein–coupled receptor GPR40, and regulates insulin secretion in pancreatic β-cells, Antioxid. Redox Signal. 23 (2015) 958–972.

P1 b/7 Mitochondrial energetic status and oxidative stress during tardigrade anhydrobiosis Andonis Karachitos1, Daria Grobys1, Milena Roszkowska1, Łukasz Kaczmarek2, Hanna Kmita1 1Department of Bioenergetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland 2Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland

Many tardigrade species are able to survive complete dehydration and this phenomenon is called anhydrobiosis. During dehydration tardigrades curl up into a little ball called a tun which can be regarded as a consequence of cytoprotective mechanisms triggered at cellular and molecular levels. Anhydrobiosis can be therefore defined as an organized state and as such it requires some form of energy supply. This imposes mitochondria involvement in successful anhydrobiosis. Accordingly, in the presence of so called “mitochondrial uncoupler” tardigrades are able to survive but lose the ability to form the tun which implies mitochondrial coupling supporting ATP synthesis based on the oxidative phosphorylation. Besides, mitochondria, the main intracellular location for ATP synthesis, are also involved in reactive oxygen species (ROS) production. Importantly, an efficient protection against dehydration stress in anhydrobiotic organisms requires ROS scavenging mechanisms. This adds to mitochondrial role in maintaining proper course of dehydration process. We decided to estimate mitochondrial coupling in active and anhydrobiotic, i.e. forming tuns, tardigrades by application of the TMRM fluorophore transported into mitochondria in the presence of the mitochondrial inner membrane potential. We also studied the levels of mitochondrial ROS production by the application of the MitoSOX Red fluorescent dye as a mitochondrial superoxide indicator. We observed that the presence of functional mitochondria in tuns correlates with successful recovery to the active stage. We were also able to detect differences in MitoSOX fluorescence between the active stage and the tun stage. These results indicate the important role of mitochondial activity and related ROS production in the mechanisms responsible for successful anhydrobiosis.

The work was supported by the research grant of National Science Centre, Poland, NCN 2016/21/B/NZ4/00131.

P1 b/8 VIRTUAL : A model of ROS production by complex II and energy metabolism Jean-Pierre MAZAT, Stéphane RANSAC IBGC-CNRS & Université de Bordeaux, France

Introduction: Virtual Mitochondrion is a project of a multilevel modelling of mitochondrial bioenergy metabolism. It involves: - A molecular/atomic level with stochastic modelling (Gillespie) of electrons and protons transfers in respiratory chain complexes and super complexes of respiratory chain. It allowed us to predict a natural bifurcation of electrons in complex III (proof of Q-cycle hypothesis of Mitchell), to clarify the antimycin inhibition constraints and to simulate the ROS production in complex I, II and III. The production of ROS by the complex II is particularly addressed in the poster - A mitochondrial level with the global modelling of the respiratory chain. The aim is to understand how local changes (pathological mutations for instance, drug effect, competition between respiratory substrates) in respiratory complexes influence the global behavior of the oxidative phosphorylation. - A cell level with the description of simple(s) model(s) of central energy metabolism easy to manipulate and to understand but nevertheless keeping the exact stoichiometry, the thermodynamics constraints and realistic rate equations. Our aim is to coherently integrate various types of data, metabolomics, fluxomics, transcriptomics and to follow, with different theoretical approaches (FBA, EFMs, dynamical system) the rerouting of metabolism, their regulations and controlling steps/targets (Metabolic Control Analysis). Conclusion: We would like to emphasize the connection between lower and upper levels: how the functioning at a given level explains (or does not explain) the functioning at the upper/integrated level? Thus, in this work, the purpose of a model is not only to fit the experimental results accurately but rather to evidence inconsistencies that will lead to unveil mechanisms/properties which were hitherto not taken into account or even unknown.

P1 b/9 Regulatory Role of Heme Oxygenase and Nitric Oxide Synthase in Macrophages Andrea Müllebner1,2, G. Dorighello4, H. Michenthaler2, M. Kames2, A. Rupprecht3, A. V. Kozlov1, J.C. Duvigneau2 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria 2Department of Biomedical Sciences, Institute for Medical Biochemistry, University of Veterinary Medicine, Vienna, Austria 3Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine, Vienna, Austria 4Dept of Structural and Functional Biology, Biology Institute, University of Campinas, Campinas, Brazil

Macrophages provide defense against invading pathogens and play a role in tissue repair and maintenance of tissue and iron homeostasis. Upon inflammation the macrophages arginine metabolism is shifted to nitric oxide (NO) production via NO synthase (NOS). Heme oxygenase (HO), the rate limiting enzyme in heme catabolism, is essential in regulating macrophage function by limiting inflammation. The aim of this study was to understand how NOS and HO contribute to the regulation of NADPH oxidase (NOX) activity and phagocytosis, key components of macrophage immune function. Resting J774A.1 macrophages were treated with hemin or vehicle and activity of NOS, HO and NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS) formation was determined by Amplex© red assay, mitochondrial function was assessed using extracellular flux analyzer (XFe96, Agilent) and phagocytosis was measured using FITC-labeled bacteria. Furthermore, the fate of intracellular heme was analyzed using electron spin resonance spectroscopy. We show that NOS activity inhibits mitochondrial respiration and stimulates NOX activity by triggering mitochondrial ROS production in a NO dependent manner but does not affect phagocytosis. In contrast, HO activity neither clearly affects NOX activity nor mitochondrial function nor phagocytosis. However, treatment of macrophages with hemin results in intracellular accumulation of ferrous heme, thereby compromises mitochondrial integrity indicated by a proton leak of the inner mitochondrial membrane and leads to an inhibition of phagocytosis. Both mitochondrial dysfunction and impaired phagocytosis were further aggravated upon inhibition of HO. In conclusion our results suggest that effective degradation of heme by HO together with NOS, which amplifies NOX activity, is essential for macrophages to exert their full bactericidal activity.

P1 b/10 Implication of mitochondrial Reactive Oxygen Species production in cardiomyocyte signaling and cardiac rhythm Audrey Sémont1,2,3, Pasdois P.1,2,3, Colin C1, Ernault A.1,2,3, Dos Santos P.1,2,3,4, Diolez P.1,2,3 1IHU Liryc, Electrophysiology and Heart Modeling Institute, fondation Bordeaux Université, Bordeaux, France 2Univ. Bordeaux, Centre de recherche Cardio-Thoracique de Bordeaux, Bordeaux, France 3INSERM, Centre de recherche Cardio-Thoracique de Bordeaux, Bordeaux, France 4Bordeaux University Hospital (CHU), Heart Failure Treatment Unit, Pessac, France

Cellular oxidative stress is defined as an imbalance in favor of the production of Reactive Oxygen Species (ROS) over antioxidant mechanisms [1]. Cellular ROS, (e.g. superoxide anion, and hydrogen peroxide) are generated under numerous in vivo conditions and in response to various endogenous and exogenous factors. Mitochondria are now equally considered as major ROS producers in cells and have been associated with numerous pathologies including cardiac diseases (for example arrhythmias) [2-4]. In addition, the production and activity of reactive oxygen species (ROS) is thought to play a key role, affecting mitochondrial membrane potential and damaging proteins of the respiratory chain [5]. The first objective of this study is to compare different substrates on ROS production. Once the substrate that increases the most the production has been identified we further analyzed Trolox antioxidant effect. We isolated cardiomyocytes and measured continuously the release of mitochondrial H2O2 (with Amphex Red) via spectrofluorometry in the presence of 3 metabolic substrates: glucose, pyruvate and succinate. The Trolox effects were then investigated on rats divided in 4 groups: control group (G1) and groups where hearts were incubated with different Trolox concentrations: 30µM (G2), 60µM (G3), 100µM (G4). Each group was stressed with succinate. Application of succinate to isolated cardiomyocytes significantly increased the mitochondrial ROS production compared to glucose and pyruvate (n=10, p<0,0001). Trolox treatment significantly reduced the production of ROS on cardiomyocytes (n=5, p<0,0001). In G2 the ROS production was reduced by 35%, G3 by 56,6%, and G4 by 70,5%. We show that succinate increased the most the ROS production as compared to glycolysis substrate. Succinate increased ROS production and the antioxidant Trolox prevented the succinate-induced impairment in cardiomyocyte function. Further experiences on myocytes stimulation with the Ionoptix are in progress.

References [1] Dai DF, Chiao YA, Marcinek DJ, Szeto HH, Rabinovitch PS Mitochondrial oxidative stress in aging and healthspan, Longev Healthspan. (2014) 2046-2395-3-6. [2] Murphy MP How mitochondria produce reactive oxygen species Biochem J. (2009) 417(1):1-13 [3] Görlach A, Bertram K, Hudecova S, Krizanova O Calcium and ROS: A mutual interplay. Redox Biol. (2015) 6:260-71 [4] Köhler AC, Sag CM, Maier LS. Reactive oxygen species and excitation-contraction coupling in the context of cardiac pathology. J Mol Cell Cardiol. (2014) 73:92-102 [5] Giorgi-Coll S, Amaral AI, Hutchinson PJA, Kotter MR, Carpenter KLH. Succinate supplementation improves metabolic performance of mixed glial cell cultures with mitochondrial dysfunction. Sci Rep. (2017) 21;7(1):1003

P1 b/11 The vesicle trap. How to measure the real activity of the phagocyte NADPH oxidase? Xavier Serfaty1; Pauline Lefrançois2; Chantal Houée-Levin1; Stéphane Arbault2; Laura Baciou1, Tania Bizouarn1 1Laboratoire de Chimie Physique, UMR 8000, Paris Sud University, CNRS, Paris Saclay University, Orsay, France 2Institut des Sciences Moléculaire UMR 5255, groupe NSysA, University of Bordeaux, France

The phagocyte NADPH oxidase plays a key role in the killing of invaders during phagocytosis and in the inflammatory processes. The phox membrane heterodimer Nox2-p22 , also called flavocytochrome b558, containing the electron transport chain made of a flavin and two hemes, in interaction with four cytosolic proteins (p67phox, p47phox, p40phox and Rac1/2) constitutes the functional enzyme catalysing the

+ ∙- reaction NADPH + 2O2  NADP + 2O2 . Electrons cross the membrane from the cytosolic donor NADPH to the extracellular or phagosomal receptor dioxygen. The community working in vitro on the functioning of NADPH oxidase, commonly follows the progress of

∙- the enzymatic reaction by the O2 cytochrome c reduction (followed by absorption at 550 nm). We found that the enzyme turnover appeared twice higher by measuring NADPH oxidation rate than the Cytc reduction rate. We decided to analyse further the stoichiometry by measuring O2 by electrochemistry with a Clark electrode. Up to now, the literature provides neither explanation nor real discussion despite the critical importance to quantify exactly the enzyme turnover, since such measurements lead to most of the interpretations of NADPH oxidase enzymology. We decided to get to the bottom of this point by checking if this discrepancy is due to artefacts linked to Cytc measurements or intrinsic to the enzyme in its biochemical context. Using ionophores, detergents, temperature change, H2O2 measurements in bulk or in microreactor (liposomes and amplex-red) by confocal fluorescence microscopy, our results

∙- show that membrane permeability, vesicle confinement and unexpected secondary reactions (O2 and H2O2 disproportionations), have a strong impact on the discrepancy. These results highlight the crucial importance of careful measurements in compartmented systems such as vesicles as well as in cells to get the right interpretations and reach true conclusions.

P1 b/12 Enhanced ROS production in mitochondria from prematurely aging mtDNA mutator mice Irina G. Shabalina1, Daniel Edgar1, Natalia Gibanova1,2, Anastasia Kalinovich1, Natasa Petrovic1, Mikhail Yu. Vyssokikh2, Barbara Cannon1, and Jan Nedergaard1 1Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden 2The Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation

Mitochondrial DNA (mtDNA) mutations and reactive oxygen species (ROS) production are implicated in mitochondrial disease and in the aging process. The mtDNA mutator mice accumulate a large amount of mtDNA mutations giving rise to defective mitochondria and an accelerated aging phenotype. ROS measured in living mtDNA mice has been found increased with age [1], in contrast when analysed ex vivo no change in production of ROS by isolated mitochondria has been reported. To reconcile these apparently contradictory findings we examined here the mode of ROS production in parallel to membrane potential and oxygen consumption in skeletal muscle, heart and liver mitochondria on different substrates supply and inhibition of respiratory chain at various points. Using standard conditions for measuring ROS production, i.e. with succinate as substrate and ROS mainly resulting from reverse electron flow, we found a marked decrease in ROS production in the mtDNA mutator mitochondria compared to wildtype. In contrast, with forward electron flow from complex I (supported by pyruvate + malate or palmitoyl CoA + carnitine), mitochondrial ROS production was enhanced. ADP reduces ROS production in wildtype mitochondria on all types of substrates; this reduction was significantly lower in mtDNA mutator mitochondria. ADP also accelerates hydrogen peroxide removal by wildtype mitochondria but does not do it in mtDNA mutator mitochondria. Looking at oxidative damage, levels of protein 4-HNE-adducts and carbonylation were increased in liver. In addition, mutator tissues had increased susceptibility to lipid peroxidation. Taken together this suggests that ROS may indeed have a detrimental effect on the mtDNA mutator mice and this effect is more evident under conditions mimicking conditions in vivo (physiological substrates and high ADP level).

1. A. Logan, I.G. Shabalina, T.A. Prime, S. Rogatti, A.V. Kalinovich, R.C. Hartley, R.C. Budd, B. Cannon, M.P. Murphy, In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice, Aging Cell 13 (2014) 765-768

P1 b/13 Preservation of Mitochondrial Bioenergetics with CAT-4001 after Peroxide Injury Alexander H. Thomson1, Mitchell E. Allen1, Justin B. Perry1, Andrew J. Nichols2, John F. Reilly2, Pradeep Bista2, Diana Lee2, Chi B. Vu2, David A. Brown1 1Virginia Tech, Department of Human Nutrition, Foods, and Exercise and the Virginia Tech Center for Drug Discovery, Blacksburg, VA 2Catabasis Pharmaceuticals, One Kendall Square, Suite B14202, Cambridge, MA

Mitochondrial dysfunction is noted across disease states and is a primary target for emerging treatments. In these studies, we determined the bioenergetic efficacy of a novel cell-permeable compound, CAT-4001. CAT-4001 is a conjugate of monomethyl fumarate (activates Nrf2) and docosahexaenoic acid (inhibits NF-kB). Once inside cells, the conjugate was hydrolyzed by intracellular enzymes to synergistically decrease inflammation (by inhibiting NF-kB) and stimulate cellular resistance to oxidative stress (by activating Nrf2). Oxygen consumption rates (OCR) were assessed in undifferentiated C2C12 myoblasts exposed to a 500µM hydrogen peroxide insult. The effects of CAT-4001 were compared to positive controls N-acetylcysteine (NAC) and catalase, using a 24-hour treatment paradigm. Peroxide treatment significantly decreased maximal OCR (90±19 pmol/min) compared to untreated control (184±34 pmol/min, P<0.05). Treatment with 1µM CAT-4001 significantly improved maximal OCR (206±19 pmol/min) to levels comparable to positive controls (192±25 pmol/min for 5mM NAC and 194±19 pmol/min for catalase). 10µM of CAT-4001 resulted in substantially higher maximal OCR (348±20 pmol/min). ATP-dependent respiration was also significantly reduced after peroxide insult, decreasing ATP-dependent OCR from 51±8 pmol/min in untreated cells to 32±5 pmol/min after hydrogen peroxide. NAC and catalase treatment led to modest improvements in ATP-dependent respiration that did not reach statistical significance (47±5 pmol/min and 49±4 pmol/min, respectively). Both 1µM and 10µM CAT-4001 evoked significantly higher ATP-dependent respiration (69±5 pmol/min and 113±5 pmol/min, respectively; P<0.05 compared to all other peroxide-treated groups). These data highlight the efficacy of CAT-4001 as a treatment to improve mitochondrial function by improving cellular tolerance to oxidative insults.

P1 b/14 The interplay between mitochondrial reactive oxygen species formation and ubiquinone reduction level in Acanthamoeba castellanii mitochondria Karolina Dominiak, Agnieszka Koziel, Wieslawa Jarmuszkiewicz Department of Bioenergetics, Adam Mickiewicz University, Poznan, Poland

Acanthamoeba castellanii is a small non-photosynthesizing amoeba, which is frequently used in studying mitochondrial respiratory chain. Coenzyme Q (Q) is an important mobile component of the mitochondrial electron transport chain that takes part in mitochondrial reactive oxygen species (ROS) production, contributing to oxidative stress and damaging mitochondria and cells. On the other hand, Q displays an antioxidant property that protects the cells from harmful ROS. A plant-type respiratory chain of A. castellanii mitochondria contains two QH2-oxidizing pathways, the classical cytochrome pathway and the alternative ubiquinol oxidase (AOX). The aim of our study was to elucidate the relationship between ROS formation and the reduction level of Q pool under different mitochondrial respiring conditions, i.e., at a diverse engagement of Q-reducing pathway (succinate dehydrogenase, complex II) and QH2-oxidizing pathways

(the cytochrome pathway and AOX) in isolated A. castellanii mitochondria. The Q reduction level was increased by inhibition of QH2- oxidizing pathways (complex III, complex IV, or AOX) or through inhibition of oxidative phosphorylation system (ATP synthase or ATP/ADP antiporter). The Q pool was shifted to a more oxidized state through inhibition of the Q-reducing pathway (substrate dehydrogenase) or by stimulating the activity of QH2-oxidizing pathways under uncoupling conditions (the cytochrome pathway) or under GMP-activation (AOX). We measured the Q reduction level under given mitochondrial oxygen consumption and membrane potential conditions in relation to H2O2 formation. Our results indicate that membranous Q reduction level is proportional to ROS formation within a defined respiratory path-dependent range.

Acknowledgments: This work was supported by the National Science Center Grants, Poland (2016/21/B/NZ3/00333) and partially by KNOW Poznan RNA Centre (01/KNOW2/2014).

P2 a/1 Acanthamoeba castellanii UCP protein expressed in yeast system; influence on viability of SOD1- and SOD2-deficient yeast under oxidative stress Nina Antos-Krzeminska, Klaudia Gradzka, Katarzyna Jasiewicz, Wieslawa Jarmuszkiewicz Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland

Uncoupling proteins (UCPs) belong to the inner mitochondrial membrane anion transporters’ family. They uncouple the oxidation of fuels via the electron respiratory chain from ATP synthesis by mediating proton leak into mitochondrial matrix. A direct consequence of the free fatty acid-stimulated UCP activity is a decrease in the oxidative phosphorylation efficacy. Therefore, UCPs dissipate energy and decrease reactive oxygen species production affecting energy cellular metabolism. The aim of this study was to analyse the role of Acanthamoeba castellanii uncoupling protein (AcUCP) that was expressed heterologously in superoxide dismutase (SOD1 and SOD2)- deficient yeast cells. The coding sequence of AcUCP was amplified and cloned to the yeast expression pYES2 vector. Yeast Saccharomyces cerevisiae do not possess UCP, so they provide an excellent model to confirm functionally AcUCP gene’s product presence. We transformed SOD1-deficient and SOD2-deficient strains of S. cerevisiae with pYES2+AcUCP vector or empty vector (pYES2) and confirmed the proper transformation by selection of mutants in the medium without uracil. SOD knockout yeast, especially SOD1-deficient strain, are particularly sensitive to oxidative stress that affects their growth. After D-galactose induction, the abundance of AcUCP in yeast cells’ extracts was confirmed by mass spectrometry analysis. Subsequently, we performed yeast viability experiments. Yeast cells were subjected to an external oxidative stress, i.e., different concentrations of H2O2 for 3h. In both SOD1- deficient and SOD2-deficient yeast, the AcUCP presence increased significantly their survival rate by about 100% and 20%, respectively. Our results indicate that AcUCP may complement an antioxidative function of yeast SOD1 and SOD2.

This work is supported by the grant from the National Science Center 2016/21/B/NZ3/00333 and partially by KNOW Poznan RNA Centre (01/KNOW2/2014).

P2 a/2 Acclimation of Danio Renio (zebrafish) embryos to the cold Clarissa S. Barthem1, José Leonardo de Oliveira², Natália M. Feitosa² and Wagner S. da Silva1 1Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 2Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé (NUPEM), Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Thermogenesis in mammals has been arduously studied in the past years. As they are endothermic animals, thermoregulation is highly regulated, having multiples proteins involving in this pathway. For example, sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) has been shown to uncouple the ATP hydrolysis to Ca2+ pumping, transducing the energy to heat. As a similarly mechanism, UCP1 uncouples the gradient of H+ from the ATP synthesis in the mitochondria, generating heat. Danio renio, or zebrafish, is a largely know model of research. Its’ external fertilization, rapid development and translucent embryo are some of the features that make zebrafish a promising model. Although it is an ectothermic animal, zebrafish also has ortholog genes of mammals’ thermogenic proteins as SERCA2 and UCP1. However the role of these genes in ectothermic animals is still unknown. Thus, the aim of this work was to evaluate the effects of cold acclimation during early phase of zebrafish development. 96 hours post fertilization, larvae were incubated during 2, 4 or 6 hours at the control temperature of 28ºC, or at 18ºC. After experiment, the mRNA of the larvae were extracted for expression analysis of atp2a1, atp2a2b (genes that encodes SERCA1 and SERCA2b respectively), ucp1 and ucp2, by rt-PCR. Cold acclimation suggests a reduction in mRNA levels of atp2a1 and atp2a2b only in 2 hours exposure (0.12-fold and 0.60- fold respectively). ucp2 levels remained unchanged in all conditions. However, ucp1 levels increased after 6h at 18ºC compared to 28ºC group (1.55-fold). In summary atp2a1 and 2b were not cold modulated in longer times of treatment, 4 and 6 hours, but ucp1 seems to be modulated at 6 hours of acclimation. This work suggests that ucp1 in ectothermic animals as zebrafish might be regulated at colder temperatures and thus needs to be further elucidated.

P2 a/3 Molecular Identity and Regulatory Mechanisms of the Mitochondrial Uncoupling Protein of Regular Somatic Tissues Ambre M. Bertholet1, Andriy Fedorenko1, Edward T. Chouchani2, Lawrence Kazak2, Alessia Angelin3, Sara Vidoni2, Joonseok Cho4, Naohiro Terada4, Bruce M. Spiegelman2, Douglas C. Wallace3, and Yuriy Kirichok1 1Department of Physiology, University of California San Francisco, San Francisco, CA, USA 2Dana-Farber Cancer Institute & Department of Cell Biology, Harvard Medical School, Boston 3Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA 4Department of Pathology, University of Florida College of Medicine, Gainesville, FL, USA

Molecular mechanisms of mitochondrial uncoupling, whereby mitochondria generate heat, remain a fundamental problem of bioenergetics. Mitochondrial uncoupling and thermogenesis are due to H+ leak across the inner mitochondrial membrane. This H+ leak short-circuits mitochondrial H+ gradient (∆p), reduces efficiency of ATP production, and makes mitochondria generate heat. In the specialized thermogenic tissue brown fat, the mitochondrial H+ leak is mediated by uncoupling protein 1 (UCP1), but the molecular mechanisms of mitochondrial H+ leak in all other tissues remain poorly understood. Here, by directly measuring H+ currents across the whole inner membrane using patch-clamp technique, we provide unambiguous identification of the mitochondrial uncoupling protein of regular somatic tissues. We also identify the mechanisms of physiological regulation of this uncoupling protein. This ubiquitously expressed mitochondrial uncoupling protein is likely to play an important role in controlling body energy expenditure and metabolic efficiency, and could be targeted in obesity, diabetes, and age-related disorders.

P2 a/4 Complete knock-out of both COX4 isoforms in HEK293 cells leads to combined complex IV and complex I deficiency Kristýna Čunátová, David Pajuelo Reguera, Marek Vrbacký, Josef Houštěk, Tomáš Mráček, Petr Pecina IPHYS CAS, Department of Bioenergetics, Prague, Czech Republic

Oxidative phosphorylation (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of respiratory chain. One of its regulatory subunits, Cox4, is an early-assembling COX component, essential for incorporation of Cox2 catalytic subunit, thus for the assembly of catalytically functional enzyme. Moreover, regulated expression of its two isoforms (Cox4i1, Cox4i2) is hypothesized to optimize respiratory chain function according to oxygen supply. However, the functional impact of the isoform switch for mammalian cells is still only partly understood. We established HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) employing CRISPR CAS9-10A paired nickase technology and characterized its impact on OXPHOS. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of Cox subunits resulting in total absence of COX holoenzyme. Moreover, content of complex I subunits as well as the assembled complex were decreased in COX4i1/4i2 KO clones. In contrast, complexes II, III, and V were not significantly affected. Pulse-chase metabolic labelling of mtDNA-encoded proteins uncovered decrease of COX and complex I subunits translation, while complexes III and V subunits were not significantly affected. Partial impairment of mitochondrial proteosynthesis correlated with decreased level of mitochondrial ribosomal proteins. As expected, mitochondrial respiration was undetected in COX4i1/4i2 KO cells, and was compensated by increased glycolytic capacity. In summary, the HEK293- based cellular model of COX4i1/4i2 KO displayed phenotype of total COX absence, making cells fully reliant on OXPHOS-independent ATP production. We hypothesise that impairment of mitochondrial proteosynthesis represents a secondary effect of respiratory chain dysfunction. Supported by Czech Science Foundation 16-13671S.

P2 a/5 Mitochondrial supercomplexes do not enhance catalysis by quinone channeling Justin G. Fedor, Judy Hirst Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom

The respiratory machinery of mitochondria are often organised into supercomplex assemblies of varying stoichiometry, for instance the

‘respirasome’ comprises complexes I:III2:IV. These supercomplex assemblies have been proposed to exist for a number of reasons, but most controversially they may channel electron carriers (cytochrome c and ubiquinone). While previous studies have demonstrated that cytochrome c is not channeled and that the quinone pool is shared between complexes I, II and III, this point continues to be controversial. Furthermore, the question remains whether there is a kinetic advantage for electron transfer via the quinone pool in supercomplexes, even if quinone is not channeled. To address this question, and to more definitively demonstrate that quinone is not channeled in supercomplexes, the cyanide-insensitive alternative oxidase (AOX) from Trypanosoma brucei brucei was introduced to a suspension of bovine heart submitochondrial particles. This is a conceptually simple and classic approach to test for substrate channeling: if a substrate is channeled the introduction of a competing enzyme for that substrate should not have activity. We find that as the amount of AOX is increased, the NADH:O2 cyanide- sensitivity decreases while the rate increases up to 4-fold [1]. After confirming that AOX is not perturbing the structural integrity of the membrane nor of the supercomplexes, we conclude that since complex I is predominantly in supercomplexes, that quinone is not channeled and quinone diffusion is not rate limiting. While our results suggest quinone channeling does not occur, we tend to believe that supercomplexes may exist to better ensure a homogenous distribution of active sites in the crowded inner mitochondrial membrane.

[1] J.G. Fedor, J. Hirst, Mitochondrial supercomplexes do not enhance catalysis by quinone channeling, Cell Metab. In Press (2018).

P2 a/6 Isolation of the yeast respiratory supercomplex using styrene-maleic acid Andrew M. Hartley1, Brigitte Meunier2, Amandine Maréchal1 1Institute of Structural and Molecular Biology, University College London, Gower Street, London, United Kingdom 2Institute for Integrative Biology of the Cell, Bat. 26, Avenue de la Terrasse, Gif-sur-Yvette, France

Saccharomyces cerevisiae offers an ideal opportunity to investigate mitochondrial respiratory chains due to its genetic amenability, and the ability to produce large quantities of wild-type and mutant proteins for detailed biophysical characterisation. In addition, yeast respiratory complexes III and IV share extensive homology with mammalian forms, and have also been shown to form supercomplexes [1]. However, use of the yeast system as a model is limited by the lack of detailed structural information available. Despite the recent publication of several mammalian respiratory supercomplex structures in recent years [2-5], a high resolution structure of the yeast respiratory supercomplex (and that of complex IV alone) is yet to be determined: to date, the best available structure of the yeast respiratory supercomplex is a 24 Å pseudo-atomic map [6]. Styrene-maleic acid (SMA) [7] has previously been used to successfully extract complex IV from yeast mitochondrial membranes [8,9], and recently has been used to extract the intact respiratory supercomplex from F. johnsoniae [10]. Here, we describe our progress towards the isolation of the S. cerevisiae respiratory supercomplex using SMA, its biochemical and biophysical characterisation, and the determination of its structure by cryo-electron microscopy.

1. H. Schägger and K. Pfeiffer, Supercomplexes in the respiratory chain of yeast and mammalian mitochondria, EMBO 19 (2000) 1777-1783 2. J.A. Letts, K. Fiedorczuk and L.A. Sazanov, The architecture of respiratory supercomplexes, Nature 537 (2016) 644-648 3. J. Gu, M. Wu, R. Guo, K. Yan, J. Lei, N. Gao and M. Yang, The architecture of the mammalian respirasome, Nature 537 (2016) 639- 643 4. J.S. Sousa, D.J. Mills, J. Vonck and W. Kuhlbrandt, Functional asymmetry and electron flow in the bovine respirasome, eLIFE 5 (2016) 21290 5. R. Guo, S. Zong, M. Wu, J. Gu and M. Yang, Architecture of Human Mitochondrial

Respiratory Megacomplex I2III2IV2, Cell 170 (2017) 1247-1257 6. E. Mileykovskaya, P.A. Penczek, J. Feng, V.K.P.S. Mallampalli, G.C. Sparagna and W. Dowhan, Arrangement of the Respiratory

Chain Complexes in Saccharomyces cerevisiae Supercomplex II2IV2 Revealed by Single Particle Cryo-Electron Microscopy, JBC 287 (2012) 23095-23103 7. J.M. Dörr, S. Scheidelaar, M.C. Koorengevel, J.J. Dominguez, M. Schäfer, C.A. van Walree and J.A. Killian, The styrene-maleic acid copolymer: a versatile tool in membrane research, Eur Biophys J 45(2016) 3-21 8. A.R. Long, C.C. O’Brien, K. Malhotra, C.T. Schwall, A.D. Albert, A. Watts and N.N. Adler, BMC Biotechnology 13 (2013) 41 9. I.A. Smirnova, D. Sjöstrand, F. Li, M. Björck, J. Schäfer, H. Östbye, M. Högbom, C. von Ballmoos, G.C. Lander, P. Ädelroth and P. Brzezinski, BBA 1858 (2016) 2984-2992 10. C. Sun, S. Benlekbir, P. Venkatakrishnan, Y. Wang, S. Hong, J. Hosler, E. Tajkhorshid, J.L. Rubinstein and R.B. Gennis, Nature 557 (2018) 123-126

P2 a/7 The well-known biocide triclosan demonstrates unusually high protonophoric activity on artificial planar BLM and bacterial cell membranes, but moderate uncoupling potency in isolated mitochondria and neuronal cells Elena A. Kotova1, Ekaterina S. Nosikova1,2, Lyudmila B. Popova1, Pavel A. Nazarov1, Lyudmila S. Khailova1, Yuri N. Antonenko1 1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 2Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia

The formerly widely used broad-spectrum biocide triclosan has recently attracted much attention due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that triclosan is an uncoupler of oxidative phosphorylation in mitochondria, there have been previously no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). In our recent publication [1], we for the first time reported data on high efficacy of triclosan in generating proton-selective electric current across planar BLM. In fact, triclosan proved to be a more effective protonophore on planar BLM, than classical uncouplers, such as CCCP. By measuring bacterial membrane potential with the voltage-sensitive dye DiSC3(5), we observed a strong depolarizing effect of triclosan on bacterial membranes, which correlated with suppression of Bacillus subtilis growth. This correlation might imply substantial contribution of triclosan protonophoric activity to its antimicrobial efficacy, along with the generally accepted mechanism associated with triclosan inhibition of bacterial fatty acids synthesis. With isolated rat liver mitochondria, triclosan exhibited protonophoric activity, as monitored by proton-dependent mitochondrial swelling. Importantly, similar to CCCP, although much less effectively, triclosan induced release of Ca2+ ions previously accumulated in isolated mitochondria. A comparison of triclosan effects on neurons from Lymnaea stagnalis with those of conventional mitochondrial uncouplers allowed us to ascribe the triclosan-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Thus, mitochondrial uncoupling could alter neuronal function through distortion of Ca2+ homeostasis.

1. L.B. Popova, E.S. Nosikova, E.A. Kotova, E.O. Tarasova, P.A. Nazarov, L.S. Khailova, O.P. Balezina, Y.N. Antonenko, Protonophoric action of triclosan causes calcium efflux from mitochondria, plasma membrane depolarization and bursts of miniature end-plate potentials, Biochim. Biophys. Acta - Biomembranes 1860 (2018) 1000-1007

P2 a/8 The impact of genipin in cells is not only confined to UCP2 Jürgen Kreiter1, Anne Rupprecht1, Lars Zimmermann1, Maria Fedorova2, Michael Moschinger1, Tatyana I. Rokitskaya3, Lars Gille1, Yuri N. Antonenko3, Elena E. Pohl1 1University of Veterinary Medicine, Vienna, Austria 2Center of Biotechnology-Biomedicine, University of Leipzig, Germany 3Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russia

Genipin is a natural cross-linker found in Gardenia jasminoides Ellis and is widely used as a specific inhibitor of uncoupling protein 2 (UCP2). UCP2 belongs to the mitochondrial carrier superfamily, mediates a regulated proton leak through the inner mitochondrial membrane and is found in rapidly proliferating cells [1]. In this work, we tested the hypothesis that genipin may simultaneously affect several proteins in cells. For that, we investigated (i) the activity of several recombinant proteins reconstituted in artificial bilayers [2] in the presence/absence of genipin (ii) mitochondrial membrane potential in neuroblastoma cells after addition of genipin using confocal microscopy and (iii) genipin-mediated modifications of proteins and lipids using mass spectrometry. Our findings outline that genipin, in addition to UCP2, inhibits its homologs UCP1 and UCP3 and complex III of the respiratory chain, but not the non-mitochondrial protein α-hemolysin. Based on the results with mutant proteins and competitive inhibition experiments, we propose the molecular mechanism which may be responsible for the multiple action of genipin.

1. A. Rupprecht et al., Uncoupling protein 2 and 4 expression pattern during stem cell differentiation provides new insight into their putative function, PLoS ONE 9 (2) (2014) e88474 2. V. Beck et al., Biochimica et Biophysica Acta 1757 (2006) 474–479

P2 a/9

Functional and Structural Characterization of Isolated Mammalian Mitochondrial Respiratory Supercomplex I+III2 James A. Letts, Karol Fiedorczuk and Leonid A. Sazanov Institute of Science and Technology Austria, Klosterneuberg, Austria

The protein complexes of the inner mitochondrial membrane electron transport chain (ETC) are responsible for catalyzing the terminal electron transfer reactions from NADH or succinate, via co-enzyme Q (CoQ) and cytochrome c (cyt c), to O2 in cellular respiration.

These energetically ‘downhill’ electron transfers are coupled to proton pumping in complexes I (CI), III2 (CIII2) and IV (CIV), conserving their energy in the form of a H+ gradient across the inner mitochondrial membrane. Finally, the ATP synthase complex (CV) harvests the H+ gradient to catalyze the production of ATP, which is then used throughout the cell to power many essential reactions. It has been shown that the ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry [1], but the physiological roles of these SCs remains unclear [2]. Here I will present the isolation, using a novel procedure, of a chromatographically pure, functional SC

I+III2 lipoprotein particle. In addition to functional characterisation, the structure of this SC has been resolved to ~4.0 Å resolution with multiple structural classes. This preparation provides a new framework for structure/function studies addressing the physiological roles of respiratory SCs.

[1] H. Schägger, K. Pfeiffer, The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes, J. Biol. Chem. 276 (2001) 37861–37867. doi:10.1074/jbc.M106474200. [2] J.A. Letts, L.A. Sazanov, Clarifying the supercomplex: the higher-order organization of the mitochondrial electron transport chain, Nat. Struct. Mol. Biol. 24 (2017) 800–808. doi:10.1038/nsmb.3460.

P2 a/10 The effect of lipid phase transitions in mitochondrial membranes on respiration and OxPhos system supercomplex formation Semen V. Nesterov1, 3, Yulia A. Skorobogatova1, 3, Lev S. Yaguzhinskiy1, 2 1Institute of Cytochemistry and Molecular Pharmacology, Moscow, Russian Federation 2Belozersky Research Institute for Physico-Chemical Biology, Moscow, Russian Federation 3Moscow Institute of Physics and Technology, Dolgoprudny, Russian Federation

The work is devoted to the study of structural rearrangements in mitochondrial membranes during the transition of mitochondrial oxidative phosphorylation (OxPhos) system to the highly coupled functioning mode (supercomplex), which formation is facilitated by hypotonic conditions [1]. Membrane-soluble aromatic fluorescent probe pyrene was used to estimate the intensity of lipid-protein interactions and lipid mobility in rat liver mitochondria. Structural transitions of annular lipids (located in the vicinity of proteins) were detected at 19 and 25°C. Literature-based analysis of lipid phase transitions in liposomes and proteoliposomes [2] made it possible to identify 19 and 25°C correspondently as pretransition and main transition temperatures of mitochondrial annular lipids. The observed changes in lipid-pyrene interaction are consistent with abrupt alterations in efficiency of tryptophan fluorescence quenching by pyrene occurring at the same temperatures. ATP synthesis rate on succinate oxidation was investigated at the same conditions and it was found a break on the Arrenius plot at 25°C indicating a reduction of apparent activation energy in the high-temperature range. It should be stressed that this functional alterations are not accompanied by decrease in efficiency and that is why they cannot be attributed to oxidative damage of mitochondria at high temperature. Obtained data indicates that hypotonic conditions stimulate the formation of specific raft-like clusters in mitochondrial membranes which promotes the OxPhos system supercomplex formation.

1. I.P. Krasinskaya, V.N. Marshansky, S.F. Dragunova, L.S. Yaguzhinsky, Relationships of respiratory chain and ATP-synthetase in energized mitochondria, FEBS Lett. 167 (1984) 176–180. 2. L. Picas, S. Merino-Montero, A. Morros et al., Monitoring pyrene excimers in lactose permease liposomes: revealing the presence of phosphatidylglycerol in proximity to an integral membrane protein, J. Fluoresc. 17 (2007) 649–654.

P2 a/11 PFOA modulates activity and gene and protein expressions of UCP1 in brown adipocytes Patrícia Reckziegel1,2, Natasa Petrovic1, Antonia Giacco3, Fernando Goglia3, Barbara Cannon1, Jan Nedergaard1 1Department of Molecular Bioscience, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden 2Department of Pharmacology, Biomedical Sciences Institute, University of São Paulo, São Paulo, Brazil 3Department of Science and Technologies, University of Sannio, Benevento, Italy

Perfluorooctanoic acid (PFOA) is a synthetic organofluoride surfactant used in a wide variety of products due to its thermal resistivity. However, it can accumulate in the environment and demonstrate toxic effects in animals. Experimentally, PFOA decreases body weight of mice and increases the respiratory rate in isolated mitochondria. Here we evaluated the direct effect of PFOA on the respiratory rate of mature adipocytes as well as its effects on the expression of thermogenic and adipogenic markers in adipocyte primary cultures. Mature brown adipocytes were isolated from wild-type or UCP1 knockout mice. PFOA stimulated oxygen consumption rate of mature adipocytes in a dose-dependent manner (2-8 mM); this effect was not observed in adipocytes devoid of UCP1. Furthermore, we evaluated if PFOA could change the UCP1 gene and protein expressions in adipocytes. Primary cultures of white (inguinal) and brown adipocytes were exposed to 200 or 400 µM PFOA or vehicle for 48 hours (from day 5 to day 7). Before harvesting, cells were stimulated with 1 µM norepinephrine for 2 or 48 hours and were used for analyses of mRNA and protein expression. PFOA increased the expression of UCP1 and other thermogenesis-related genes (PPARGC1A, CPT1) in both white and brown adipocytes. In addition, PFOA increased the expression of FABP4 and changed the gene expressions of PPARG and PPARG2 (general adipogenic marker genes). PFOA also increased the expression of PPARA. Moreover, the protein levels of UCP1 and FABP4 were increased in adipocytes exposed to PFOA (thus were positively correlated to their respective gene expression). The mechanism by which PFOA increases the expressions of thermogenic and adipogenesis-related genes/proteins is under investigation. In summary, PFOA exposure may affect thermogenesis by stimulating the activity and gene and protein expressions of UCP1 in brown adipocytes. Funding: São Paulo Research Foundation (FAPESP, grant#2017/08850-4) and Formas.

P2 a/12 Molecular bases of mitochondrial uncoupling protein 1 induced thermogenesis Mathilde S. Piel1,2, Sandrine Masscheleyn1,2, Fabrice Giusti1,2, Florian Accettella1,2, Manuela Zoonens1,2, Karine Moncoq1,2, Bruno Miroux1,2 1 Université Paris-Diderot, Paris, France 2 Institut de Biologie Physico Chimique, Paris, France

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocyte and belongs to the mitochondrial carrier family (SLC25). In the presence of long-chain fatty acids (LCFA), UCP1 increases the H+ conductance to ‘short-circuit’ the proton-motive force, which, in turn, increases fatty acid oxidation and energy release as heat. The precise location of the LCFA binding site(s) has not been determined. Different models of UCP1 and UCP2 have been obtained by NMR in dodecylphosphocholine (DPC) and Zhao et al. proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation in proteoliposomes [1]. However, DPC has been shown to inactivate UCP1 [2]. Therefore, we revisited those mutants in a mitochondrial environment using a robust expression system previously validated for UCP1 function. Wild type UCP1 and four mutants (R54S, K56S, K269S and K56S/K269S) were expressed in S. cerevisiae and mitochondrial respiration was assayed on permeabilized spheroplast. The highest UCP1 LCFA dependent stimulation of respiration was obtained with a lauric acid/BSA ratio of 4. At this ratio, all four mutants were activated by LCFA similarly to the wild type protein. To address the identification of the fatty acid binding site in UCP1, we have synthesized LCFA based ligands functionalized with active probes and tested their ability to activate UCP1 in yeast mitochondria. We used β-lactoglobulin as a template to optimize crosslinking conditions either in solution or in crystals. Crosslinking experiments on UCP1 are under progress.

1. L. Zhao, S. Wang, Q. Zhu, B. Wu, Z. Liu, B. OuYang, J.J. Chou, Specific Interaction of the Human Mitochondrial Uncoupling Protein 1 with Free Long-Chain Fatty Acid, Structure 25 (2017) 1-9

2. M. Zoonens, J. Comer, S. Masscheleyn, E. Pebay-Peyroula, C. Chipot, B. Miroux, F. Dehez, Dangerous Liaisons between Detergents and Membrane Proteins. The Case of Mitochondrial Uncoupling Protein 2, J. Am. Chem. Soc. 135 (2013) 15174-15182

P2 a/13 The role of Rcf1 as a modulator of the respiratory chain in yeast Jacob Schäfer, Hannah Dawitz, Tobias Nilsson, Martin Ott, Pia Ädelroth, Peter Brzezinski Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden

In S. cerevisiae respiratory supercomplexes are stabilized by cardiolipin and small proteins. One class of these proteins is the respiratory supercomplex factors (Rcf) [1–3]. Rcf1 directly interacts with cytochrome c oxidase (CytcO) [4]. Here, CytcO from S. cerevisiae was isolated in two sub-states, one with spectral and functional properties similar to those of CytcOs characterized earlier and one with an altered structure and lower activity. The fraction of the latter sub-state increased upon removal of Rcf1. The structurally altered form displayed a lower midpoint potential of heme a3 as well as accelerated ligand binding, suggesting structural changes around the catalytic site. We showed that the activity of CytcO lacking Rcf1 could be restored to that of the wild-type CytcO by co- reconstitution of the modified CytcO sub-state with pure Rcf1, expressed in E. coli, in liposomes.

References: [1] V. Strogolova, A. Furness, M. Robb-McGrath, J. Garlich, R.A. Stuart, Rcf1 and Rcf2, members of the hypoxia-induced gene 1 protein family, are critical components of the mitochondrial cytochrome bc1-cytochrome c oxidase supercomplex, Mol. Cell. Biol., 32 (2012) 1363–73. [2] Y.-C.C. Chen, E.B. Taylor, N. Dephoure, J.-M.M. Heo, A. Tonhato, I. Papandreou, N. Nath, N.C. Denko, S.P. Gygi, J. Rutter, Identification of a protein mediating respiratory supercomplex stability, Cell Metab., 15 (2012) 348–360. [3] M. Vukotic, S. Oeljeklaus, S. Wiese, F.N. Vögtle, C. Meisinger, H.E. Meyer, A. Zieseniss, D.M. Katschinski, D.C. Jans, S. Jakobs, B. Warscheid, P. Rehling, M. Deckers, Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex, Cell Metab., 15 (2012) 336–347. [4] J. Garlich, V. Strecker, I. Wittig, R.A. Stuart, Mutational analysis of the QRRQ motif in the yeast hig1 type 2 protein Rcf1 reveals a regulatory role for the cytochrome c oxidase complex, J. Biol. Chem., 292 (2017) 5216–5226.

P2 a/14 Purification and analysis of a respiratory supercomplex in the Firmicutes Bacillus subtilis and Geobacillus stearothermophilus Nathalie Sisattana1, Lucie Bergdoll2, Elodie Point1, Daniel Picot1 1Institut de Biologie Physico-Chimique, UMR 7099 CNRS/Université Paris Diderot, Paris, France 2Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA

Rieske/cytochrome b complexes are important components of electron transfer chains, they increase the yield of translocated protons per transferred electron through the use of a Q-cycle, but at the cost of introducing a rate-limiting step. The best studied complexes of this family are cytochromes bc1 from proteobacteria and mitochondria and cytochromes b6f in oxygenic photosynthesis, they use high potential quinones: ubiquinone or plastoquinone. Low GC gram positive bacteria (Firmicutes) use a complex b6c that belongs to the same group as the photosynthetic b6f complex (the green clade) [1], but use a low potential quinone, menaquinone. The b6c has a split cytochrome b, a haem ci but unlike the b6f has a C- type cytochrome anchored to the subunit IV. The b6c complexes are associated with the cytochrome c oxidase caa3 and the cytochrome c550 to form a supercomplex. The study of the supercomplex b6c:c550:caa3 from Geobacillus stearothermophilus allowed us to investigate the thermodynamic constraints on the Q-cycle by comparing high and low potential quinones [2]. B. subtilis has a respiratory chain composed of several dehydrogenases such as NDH-II, SDH and GPDH that use respectively NADH, succinate and glycerol phosphate as substrates. They reduce menaquinone that can then be reoxidised by the quinol oxidase aa3 or by the supercomplex b6c:c550:caa3 depending on the growth conditions. We will present and discuss a protocol to purify and analyse these supercomplexes from B. subtilis.

1. W Nitschke W, R. van Lis, B. Schoepp-Cothenet, F. Baymann, The "green" phylogenetic clade of Rieske/cytb complexes, Photosynth Res. 104 (2010) 347-55. 2. L. Bergdoll, F. Ten Brink, W. Nitschke, D. Picot, F. Baymann, From low- to high-potential bioenergetic chains: Thermodynamic constraints of Q-cycle function, Biochim Biophys Acta 1857 (2016) 1569-79.

P2 a/15 Protein turnover within mitochondrial complexes Ilka Wittig, Juliana Heidler, Valentina Strecker Functional Proteomics, Goethe University, Frankfurt, Germany

Many patients with mitochondrial disorders suffer from impaired assembly of mitochondrial protein complexes due to defects in genes encoding assembly factors. The sequence of protein complex assembly was intensively studied in cell culture with proliferating cells. These studies reflect mostly the de-novo assembly and give only limited information of the protein complex dynamics in differentiated cells and tissues. Blue-native electrophoresis (BNE) has become a popular tool to study steady state levels and assembly of mitochondrial complexes in cells from many model organisms and from patients. In combination with quantitative mass spectrometry even scarce sub-complexes, assembly intermediates and complex remodeling can be studied. In this study, we combined complexome profiling and pulse stable isotope labeling of amino acids in cell culture (Pulsed-SILAC) to study turnover of single proteins within protein complexes in proliferating and differentiated cells. The results show that mitochondrial protein complexes contain modules with higher turnover rates than other parts of the complexes indicating that de-novo assembly and repairing mechanisms ensure bioenergetics function in proliferating and differentiated cells.

P2 a/16 Erich Gnaiger1,2, on behalf of COST Action CA15203 MitoEAGLE consortium3 1D. Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria 2Oroboros Instruments, Innsbruck, Austria 3http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2018-02-08

Mitochondrial respiratory states and rates: building blocks of mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow IUPAC guidelines on terminology in physical chemistry, extended by considerations on open systems and irreversible thermodynamics. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols to the nomenclature of classical bioenergetics. In the frame of COST Action MitoEAGLE open to global bottom- up input, we endeavour to provide a balanced view on mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes [1]. Uniform standards for evaluation of respiratory states and rates will ultimately support the development of databases of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.

Reference: 1. MitoEAGLE preprint 2018-06-10(39) Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology Part 1. - http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2018-02-08

P2 a/17 Assunta Lombardi2, Elena Silvestri1, Alessandra Gentile2, Giuseppe Petito3, Antonia Lanni3, Fernando Goglia1 1Department of Science and Technology University of Sannio, Italy 2Department of Biology University of Naples “Federico II”, Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”

Absence of UCP3 impacts mitochondrial glycerol-3-phosphate dehydrogenase activity and enzymatic antioxidant capacity in brown adipose tissue of mice acclimated at thermoneutrality

Brown adipose tissue (BAT) expresses both uncoupling protein-1 and -3 (UCP1, UCP3). Despite the role played by UCP1 in non- shivering thermogenesis is well established, the function of UCP3 has not received universal acceptance and is still under debate. In the present study we gain further insight into this aspect by evaluating the impact of the absence of UCP3 on BAT mitochondria functionality. We used wild type (WT) and UCP3 knockout (KO) female mice, housed at thermoneutrality. The lack of UCP3 did not influence mitochondrial respiration rate when using piruvate (+ malate) as respiratory substrate, while a significant reduction was observed when using glycerol-3-phosphate (G3P). When evaluating the kinetic response of the reactions involved in the oxidation of the G3P and in the formation of proton motive force to a change in membrane potential, we found an inhibition of the above reactions. No differences in the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH) protein levels were observed between WT and KO mice, on the other hand, G3PDH in gel activity was significantly reduced in KO mice, thus indicating the involvement of a post-trasductional mechanism in such a reduction. In the presence of G3P as respiratory substrate, BAT mitochondria of KO mice showed an increase in the percentage of electrons that, during their transport by respiratory chain, leaks and reduces oxygen to superoxide; this process is also associated with a damage to mitochondrial membrane lipids as evident by the measured increase of lipid hydroperoxides levels. In addition, the expression of enzymes involved in the mitochondrial antioxidant defense such as superoxide dismutase -2 and catalase were significantly enhanced in KO mice. As a whole, these data indicate that the absence of UCP3 in BAT of mice housed at thermoneutrality influences mG3PDH activity and mitochondrial oxidative stress.

P2 b/1 Nitrite lowers the oxygen cost of ATP supply in skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis Charles Affourtit, Anthony Wynne School of Biomedical Sciences, University of Plymouth

Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from the stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate species. Here we report acute effects of sodium nitrite on the bioenergetic behaviour of L6 myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a ½-hour exposure to NaNO2 (0.1 – 5 µM) significantly decreases mitochondrial coupling efficiency in static myoblasts and tends to lower it in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, highly significantly so in myoblasts (P <0.001) and approaching significance in myotubes (P = 0.074).

Notably, NaNO2 exposure shifts the myocytes to a more glycolytic phenotype. Mitochondrial oxygen consumption changes comparatively little after NaNO2 exposure, whilst non-mitochondrial respiration decreases. When total ATP synthesis rates are normalised to cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply both in myoblasts and myotubes.

P2 b/2 Molecular mechanisms of ischemic kidney injury and protection: the role of mitochondria Nadezda V. Andrianova1, Stanislovas S. Jankauskas2, Ljubava D. Zorova2, Irina B. Pevzner2, Vasily A. Popkov1,2, Denis N. Silachev2, Dmitry B. Zorov2, Egor Y. Plotnikov2 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia 2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

There are not so many approaches for treatment of acute kidney injury (AKI) which is a high-mortality disease affecting approximately a third of intensive care units patients. We investigated the nephroprotective possibility of two different methods, namely ischemic preconditioning (IPC) and dietary restriction (DR), and evaluated the role of autophagy and mitophagy in mechanisms of damage and protection of ischemic kidney. While the majority of patient with AKI are elderly, all parts of the research were performed in young and old animals. IPC consisted of 4 cycles, including 15 seconds of ischemia and 15 seconds of reperfusion, immediately before the 40 minutes ischemia. DR was performed for 4 weeks by limiting the amount of food to 65% of the daily intake. Both methods, IPC and DR, attenuated AKI induced by ischemia/reperfusion (I/R), but only in young rats. In old animals, those approaches were unable to decrease the levels of serum creatinine and blood urea nitrogen. To unravel mechanisms underlying those effects, we evaluated the activation of the autophagic-lysosomal system in kidney tissue. We have shown that in young rats with IPC, fluorescence intensity of LysoTracker Green was not as increased as in the group with I/R alone. However, in old rats we observed the increase only in the group with IPC. Similarly, DR significantly increased LysoTracker Green fluorescence intensity and LC3II/LC3I ratio in young rats, but there were no such alterations in old rats. Impaired mitochondrial quality control was demonstrated by analysis of mitochondrial transmembrane potential by flow cytometry. A fraction of low-potential mitochondria was found in old kidneys and this de-energized population was even increasing after IPC in old, but not in young rats. Ineffectiveness of mitophagy was also suggested by the evaluation of the PINK-1 level in mitochondria.

Supported by RSF grants 18-15-00058 (analysis of AKI, DR and mitophagy) and 14-15-00147 (study of IPC).

P2 b/3 Short term cultivation of human amniotic mesenchymal stromal cells at atmospheric oxygen causes metabolic switch to oxidative phosphorylation Asmita Banerjee1,2, Adelheid Weidinger1,2, Andrea Lindenmair2,3, Simone Hennerbichler2,4, Ralf Steinborn5, Heinz Redl1,2, Susanne Wolbank1,2, Andrey V. Kozlov1,2 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria 2Austrian Cluster for Tissue Regeneration, Vienna, Austria 3Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Linz, Austria 4Red Cross Blood Transfusion Service for Upper Austria, Linz, Austria 5Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria

Recently, vital human amniotic membrane (hAM), containing cells with distinct stem cell properties, has come into focus for clinical applications in regenerative medicine. Most stem cells reside in low oxygen niches (1 – 3%), and generate energy predominantly via glycolysis. Common cell culture laboratories, usually set up at 20% oxygen, pose a challenge for stem cell cultivation, since a possible metabolic switch from glycolysis to oxidative phosphorylation could impact stem cell fate and cell function. We tested the impact of short term cultivation (4 days) at either 3% or 20% oxygen on human amniotic mesenchymal stromal cells (hAMSCs), regarding mitochondrial and inflammatory parameters, in order to clarify whether the energy metabolism is substantially different at these two oxygen levels. HAMSCs were isolated from two sub-regions of hAM, placental and reflected. Measurement of mitochondrial activity was performed with high-resolution respirometry (Oxygraph-2k, Oroboros Instruments, Austria). Nitric oxide (NO) was detected with Sievers 280i-NO Analyzer (General Electrics), and levels of reactive oxygen species (ROS) were analysed with electron paramagnetic resonance spectroscopy (EPR). We found 20% oxygen to increase mitochondrial oxidative phosphorylation, and the release of IL-6, especially in placental hAMSCs. Interestingly, oxygen tension did not influence ROS levels. The release of NO was elevated at 20% oxygen but independent of the amniotic region. Our data show that exposure of hAM to atmospheric oxygen tension elevates oxidative phosphorylation, which may alter cellular differentiation and proliferation potential. We propose that the full therapeutic potential of viable hAM for tissue engineering and regenerative medicine can only be exploited if its microenvironment is carefully chosen and monitored.

P2 b/4 MODULATING CELLULAR FLAVIN COFACTOR LEVELS IN HUMANS VIA THE BI-FUNCTIONAL AND MONOFUNCTIONAL FLAD1 GENE PRODUCTS Maria Barile1, Piero Leone1, Maria Tolomeo1, Michele Galluccio2, Cesare Indiveri2 1Department of Biosciences, Biotechnology, and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy 2Department DiBEST Unit of Biochemistry and Molecular Biotechnology, University of Calabria Arcavacata di Rende, Italy

FAD synthase (2.7.7.2) is an ubiquitous enzyme essential for cellular supply of FAD. In humans this enzyme is encoded by FLAD1 gene (MIM 610595), whose clinically relevant variants are the cause of multiple Acyl Co A dehydrogenase deficiency or MADD (OMIM 231680) and respiratory chain dysfunction, sometimes treatable with riboflavin [1]. We have characterized three different isoforms of hFADS: two of them are composed of two domains, a Molybdo-pterin resembling binding domain and a PAPS reductase domain, stabilized by a redox sensitive SS/SH network [1]. The third isoform of hFADS characterized so far is composed by the sole PAPS domain. It is crucial to ensure survival of patients with frameshift mutations in FLAD 1 gene [2]. The PAPS reductase domain, that we modeled in its FAD bound conformation, is per se able to catalyse the Mg++ dependent FAD synthesis, performing an ordered bi-bi mechanism, in which FAD release is the limiting step of the catalytic cycle. In the Molybdo-pterin binding domain of the bi-functional isoforms of hFADS, a Co++ dependent FAD hydrolytic activity resides, which is stimulated by K+ and inhibited by pyridine nucleotides. When incubated with appropriate apo-flavoprotein hFADS exerts a FAD chaperoning effect, contributing to the biogenesis of cellular flavoproteome. Using cellular and worm models we mimicked alteration of FLAD1 expression level and observed alteration of mitochondrial function, compensated by riboflavin supplementation.

1 Barile M., Giancaspero T. A., Leone P, Galluccio M., Indiveri C., Riboflavin transport and metabolism in humans, J Inherit Metab Dis (2016) 39:545-557 2 Leone P., Galluccio M., Barbiroli A., Eberini I., Tolomeo M., Vrenna F., Gianazza E., Iametti S., Bonomi F., Indiveri C., Barile M., Bacterial Production, Characterization and Protein Modeling of a Novel Monofuctional Isoform of FAD Synthase in Humans: An Emergency Protein?, Molecules (2018) 23: E116

P2 b/5 Ubiquitin-dependent degradation of mitochondrial proteins regulates energy metabolism Giovanni Bénard1,2,, Julie Lavie1,2, Harmony De Belvalet1,2, Sessinou Sonon1,2, Ana Madalina Ion1,2,3, Elodie Dumon1,2, Su Melser2,4, Didier Lacombe1,2,5,Jean-William Dupuy2,6, Claude Lalou1,2 1Laboratoire Maladies Rares: Génétique et Métabolisme- INSERM U1211, Bordeaux, France 2Université de Bordeaux, Bordeaux cedex, France 3Molecular Mechanisms of Disease, Radboud University, 65000 HC Nijmegen, The Netherlands 4INSERM, U1215 NeuroCentre Magendie, Bordeaux, France. 5CHU Bordeaux, Service de Génétique Médicale, Bordeaux, France 6Plateforme Protéome, Centre de Génomique Fonctionnelle, Université de Bordeaux, Bordeaux Cedex, France

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions remains uncharted. Here, we showed that ubiquitination occurs in different mitochondrial compartments including in inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS-dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrated that SDHA ubiquitination occurs inside the organelle. In addition, we showed that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases adenosine triphosphate, malate and citrate levels. These findings revealed that, despite their embedded localization, mitochondrial metabolic machinery is also regulated by the UPS.

P2 b/6 ATP, the forgotten nucleotide in relationship between metabolism and body mass of individuals Mélanie Boël, Yann Voituron, Caroline Romestaing, Damien Roussel Laboratoire d'Ecologie des Hydrosystèmes Naturels et Anthropisés, Université de Lyon, ENTPE, Lyon, France

A lot of interspecific studies have been conducted on the relationship between metabolism and individual body mass and showed that specific oxygen consumption (expressed in mass units) negatively correlates with body mass in mammals. In cells, oxygen consumed by organism is transformed by mitochondrial oxidative phosphorylation into cellular energy, essential to growth, survival and fitness: ATP. However, all the oxygen consumed is not totally converted in ATP. 20 – 30% of individual oxygen consumption are dissipated as heat to compensate energetic losses occurring during this biochemical process, known as “proton leak”. Thus, the use of oxygen consumption without the inclusion of the proton leak intensity does not provide the best estimation of the real quantity of ATP produced that can be invested in individual performances. One of methods allowing to take proton leak into account is the determination of oxidative phosphorylation efficiency (ATP/O), by measuring both mitochondrial oxygen consumption and ATP synthesis. Many authors demonstrated that proton leak negatively correlates with body mass in mammals, suggesting that larger ones have highest mitochondrial efficiency and thus, best ATP synthesis. The aim of our study is to revisit the relationship between metabolism and body mass in mammals, considering main mitochondrial components – oxygen consumption, ATP production and mitochondrial efficiency – in muscle tissue. Even after phylogenetic correction (using Phylogeny Independent Contrasts), our results show that both oxygen consumption and ATP synthesis of muscle mitochondria negatively correlate with body mass. However, mitochondrial efficiency remains surprisingly constant, suggesting that mitochondrial efficiency is probably one of rarely metabolic functions which are independent with body mass.

P2 b/7 Aerobic metabolism of endothelial cells chronically exposed to statins Izabela Broniarek, Wieslawa Jarmuszkiewicz Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland

Statins belong to cholesterol-lowering medications, because they act as inhibitors of 3-hydroxy-3-methyl- glutaryl-CoA reductase. They are widely used to prevent atherosclerosis and other cardiovascular diseases, which can result from endothelial dysfunction. Endothelial cells line the entire circulatory system. Because they have permanent contact with blood and blood-transported drugs, we hypothesize that statins influence aerobic metabolism of endothelial cells. To verify this hypothesis, endothelial cells (cell line EA.hy926) were cultivated 6 days in medium containing pravastatin (100 nM) or atorvastatin (100 nM). Then, following parameters were measured: oxygen consumption rate with different respiratory substrates, mitochondrial enzymes activity (citrate synthase, lactate dehydrogenase and cytochrome c oxidase), cell viability, coenzyme Q10 concentration in mitochondria and reactive oxygen species (ROS) production. According to the results, exposure to statins leads to slight but statistically significant changes in aerobic metabolism of endothelial cells. It is manifested, among others, by an increased cellular ROS production, changes in oxygen consumption rate, decreased level of coenzyme Q10 in mitochondria and reduced activity of lactate dehydrogenase. It is worth mentioning that effects depend on the used statin and generally they are stronger in the case of atorvastatin than pravastatin. Acknowledgements: This work was supported by the National Science Centre, Poland (2016/21/N/NZ1/00018) and partially by KNOW Poznan RNA Centre (01/KNOW2/2014). Publication resulted from a fellowship obtained from Jagiellonian Medical Research Center Foundation and Adam Mickiewicz University Foundation Scholarship for 2017/2018.

P2 b/8 Role of mitochondrial Ca2+ in the interplay between circadian core clock genes and cellular energetic metabolism Olga Cela1, Rosella Scrima1, Gianluigi Mazzoccoli2, Nazzareno Capitanio1 1Dept. of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy 2Dept. of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy

Mounting evidences are disclosing the tight correlation between circadian rhythms and cell metabolism to align bioenergetic demand to environmental variants. In the last few years we focused on the interplay between the clock gene machinery and the mitochondrial physiology. Using well-established in-vitro-synchronized cultured cells, we demonstrated a BMAL1-dependent ultradian oscillation of the mitochondrial respiratory activity [1] as well as of the glycolytic activity. This translated in a rhythmic change of the cellular energy charge. The rhythmic respiratory activity was associated with: i) oscillation in cellular NAD content; ii) clock-genes-dependent expression of NAMPT, NMNAT3 and Sirtuins 1/3; iii) reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I; iv) reversible phosphorylation of the pyruvate dehydrogenase (PDH). Notably, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the clock-gene expression in synchronized cells and a similar result was attained with mtDNA depleted (Rho0) cells [2]. In this context the mitochondrial-endoplasmic reticulum (ER) calcium homeostasis appears to be involved as inhibition of either of the mitochondrial calcium uniport, the ER Ca2+-channel(s), the cyclic ADP-ribose (cADPR) synthesis resulted in alteration of the rhythmic respiratory activity whereas chelation of extracellular Ca2+ was ineffective. All together our findings provide novel levels of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork.

References 1. Cela et al. BBA-BIO (2016) 1863(4):596-606. 2. Scrima et a. BBA-BIO (2016) 1857(8):1344-51.

P2 b/9 Real-time imaging of mitochondrial ATP dynamics discloses the metabolic setting of single cells Maria R. Depaoli, Felix Karsten, Corina T. Madreiter-Sokolowski, Christiane Klec, Benjamin Gottschalk, Helmut Bischof; Emrah Eroglu, Markus Waldeck-Weiermair, Wolfgang F. Graier, Roland Malli Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical , Graz, Austria

Recent research revealed that reprogramming of metabolic pathways determines cell functions and fate. In our work, we have used organelle-targeted ATP biosensors [1–3] to evaluate cellular metabolic settings with high resolution in real-time. Our data show that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. Unexpectedly, this hexokinase- dependent process was reversed upon the removal of glucose or other hexose sugars. Our data further corroborate that mitochondria of cancer cells predominately consume ATP. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). On the other hand, pancreatic β-cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased ATP production via mitochondrial respiration. Our findings add new perspectives on cellular bioenergetics and demonstrate that live-cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate the metabolic flexibility and heterogeneity of cells and cell populations.

[1] H. Imamura, K.P.H. Nhat, H. Togawa, K. Saito, R. Iino, Y. Kato-Yamada, T. Nagai, H. Noji, Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 15651–15656. doi:10.1073/pnas.0904764106. [2] N. Vishnu, M. Jadoon Khan, F. Karsten, L.N. Groschner, M. Waldeck-Weiermair, R. Rost, S. Hallström, H. Imamura, W.F. Graier, R. Malli, ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release, Mol. Biol. Cell. 25 (2014) 368–379. doi:10.1091/mbc.E13-07-0433. [3] T. Yoshida, S. Alfaqaan, N. Sasaoka, H. Imamura, Application of FRET-Based Biosensor “ATeam” for Visualization of ATP Levels in the Mitochondrial Matrix of Living Mammalian Cells, Methods Mol. Biol. Clifton NJ. 1567 (2017) 231–243. doi:10.1007/978-1-4939-6824- 4_14.

P2 b/10 Analysis of mitochondrial ATP synthase cluster formation in insulin-secreting INS-1E cells as visualized by 3D super- resolution dSTORM microscopy

Andrea Dlasková, Anežka Kahancová, Hana Engstová, Tomas Špaček, Petr Ježek Institute of Physiology, Department of Mitochondrial Physiology, Prague, Czech Republic dSTORM microscopy represents a powerful super-resolution method for accessing ultrastructural changes of objects that could not be resolved by conventional confocal microscopy. In this work, we have employed dSTORM microscopy to examine clustering of mitochondrial ATP synthase in the context of mitochondrial cristae changes and ATP synthase oligomerization status. Insulin secreting INS-1E cells were incubated for 2 hours at 3 to 20 mM glucose, fixed and labeled with primary antibody against F1-alpha directly conjugated to Alexa-647. Obtained 3D images were analyzed with the help of Ripley’s K-function and from it derived distance distribution function. Resulting histograms provided the most frequent distances (MFD) between the localized single antibody molecules [1]. Interestingly, in fasting state (0-3 mM glucose) MFD between F1-alpha were ~80 nm, while at 11and 20 mM glucose decreased to 61 nm and 57 nm, respectively. These changes well corresponded to a decrease in cristae width upon GSIS and also concomitant increase in ATP synthase oligomerization. Of note, knock-down of FO-subunit F, which is essential for ATP synthase oligomerization, caused also noticeable rearrangement of ATP synthase clusters. We conclude that the changes in the ATP synthase spatial organization might represent important regulatory mechanism of mitochondrial OXPHOS efficiency and might be critical for appropriate glucose sensing by pancreatic β-cells.

Supported by GACR 17- 08565S grant.

[1] A. Dlasková, H. Engstová, T. Špaček, A. Kahancová, K. Smolková, J. Špačková, L. Plecitá-Hlavatá, and P. Ježek, 3D super- resolution microscopy reflects mitochondrial cristae alternations and mt DNA nucleoid size and distribution. Biochim. Biophys. Acta special issue EBEC 2018.

P2 b/11 Respiratory mapping of mitochondrial pathways for establishing a database of mitochondrial physiology Carolina Doerrier1, Zuzana Sumbalova1,2, Gerhard Krumschnabel1, Luiz F Garcia-Souza1,3, Erich Gnaiger1,4 1Oroboros Instruments, Innsbruck, Austria 2Pharmacobiochem Lab, Fac Med, Comenius Univ, Bratislava, Slovakia 3Inst Sport Science, Univ Innsbruck, Austria 4Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria

Mitochondria (mt), the powerhouses of the cell, play a key role under many physio-pathological conditions. Therefore, the study of mitochondrial function is crucial for understanding such conditions, particularly in clinical applications. However, despite of the growing relevance of mitochondrial research, there is no database which provides comprehensive information about mitochondrial physiology. As a first step towards closing this gap, we developed the substrate-uncoupler-inhibitor titration (SUIT) reference protocol (RP) [1]. RP comprises two complementary and harmonized SUIT protocols (RP1 and RP2) to investigate mt-pathways converging at the Q-junction from fatty acid oxidation (FAO or F), NADH-linked substrates (N), succinate (S) and alpha-glycerophosphate (Gp), allowing the identification of specific metabolic profiles and mt-related injuries. Here, we explain key points for SUIT protocol development and application of High-Resolution FluoRespirometry (HRFR) with a wide spectrum of mitochondrial preparations from different model organisms, tissues and cells. We highlight the use of a low malate concentration (0.1 mM) as a co-substrate with fatty acid(s), avoiding a significant overestimation of FAO encountered at 2 mM malate if anaplerotic pathways are involved and saturate the N-pathway with 2 mM malate only. An application of the SUIT-RP reveals defects in the N- and F-pathways and loss of mt-outer membrane integrity as a result of ischemia-reperfusion injury of mouse heart mitochondria. Respiratory OXPHOS analysis by HRFR combining the evaluation of mitochondrial respiration with additional bioenergetic parameters (i.e., hydrogen peroxide production) provides a sensitive tool for comprehensive analysis of mitochondrial function in health and disease. Taken together, reproducibility, accuracy, application of comparable SUIT protocols and standardized normalization are required for functional mt-mapping as for a mt-database of mitochondrial physiology [2].

References 1. C. Doerrier, L.F. Garcia-Souza, G. Krumschnabel, Y. Wohlfarter, A.T. Mészáros, E. Gnaiger, High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria, Mitochondrial Bioenergetics 1782 (2018) 31-68. 2. MitoEAGLE preprint 2018-06-10(39) Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology Part 1. - http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2018-02-08 Support K-Regio project MitoFit and European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.

P2 b/12 Activation of microglial cells requires metabolic shifts towards glycolysis Amalia M. Dolga1, Angelica Sabogal1, Birgit Honrath1,2, Maike Gold3, Goutham Ganjam2, Nunzianna Doti4, Carsten Culmsee2 1Dept. of Molecular Pharmacology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands 2Inst. für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, Marburg, Germany 3Dept. of Neurology, Philipps-Universität Marburg, Marburg, Germany 4Institute of Biostructures and Bioimaging (IBB)-CNR, CIRPEB, Naples, Italy

Recent studies revealed that small conductance calcium-activated potassium (SK) channel opening prevented mitochondrial fission, oxidative stress, and ultimately neuronal cell death. However, little is known about the function of SK channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. In this study, we addressed the question whether SK channel activation affected inflammatory responses of primary mouse microglia upon α-synuclein challenge. We used real-time cell impedance to detect the degree of microglial morphological alterations, ELISA measurements for cytokine release, electrophysiological studies to analyze the SK channel activity, and Seahorse system to assess the mitochondrial function. We determined that by SK channel activation, alpha-synuclein-activated microglial cells have a decreased cytokine release, NO production, AMPK activation and an overall attenuated microglial activation. Interestingly, opening of SK channels promotes an immediate glycolytic activity by increasing pyruvate and lactate production, followed by a decrease in glycolysis, as indicated by analysis of metabolite profile and extracellular acidification rate. Although pyruvate facilitates cytokine IL-6 and TNF-a release in response to alpha-synuclein, only IL-6 promotes glycolytic activity, suggesting that IL-6 is tightly linked to mitochondrial metabolism. AMPK inhibition sustains the metabolic profile of non-activated microglia and completely prevents alpha-synuclein microglial activation. The initial metabolic shift followed by a decrease in glycolytic activity induced by SK channel activation provides a mechanistic explanation for the reduction of microglial activation in conditions of inflammation. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson’s disease, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease.

P2 b/13 Bioenergetic basis for the effects of arginine and alpha-ketoglutarate on lifespan Dmytro Gospodaryov, Vitaliy Balatskiy, Maria Bayliak Department of Biochemistry and Biotechnology, Vasyl Stefanyk Precarpathian National University, Ivano-Frankivsk, Ukraine

Alpha-ketoglutarate (AKG) was found to extend lifespan of nematode Caenorhabditis elegans [1]. The pro-longevity effect of AKG is supposedly based on its ability to inhibit ATP synthase, leading to down-regulation of target-of-rapamycin signalling pathway [1]. Our studies in yeast and fruit flies suggest common mechanisms for the extension of lifespan by AKG in different organisms [2]. Now, using isolated mitochondria from fruit flies and mouse cerebral cortex, we have shown strong inhibition of respiration by AKG in concentrations > 5 mM. Another health-promoting metabolite, arginine, was recently shown to accelerate fruit fly development, simultaneously causing oxidative stress and lifespan shortening [3]. Here, we show that arginine in concentrations > 10 mM inhibits NADH-dependent oxygen consumption in mitochondria isolated from fruit flies and mouse brain cortex. The latter effect likens arginine to guanidine-containing compound metformin, an anti-diabetic and anti-aging drug, which was shown to inhibit complex I of mitochondrial respiratory chain [4]. It also explains our recent finding on ability of arginine to drastically lower glucose levels in fruit flies [3].

1. R.M. Chin, X. Fu, M.Y. Pai, et al., The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR, Nature 510 (2014) 397–401. 2. N. Burdyliuk, M. Bayliak, Effects of long-term cultivation on medium with alpha-ketoglutarate supplementation on metabolic processes of Saccharomyces cerevisiae. J Aging Res 2017 (2017) 8754879. 3. M.M. Bayliak, M.P. Lylyk, O.V. Maniukh, et al., Dietary L-arginine accelerates pupation and promotes high protein levels but induces oxidative stress and reduces fecundity and life span in Drosophila melanogaster, J Comp Physiol B 188 (2018) 37–55. 4. O. Lushchak, D. Gospodaryov, Mimetics of caloric restriction, in: A.M. Vaiserman (Ed.), Anti-Aging Drugs: From Basic Research to Clinical Practice, The Royal Society of Chemistry, Croydon, 2017, pp. 229–271.

P2 b/14 How Mitochondrial Src Kinase Modulates Mitochondrial Functions? Hala Guedouari1,2, Nicolas, Pichaud3, Etienne Hebert-Chatelain1,2 1Department of Biology, Université de Moncton, NB, Canada 2Canada Research Chair in Mitochondrial Signaling and Physiopathology 3Department of Chemistry and Biochemistry, Université de Moncton, NB, Canada

Protein phosphorylation is a major regulatory mechanism of most cellular processes such as protein synthesis, cell division, signal transduction and aging. Protein kinases are known to regulate most cellular pathways, and their alteration can be associated to several diseases. Mitochondria are increasingly recognized as a hub for cell signaling. Many kinases localize into mitochondria where they modulate mitochondrial functions, including ATP production, calcium buffering and apoptosis, in response to specific stimuli. Among these kinases, Src-Tyrosine kinases are known to be major agents in mitochondrial tyrosine phosphorylation. The precise mechanism by which the intra-mitochondrial Src kinases affect mitochondrial physiology is however poorly understood. The aim of our work is to understand how the intra-mitochondrial c-Src kinase (mtSrc) modulates mitochondrial functions. First, our results showed that mtSrc is present in several mouse tissues, in which its activity is modulated by ATP and the energetic status of mitochondria. Deletion of c-Src alters cellular respiration, generation of reactive oxygen species and expression of different mitochondrial proteins. These results suggest that mtSrc can influence cellular fate through different mitochondrial functions. To investigate the mitochondrial protein interactors of mtSrc, we generated a c-Src specifically targeted to mitochondria and fused to the biotin ligase BioID2. The proximity- dependent biotin identification (BioID) method, coupled to LC-MS and in silico analyses identified eight mitochondrial proteins potentially targeted by mtSrc. Based on these results, generation of phosphomutants of these mtSrc targets will allow us to understand the role of mtSrc in mitochondria and cellular homeostasis. This study will therefore characterize mechanisms by which mtSrc regulates mitochondrial physiology and their role in the development of mitochondria-related disorders.

P2 b/15 Mitochondria impacts DNA methylation and gene expression through modulation of methionine and polyamine metabolism Janine H. Santos1, Oswaldo A. Lozoya1, Inmaculada Martinez-Reyes2, Tianyuan Wang1, Dagoberto Grenet1, Pierre Bushel1, Jianying Li1, Navdeep Chandel2, Richard P. Woychik1 1National Institute of Environmental Health Sciences, NIH 2Northwestern University

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial NADH oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechanistic link between mitochondrial function and epigenetic regulation of gene expression, highlighting the broad scope of cellular outcomes associated with changes in mitochondrial function.

P2 b/16 Hibernating mitochondria, the way to confer cell stress? Koen D.W. Hendriks, Rob H. Henning Department of Clinical Pharmacy and Pharmacology, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands

Hibernators are well-known to initiate safe metabolic suppression and resist ischemia and hypothermia. Earlier, we showed that isolated cultured hamster cells show these features including cold-resistance[1], suggesting cell-autonomous protecting mechanisms. These mechanisms are promising strategies to mitigate cellular damage in various conditions, such as organ transplantation. We explored the role of mitochondria herein by analysing mitochondrial (mal)function (ATP production, ROS formation, mitochondrial membrane potential [MMP] and mitochondrial morphology) in normal, cooled (4oC) and rewarmed (37oC) cells and isolated mitochondria from a hibernator (HaK) and non-hibernator (HEK293) kidney epithelial cell-line. In HEK293, cooling induced a rapid loss of MMP with decreased ATP levels. In contrast, HaK maintained MMP and ATP levels during cooling and rewarming with lower ROS damage. By measuring ATP production in isolated mitochondria at 4oC, we confirmed mitochondrial activity in HaK, whereas HEK293 mitochondria failed to do so. Further, HEK293 in normal conditions showed a fused network, with cooling provoking an extensive fission of mitochondria. HaK displayed a dispersed network both in normal and cold conditions. To verify this alternative fission/fusion homeostasis in vivo, we analysed liver mitochondria during hibernation in hamster by electron microscopy. During torpor, we found mitochondria to be shifted to a more fission like network, suggesting the activation of mitophagy during rewarming. Absence of detrimental cooling effects in hibernators may be related to the usage of an alternative electron donor and/or a dispersed state of the hibernator’ mitochondrial network, possibly preventing spreading of damaging molecules such as ROS and/or facilitating mitophagy.

References 1. K.D.W. Hendriks et al. Differences in mitochondrial function and morphology during cooling and rewarming between hibernator and non-hibernator derived kidney epithelial cells, Sci Rep. (2017)

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Branched chain 2-Oxoacid Metabolism Supplies mitochondrial H2O2 and ATP to Stimulate Insulin Secretion Blanka Holendová, Lydie Plecitá-Hlavatá, Jan Tauber, Petr Ježek Department of Mitochondrial Physiology, Institute of Physiology, AS CR, Prague, Czech Republic

Different types of nutrients like glucose, amino acids, free fatty acids as well as intestinal hormones and neuropeptides are known to contribute to insulin secretion. Among these α-ketoisocaproate (KIC), a product of leucine metabolism, is one of the most potent secretagogues stimulating insulin secretion. The secretion, in general, is achieved by generation of metabolic coupling factors e.g. ATP,

NADPH or H2O2 etc., but the exact mechanism of their interplay is unknown. Here we show that KIC is a strong inducer of insulin secretion and that the secretion is achieved by signaling factors of mitochondrial origin. The production of insulin by insulinoma INS1-E cells, pancreatic islets isolated from C57B6/J mouse and mouse itself was 3-fold higher in response to KIC compared to stimulation by sole glucose. Moreover, metabolic coupling factors such as ATP and reactive oxygen species (ROS) were elevated in response to KIC treatment of INS1-E cells. Our data suggest that the mitochondrially derived H2O2 is essential for KIC–stimulated insulin secretion, as inferred from nearly complete SkQ1 inhibition of insulin secretion. Mitochondrial H2O2 originates from the MnSOD–converted superoxide, which arises at the EF site of electron-transferring flavoprotein: Q oxidoreductase system (ETF:QOR) upon isovaleryl-CoA oxidation. Isovaleryl-CoA is produced from KIC by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex. Indeed, BCKDH silencing led to ~70% suppression of KIC–stimulated insulin release and prevented the accompanying redox signal.

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Electron supply to the Q-junction: assessment of mitochondrial respiration, H2O2 flux and the redox state of the Q-pool Timea Komlodi1, Miriam Hunger1, Anthony L. Moore2, Erich Gnaiger1,3 1Oroboros Instruments, Innsbruck, Austria 2Biochemistry and Medicine School of Life Sciences, University of Sussex, Brighton, United Kingdom 3Daniel Swarovski Research Laboratory, Mitochondrial Physiology, Department of Visceral, Transplant and Thoracic Surgery Medical University of Innsbruck, Innsbruck, Austria

The coenzyme Q (Q)-junction is a key point of convergent electron flow in the electron transfer system from mt-dehydrogenases via Complex I (CI), from succinate (S) via Complex II, glycerol-3-phosphate (Gp) via mt-glycerophosphate dehydrogenase to Complex III. Deficiency in the Q-junction can impair electron transfer and OXPHOS capacities, while the extent of the inhibition is determined by the nature of the substrates and electron transfer pathways, which results in an altered redox state of the Q-pool [1]. We aimed at investigating the relationship between the Q redox state, H2O2 and O2 fluxes with various fuel substrates.

Mitochondrial respiration and H2O2 production were determined simultaneously by High-Resolution FluoRespirometry (HRFR; Oroboros

Instruments, Innsbruck, Austria), whereas the redox state of Q (Qreduced/Qtotal reduced= Qr/Qt) was detected using a three-electrode system [2,3] inserted into the Oroboros Q2k. Experiments were carried out on mitochondria isolated from mouse brain respiring with Gp (20 mM), S (0.2, 10 or 50 mM), or with their combinations, either in the absence (LEAK) or in the presence of saturating ADP (OXPHOS).

We found a linear relationship between Qr/Qt and O2 flux in OXPHOS, between H2O2 flux in LEAK and O2 flux in OXPHOS and all these parameters increased as a function of S concentration both in LEAK and OXPHOS. The highest H2O2 flux was measured using Gp&S50 and Gp&S10 in LEAK, while respiration was highest with the same substrates in OXPHOS. In contrast, Q was most reduced in LEAK with S50. Taken together, combining various substrates had an additive effect both on ROS generation and respiration, but not on Qr/Qt.

In summary, S-evoked H2O2 flux, Qr/Qt and respiration were dependent on S concentration and S&Gp combination: Gp < S0.2 < S10 < S50 and GpS0.2 < GpS10 ~ GpS50. These data suggest that the electron pressure generated by S- and Gp or S&Gp on the Q-junction controls respiration and regulates H2O2 flux by reversed electron transfer through the Q pool to CI.

[1] E. Gnaiger, Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology, Int J Biochem Cell Biol 41 (2009), 1837-45 [2] Patent of Q-electrode (1988): Dr P.R. Rich, Glynn Res. Ph., Bodmin; European Patent no.85900699.1 [3] A.L. Moore, I.B. Dry, J.T. Wiskich, Measurement of the redox state of the ubiquinone pool in plant mitochondria, FEBS 235 (1988), 76-80

P2 b/19 Endogenous quinones sustain NADH oxidation by complex I during anoxia, supporting substrate-level phosphorylation in mouse liver mitochondria Timea Komlódi4, Dora Ravasz1, Alex Kitayev2, Collin Hill, Michael Kiebish2, Carolina Doerrier4, Anthony L Moore3, Erich Gnaiger4,5, Niven Narain2, Thomas N Seyfried6, Vera Adam-Vizi1, Christos Chinopoulos1 1Department of Medical Biochemistry, Semmelweis University, Budapest 1094, Hungary 2BERG LLC, Framingham, MA, USA 3Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Brighton, UK 4Oroboros Instruments, Innsbruck, Austria 5Daniel Swarovski Research Laboratory, Mitochondrial Physiology, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria 6Biology Department, Boston College, Chestnut Hill, Boston, MA, USA

Anoxia leads to over-reduction of mitochondrial coenzyme Q (Q, quinone) pools rendering Complex I unable to oxidize NADH, leading to a profound decrease in the matrix NAD+/NADH ratio. As a consequence, the function of matrix dehydrogenases is impaired. Yet, under certain anoxic conditions catabolism of metabolites converging through the α-ketoglutarate dehydrogenase Complex (KGDHC) is known to occur, yielding succinyl-CoA, in turn supporting substrate-level phosphorylation substantiated by succinate-CoA ligase [1]. Mitochondrial respiration and NADH autofluorescence were measured simultaneously with the NextGen-O2k (Oroboros Instruments, Innsbruck, Austria), and the redox state of the Q-pool was detected with a three-electrode system implanted into the O2k (Oroboros Q2k). We show that in isolated mouse liver mitochondria Complex I utilizes endogenous quinones oxidizing NADH during anoxia. Untargeted metabolomic analysis of matrix metabolites of mitochondria subjected to respiratory arrest due to anoxia and in the presence of specific inhibitors of respiratory complexes infer showed that NAD+ arising from Complex I is utilized by KGDHC yielding succinyl-CoA for succinate-CoA ligase, thus maintaining substrate-level phosphorylation during anoxia. Finally, by using custom-made 3D-printed plugs designed for standard fluorometric cuvettes, we show that under no conditions of respiratory arrest due to anoxia and/or pharmacological inhibition of the complexes did the mitochondria undergo swelling, which could potentially confound matrix metabolite estimations or bioenergetic parameters due to permeability transition. Our results highlight the importance of the availability of quinones in conjunction with the operation of Complex I in maintaining substrate-level phosphorylation during anoxia.

[1] G. Kiss, C. Konrad, J. Doczi, A.A. Starkov, H. Kawamata, G. Manfredi, SF Zhang, G.E. Gibson, M.F. Beal, V. Adam-Vizi, C. Chinopoulos, The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation, FASEB J. 2013 Jun;27(6):2392-406

P2 b/20 mTORC1 inhibition affects mitochondrial morphology Hana Nuskova, Deniz Senyilmaz Tiebe, Aurelio A. Teleman Division of Signal Transduction in Cancer and Metabolism, German Cancer Research Center (DKFZ), Heidelberg, Germany

The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and plays a key role in the regulation of cell metabolism, growth, proliferation and survival [1, 2]. mTORC1 (mTOR complex I) serves, among others, as a nutrient sensor. Upon starvation, mTORC1 is inactive and cells use autophagy to compensate for a lack of nutrients. To avoid loss of mitochondria via mitophagy, mitochondria fuse and create highly interconnected networks due to inhibition of mitochondrial fission [3, 4]. However, we find that pharmacological inhibition of mTORC1 by rapamycin leads to rapid mitochondrial fragmentation. The aim of the presented project is to identify the molecular mechanisms underlying this phenomenon and to investigate its physiological context.

[1] A. Gonzalez, M.N. Hall, Nutrient sensing and TOR signaling in yeast and mammals, EMBO J, 36 (2017) 397-408. [2] R.A. Saxton, D.M. Sabatini, mTOR Signaling in Growth, Metabolism, and Disease, Cell, 168 (2017) 960-976. [3] L.C. Gomes, G. Di Benedetto, L. Scorrano, During autophagy mitochondria elongate, are spared from degradation and sustain cell viability, Nat Cell Biol, 13 (2011) 589-598. [4] A.S. Rambold, B. Kostelecky, N. Elia, J. Lippincott-Schwartz, Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation, Proc Natl Acad Sci U S A, 108 (2011) 10190-10195.

P2 b/21 Development of a New Chemical Inhibitor of OPA1 Activity to Increase Cancer Cell Sensitivity Toward Apoptosis Anna Pellattiero1,2, Charlotte Quirin1,2, Nikolaos Biris3, Evripidis Gavathiotis3, Luca Scorrano1,2 1Department of Biology, University of Padua, Padua, Italy 2Venetian Institute of Molecular Medicine, Padua, Italy 3Departments of Biochemistry and Medicine, Albert Einstein College of Medicine, Bronx, NY, United States

Optic Atrophy 1 (OPA1) is a large dynamin-related GTPase protein of the inner mitochondrial membrane. It exists in two forms: a long integral membrane form (L-OPA1) and a soluble one, facing the intermembrane space (S-OPA1). OPA1 independently promotes mitochondrial fusion and regulates apoptosis via cristae remodeling process. OPA1 oligomers maintain the tightness of cristae junctions, thus controlling cytochrome c redistribution and apoptosis. Resistance to apoptosis is a known hallmark of cancer that alters the sensitivity of tumor cells toward cytotoxic drugs. It has been demonstrated that mitochondria participate as signaling units in numerous cancer-related processes. Interestingly, OPA1 is overexpressed in different type of cancers and it correlates with a poor prognosis and an increased chemotherapy resistance. On the other hand, OPA1 downregulation shows improved drug sensitivity, and mutations that abolish OPA1 catalytic activity impair its antiapoptotic function. In this work, inhibitors of OPA1 have been explored as a tool, to be employed in combination with chemotherapy, to sensitize tumor cells resistant to apoptosis by altering their thresholds through apoptosis induction. We performed a high-throughput screening (HTS) of a library of drug-like small molecules using a GTPase colorimetric assay. Then, OPA1-inhibitor interactions were further characterized with kinetics assays and by NMR. Among all, MYLS22 was identified as a potent and selective OPA1 inhibitor molecule that increases apoptotic release of cytochrome c. Further experiments confirmed its non-mitochondriotoxicity. We evaluated apoptosis of treated cells and mitochondrial functionality through real time membrane potential, respiration and cytochrome c release measurements. A chemical inhibition of OPA1 would represent a novel mitochondrial therapy to limit cancer growth and metastasis by increasing sensitivity to apoptosis, and counteract chemotherapy resistance of cancer cells.

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Dietary supplementation with MicroActive Coenzyme Q10 increases plasma and skeletal muscle Coenzyme Q10 levels in

Thoroughbred horses that are genetically variable for basal Coenzyme Q10 activity levels Mary F Rooney1, Caitriona E Curley1, Michael E Griffin2, Lisa M Katz3, Richard K Porter1, Emmeline W Hill2 1School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, Dublin, Ireland 2Plusvital Ltd., Dun Laoghaire Industrial Estate, Dublin, Ireland 3UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland

Myostatin (MSTN) genotype variation is associated with multiple phenotypes in the Thoroughbred horse including best race distance, body composition and skeletal muscle fibre proportions, driven by the presence of a promotor region ERE-1 SINE insertion 227bp polymorphism, which is tagged by the g.66493737T/C SNP. Skeletal muscle Coenzyme Q10 (CoQ10) levels in Thoroughbred horses have also been observed to be associated with MSTN genotype. Evaluation of combined mitochondrial complex 1+3 and 2+3 activity in skeletal muscle revealed significantly higher activities in the C/C compared to the T/T cohort (P ≤ 0.05, N=82; n=37 C/C, n=34 C/T, n=11 T/T) in untrained Thoroughbred horses (21 ± 3 months). The restoration of combined complex 1+3 and 2+3 activities in the T/T cohort, following addition of exogenous CoQ1 (ubiquinone1) in vitro, suggested lower endogenous production of CoQ10 in this cohort.

Here, we tested the hypothesis that daily dietary supplementation in vivo with 1.5 mg/kg of Microactive CoQ10 (Maypro Industries, USA) would increase CoQ10 activity in skeletal muscle and plasma. Following nine weeks of supplementation in a cohort of healthy untrained

Thoroughbred horses (N = 19, 1-3 years, mixed sex and MSTN genotype) there was a significant (P ≤ 0.01) increase in plasma CoQ10 levels (0.126 ± 0.073 µg/ml vs 0.250 ± 0.103 µg/ml, mean ± SD, N=12, measured by high performance liquid chromatography). Furthermore, increased (P ≤ 0.01) skeletal muscle mitochondrial electron transport chain combined complex 1+3 enzyme activity

(indicative of CoQ10) was observed post-supplementation compared to pre-supplementation (0.42 vs 0.58 pmol/min/mg of protein normalised to citrate synthase activity, mean, N=19). These data demonstrate the bioavailability of the Microactive CoQ10 supplement and its capacity to increase CoQ10 concentrations in the functionally relevant tissue.

P2 b/23 Searching of metabolic markers of successful anhydrobiosis in tardigrades Milena Roszkowska1,2, Andonis Karachitos1, Daria Grobys1, Adam Kulpa1, Tomasz Bartylak1, Łukasz Kaczmarek2, Hanna Kmita1 1Department of Bioenergetics, Adam Mickiewicz University, Poznań, Poland 2Department of Animal Taxonomy and Ecology, Adam Mickiewicz University, Poznań, Poland

Anhydrobiosis is defined as desiccation tolerance that denotes the ability to survive almost complete dehydration without sustaining damages. This phenomenon has been reported for some invertebrates including tardigrades. Responding to dehydration, tardigrades form so-called ‘tun’ and enter the state of anhydrobiosis. However, the only true evidence of successful tardigrade anhydrobiosis attainable at present is successful recovery from the tun to the active stage. The analysis of available data suggests that anhydrobiosis success requires proper carbohydrate and lipid metabolism. Therefore we decided to compare metabolic profiles of active and anhydrobiotic tardigrades differing in anhydrobiosis capability. To follow putative alterations of metabolism we applied a metabolomic approach, i.e. untargeted metabolomic profiling based on gas chromatography-mass spectrometry (GC-MS). The detected unscrambled metabolites represented mainly amino acids, monosaccharides, carboxylic acids, membrane lipids and some products of the tricarboxylic acid (TCA) cycle. Moreover the metabolites are mainly involved in oxidation processes localized in mitochondria. Thus the settled methodology leads to detection of different metabolites and allows for determination of metabolic differences between the active and tun stages. This in turn could provide metabolic markers allowing discrimination between tardigrades differing in anhydrobiosis capability being of a great importance for the explanation how to live without water.

The work was supported by the research grant of National Science Centre, Poland, NCN 2016/21/B/NZ4/00131.

P2 b/24 Dysfunction of the immune system in conditional IF1 (Atp5if1-/-) knockout mice in colon Fulvio Santacatterina, Sonia Domínguez-Zorita, Pau B. Esparza-Moltó, Cristina Nuevo-Tapioles, José M. Cuezva Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas- Universidad Autónoma de Madrid (CSIC-UAM); Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Universidad Autónoma de Madrid, Madrid, Spain.

The mitochondrial ATP synthase is a key transducer of energy metabolism and cell fate. The enzyme is inhibited by the ATPase Inhibitory Factor 1 (IF1), which binds reversibly to the ATP synthase upon changes in physiological conditions of the cell [1]. Phosphorylation of S39 (S14 in the native protein) impedes binding of pIF1 to the ATP synthase and prevents inhibition of the enzyme [2]. Recently, we have demonstrated that the overexpression of human IF1 in the colon of transgenic mice triggers metabolic reprogramming of the tissue to an enhanced glycolysis and the activation of the canonical NFκB pathway [3]. The metabolic preconditioned tissue is manifested by an anti-inflammatory phenotype in response to DSS-induced inflammation and is mediated by the ROS-dependent activation of NFκB pathway [3]. In fact, quenching mtROS by the utilization of the mtROS scavenger MitoQ obliterates the NFκB-guided anti-inflammatory phenotype [3]. These findings establish a relationship between the activity of the ATP synthase and the immune response of the tissue. With the purpose of investigating this link we have developed a conditional IF1 knockout (IF1-KO) mouse in colon. In this poster we will summarize the main findings that illustrate the successful knockout of IF1 in colonocytes confirming the implication of mitochondrial activity mediated by IF1 in the immune response of the colon.

References: [1] J. Garcia-Bermudez et al., PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H-ATP Synthase, Cell Rep, 12 (2015) 2143-2155. [2] P.B. Esparza-Molto et al., Regulation of the H+-ATP synthase by IF1: a role in mitohormesis, Cell. Mol. Life Sci., 74 (2017) 2151- 2166. [3] L. Formentini et al., Mitochondrial ROS Production Protects the Intestine from Inflammation through Functional M2 Macrophage Polarization, Cell Rep, 19 (2017) 1202-1213.

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Age dependent loss of mitochondrial function in epithelial tissue can reversed by Coenzyme Q10 Daniel Schniertshauer, Daniel Gebhard, Jörg Bergemann Department of Life Sciences, Albstadt-Sigmaringen University of Applied Sciences, Sigmaringen, Germany

The process of ageing is characterized by the increase of age-associated disorders as well as severe diseases. Due to their role in the oxidative phosphorylation and thus the production of ATP which is crucial for the many cellular processes, one reason for this could be found in the mitochondria [1,2]. The accumulation of reactive oxygen species damaged mitochondrial DNA and proteins can induce mitochondrial dysfunction within the electron transport chain. According to the ’mitochondrial theory of ageing’, this is a major cause for cellular ageing, tissue dysfunction and degeneration [3]. This study aimed to evaluate the effect of Coenzyme Q10 on mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies. Therefore, we established a Seahorse Bioscience XF24 Extracellular Flux Analyzer method to measure the mitochondrial respiration ex vivo directly in epidermis. We observed an decrease in mitochondrial respiration and ATP production with donor age and also a regeneration of mitochondrial respiratory parameters if the reduced form of CoQ10, ubiquinol, was administered. In conclusion, an age-related decrease in mitochondrial respiration and ATP production in accordance with the mitochondrial therapy of ageing was confirmed. Likewise, an increase in the respiratory parameters by the addition of CoQ10 could also be shown. The fact that there is a significant effect of administered CoQ10 on the respiratory parameters leads to the assumption that this is mainly caused by an increase in the electron transport chain.

[1] D.C. Wallace, A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine, Annu. Rev. Genet. 39 (2005) 359–407. [2] C. Desler, M.L. Marcker, K.K. Singh, L.J. Rasmussen, The Importance of Mitochondrial DNA in Aging and Cancer, Journal of Aging Research 2011 (2011) 1–9. [3] D. Harman, The biologic clock: the mitochondria?, J Am Geriatr Soc 20 (1972) 145–147.

P2 b/26 Metabolic Profiling and Myoglobin Expression in Human Neonatal and Adult Mesenchymal Stem Cells Rosella Scrima1, Francesca Agriesti2, Consiglia Pacelli1, Tiziana Tataranni2, Carmela Mazzoccoli2, Lucia Lecce1, Olga Cela1, Luigi Nappi3, Pietro Formisano4, Claudia Piccoli1,2, Nazzareno Capitanio1 1Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy 2Laboratory of Pre-Clinical and Translational Research, IRCCS CROB, Rionero in Vulure, Italy 3Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy 4Department of Translational Medical Sciences, University of Napoli “Federico II”, Napoli, Italy

Metabolic phenotype and control of O2-related metabolism is emerging as fundamental in controlling the biology of stem cells. In an earlier study we found that hematopoietic progenitor/stem cells (HP/SC) express detectable amount of myoglobin (Mb), likely involved in detoxification of reactive oxygen/nitrogen species [1]. In this study we extended such an analysis to human mesenchymal stem cells (MSC) isolated for comparative purposes from amniotic membrane (hAMSC) and from adipose tissue of the mammary gland (hATMSC). Assessment of metabolic fluxes on adherent cells by SeaHorse platform resulted in a significantly different bioenergetic profile between hAMSC and hATMSC, with the former exhibiting both higher OCR (mitochondria-dependent oxygen consumption rate) and ECAR (glycolysis-related extracellular acidification rate). Evaluation of the relative contribution of different mitochondrial respiratory substrates as well as confocal microscopy analysis of the ∆Ψm and organelle morphology confirmed differences between the two MSC samples. Intriguingly, respirometry on cell-suspension did not result in large differences in OCR. As for HP/SC, also hAMSC and hATMSC expressed a Mb transcript variant, which is under control of hypoxia responsive elements. WB analysis confirmed the expression of Mb at the protein level and, insightlully, its selective enrichment in mitochondrial subcellular fraction of hAMSC. Flow cytometry analysis unvealed a higher amount and better co-localization with the stemness marker CD73 of Mb in hAMSC. The possible distinctive role of Mb, as oxygen deliverer to the mitochondrial compartment, under hypoxic conditions, in neonatal MSC will be discussed.

1. A. D'Aprile, R. Scrima, G. Quarato, T. Tataranni, F. Falzetti, M. Di Ianni, M. Gemei, L. Del Vecchio, C. Piccoli, N. Capitanio, Hematopoietic stem/progenitor cells express myoglobin and neuroglobin: adaptation to hypoxia or prevention from oxidative stress? Stem Cells 32 (2014) 1267-77.

P2 b/27 Mitochondrial activity differs in two sub-regions of the human amniotic membrane Adelheid Weidinger1,2, Asmita Banerjee1,2, Andrea Lindenmair2,3, Simone Hennerbichler2,4, Ralf Steinborn5, Heinz Redl1,2, Andrey V. Kozlov1,2, Susanne Wolbank1,2 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Vienna, Austria 2Austrian Cluster for Tissue Regeneration, Vienna, Austria 3Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Linz, Austria 4Red Cross Blood Transfusion Service for Upper Austria, Linz, Austria 5Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, Austria

Clinicians widely use the human amniotic membrane (hAM), the innermost of the fetal membranes, for coverage of wounds and burn injuries, mostly in a de-cellularized form. The use of vital hAM opens new perspectives for regenerative medicine by taking advantage of its versatile cells and cell organelles. However, there is no assay to determine the quality of hAM, particularly of the different sub-regions of hAM. It is well known that mitochondria are essential for tissue regeneration. In this study, we investigated parameters linked to mitochondria in hAM biopsies and its freshly isolated cells in two amniotic sub-regions, reflected and placental amnion, in order to estimate vitality of hAM. Measurement of mitochondrial activity was performed with high-resolution respirometry (Oxygraph-2k, Oroboros Instruments, Austria), and levels of reactive oxygen species (ROS) were analyzed with electron paramagnetic resonance spectroscopy (EPR). We found significantly different rates of several mitochondrial respiration states and ROS levels in biopsies and isolated human amniotic epithelial cells and human amniotic mesenchymal stromal cells, in the two sub-regions. Interestingly, differences in metabolic activities were inversely related to mitochondrial DNA copy numbers. Inhibition of ATP synthase with oligomycin led to increased release of lactate in cells of placental amnion, compared to cells of reflected amnion. Since alterations in mitochondrial function impact cell differentiation and cell rescue, we propose that amniotic sub-regions may have different potential for tissue regeneration. Consideration of region-specific properties could support optimization and fine-tuning of the application of hAM for regenerative medicine.

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KMADP For Oxidative Phosphorylation Depends On Substrate Oxidative Capacity Wayne T. Willis1, Elizabeth A. Willis2, Jamie Hudgens3, Lawrence J. Mandarino1 1Department of Medicine, University of Arizona, Tucson, USA 2College of Medicine, University of Kentucky, Lexington, USA 3College of Pharmacy, Midwestern University, Glendale, Arizona, USA

The apparent KMADP for oxidative phosphorylation (OxPhos) is often interpreted to reflect the sensitivity of mitochondria (MITO) to respiratory control signals. We have recently developed a luciferase-based procedure for the evaluation of OxPhos ADP kinetic parameters. Here we use this assay to determine the influence of the oxidative fuel supply on the experimentally assessed KMADP for OxPhos. The microplate-based assay offers high throughput (assays at multiple [ADP] measured simultaneously), sensitivity (<0.2 µg MITO protein/assay), and stability (linear ATP accumulation progress curves for at least 10 min), and is also very reproducible. MITO adenylate kinase activity is inhibited with 20 µM diadenosinepentaphosphate. MITO isolated from mouse quadriceps muscle (n=5) were incubated at 8 levels of [ADP]: 0, 1.5, 3, 6, 12, 25, 50, and 125 µM, while they oxidized one of 9 different substrate combinations: Glutamate+Succinate (G+S), G+Malate, G+Malate+Arsenite, Pyruvate+Malate, 2-Oxoglutarate, Palmitoyl-Carnitine+Malate, Acetyl-

Carnitine+Malate, Succinate+Rotenone, and Glycerol-3-Phosphate (G3P). OxPhos Vmax strongly depended on the oxidative substrate combination and spanned a 7-fold range; in all preparations G+S elicited the highest Vmax and G3P the lowest. Correspondingly, the apparent KMADP spanned a roughly 5-fold range, from the G+S KMADP = 12.1 ± 0.6 µM down to the G3P value of 2.3 ± 0.2 µM. The 2 relationship between Vmax and KMADP was linear, R = 0.929, and nearly regressed through the origin. We conclude that substrate supply critically and dramatically influences the experimentally determined KMADP for OxPhos and should be taken into account in assessments of respiratory control sensitivity. Underlying mechanisms related to how fuel oxidation establishes and defends the proton motive force, which, in turn, provides the driving force for the synthesis and export of ATP by Complex V and the ATP/ADP carrier, respectively, will be discussed.

P2 b/29 Development of a Kit to Measure Absolute Millivolts of Mitochondrial and Plasma Membrane Potentials in Cell Cultures Chad Lerner1,2, Yufeng Zhang1, Martin D. Brand1, Akos A. Gerencser1,2 1Buck Institute for Research on Aging, Novato, CA, United States 2Image Analyst Software, Novato, CA, United States E-mail: [email protected]

Abnormal cellular energy production contributes to human disease, and possibly to aging. Dysfunction may arise from altered regulation, feedback and fine-tuning besides overt deficits in bioenergetic capacities. Detection of such subtle dysfunction requires quantitative approaches, applicable to often small amounts of primary cultures or ex vivo tissue. Mitochondrial membrane potential (ΔψM) is a central mediator of oxidative energy production. A technology is presented here for its unbiased and quantitative assay in micro-scale cell cultures. Commonly used ΔψM assays often lead to data misinterpretation. This is because all ΔψM probes are influenced by multiple properties of cells other than ΔψM. Previously we have designed a technology to calculate ΔψM from fluorescence time courses by accounting for all identified factors influencing ΔψM probe fluorescence. The technology relies on recording fluorescence time courses using a ΔψM probe (TMRM) and a plasma membrane potential probe. This requires wide-field, confocal or two-photon microscopy. Millivolt values are calculated by software based on a biophysical model. The potentiometric calibration now has been simplified by a kit-based formulation. The technology has been optimized in multiple cell lines and primary cultures such as dispersed neuronal and pancreatic β-cell cultures. Image analysis pipelines have been developed to automate analysis. Software has been developed to support routine work with parallel conditions, technical and experimental replicates. Breakthrough technologies led to quantum leaps in understanding, and in cellular energetics such was the Seahorse cell respirometer a decade ago. The ability to precisely monitor ΔψM in cells, and also to combine cell respiration and ΔψM data in the framework of metabolic control analysis may bring about a similar advance.

P2 b/30 Control Analysis of Cellular Energy Metabolism in Intact, Adherent Cell Cultures Chad Lerner1,2, Martin D. Brand1, Akos A. Gerencser1,2 1Buck Institute for Research on Aging, Novato, CA, United States 2Image Analyst Software, Novato, CA, United States E-mail: [email protected]

Abnormal cellular energy production contributes to human disease, and possibly to aging. Dysfunction may arise from altered regulation, feedback and fine-tuning besides overt deficits in bioenergetic capacities. In certain physiological processes of interest, e.g. neuronal or pancreatic β-cell signaling, virtually all variables of cellular energy metabolism change. This makes it challenging to define which processes drive observed changes, and how effects of disease-related molecular changes propagate and cause systems-level disturbances. Metabolic control analysis with appropriate modularization is a powerful method for understanding this through simplification. Novel mitochondrial assay technology developed by us now allows mechanistic studies of cellular energy metabolism in intact cells using micro-scale primary cell cultures. Previously we have designed a technology to calculate ΔψM in millivolts from fluorescence microscopy time courses by accounting for all identified factors influencing ΔψM probe fluorescence, such as plasma membrane potential and geometric factors. Here we combine this technology with Seahorse cell respirometry to determine ΔψM and cell respiration in parallel experiments in close to identical conditions. We demonstrate the use of modular kinetic analysis and metabolic control and regulation analysis to characterize cellular energy metabolism in cultures of human neuronal stems cells and human primary pancreatic β-cells. This approach allows isolation of effects on bioenergetic supply and demand, the former including glycolysis and mitochondrial substrate oxidation and the latter including ATP turnover and proton leak in intact cells.

P2 b/31 Unravelling the roles of Fission Protein 1: a forgotten mitochondrial fission factor with pleiotropic functions Branco FT; Barbieri E; Herkenne S and Scorrano L Venetian Institute of Molecular Medicine, Padova, Italy E-mail: [email protected]

Mitochondrial fission regulates a myriad of cellular functions, including mitochondrial clearance, and its ultimate physiological importance is underscored in devastating human disorders that arise from mutations in mitochondrial fission proteins. The machinery driving mitochondrial fission consists of the master regulator Dynamin-related protein 1 (Drp1) and its outer mitochondrial membrane adaptors, namely Fission Protein 1 (FIS1). Despite being the first proposed Drp1 receptor, FIS1 role in recruiting and regulating Drp1- mediated mitochondrial fission is highly controversial. To elucidate FIS1 role in mitochondrial dynamics, we generated a FIS1 hypomorphic mouse model, in which FIS1 levels are constitutively downregulated. FIS1 hypomorphic mice die at weaning age, due to a pleiotropic phenotype that is characterised by a defective growth and disseminated haemorrhages. Using a conditionally ablation system to deplete FIS1 from platelets or from specific tissues will help us pinpoint FIS1 essential roles in vivo.

Key words: mitochondrial fission, FIS1, Hypomorphism

P3 a/1 Self-assembled proteolipossomes to functionally characterize the alternative oxidase from Moniliophthora perniciosa Mario R. O. Barsottini1, Alice Copsey2, Fei Xu2, Gonçalo A. G. Pereira1, Anthony L. Moore2 1Institute of Biology, State University of Campinas, Campinas, Brasil 2Biochemistry & Biomedicine, University of Sussex, Brighton, United Kingdom

Moniliophthora perniciosa is a basidiomycete fungus and responsible for the witches’ broom disease of cocoa, a major threat for chocolate production. However, M. perniciosa Alternative Oxidase (MpAOX) is potential target for fungal control [1]. AOX is a terminal oxidase located in the matrix side of the internal mitochondrial membrane and a member of the non-haem di-iron carboxylate protein family. There is increasing evidence that fungal AOX is critical for the success of human and plant pathogens in different scenarios, however little is known about fungal AOXs and potential inhibitors. Here, we present the heterologous expression, purification and functional characterization of rMpAOX. rMpAOX was successfully purified, but with remarkably low activity towards Q1H2 in solution.

Preliminary results show that incorporation of rMpAOX into proteolipossomes (PLM) in the presence of ~10mM Q10 and NDH-2 from C. thermarum is essential for reaching maximal rMpAOX activity. This was achieved after optimization of the rMpAOX to NDH-2 ratio to ensure saturating levels of Q10H2 in the PLMs and maximal rMpAOX activity. Dose-response assays with AOX specific inhibitors will be presented. This work was supported by grants 2015/07653-5 & 2017/12852-2, São Paulo Research Foundation (FAPESP) & BBSRC (BB/E015328/1 & BB/L022915/1).

[1] D.P.T. Thomazella et al., The hemibiotrophic cacao pathogen Moniliophthora perniciosa depends on a mitochondrial alternative oxidase for biotrophic development., New Phytol. 194 (2012) 1025–34. [2] B. May, L. Young, A.L. Moore, Structural insights into the alternative oxidases: are all oxidases made equal?, Biochem. Soc. Trans. 45 (2017) 731–740.

P3 a/2

Role of the 2Fe-2S Rieske cluster in protection against ROS generation by cytochrome bc1 complex Łukasz Bujnowicz, Marcin Sarewicz, Arkadiusz Borek, Artur Osyczka Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

Molecular mechanism of the cytochrome (cyt) bc1 catalysis involves two processes taking place in one of the catalytic sites (Qo site): i) electronic bifurcation, in which one electron from QH2 is transferred to the Rieske cluster (FeS) while the second to hemes b and ii) large-scale movement of the domain housing FeS (HD) to shuttle electrons between Qo site and cyt c1. It is known that certain conditions i.e. in the presence of inhibitor ANT (ANT) induces a release of superoxide radical (ROS) which is commonly explained as a reaction of O2 with semiquinone (SQ) formed during the bifurcation process. Previously we showed that in ANT-inhibited enzyme a significant amount of SQ spin-coupled to the reduced FeS (SQ-FeS) is formed and a maximum occupancy of SQ-FeS depends on the equilibrium distribution of HD between cyt c1 and the Qo site, thus it may change in different mutants with altered mobility of HD. In the present study we analyzed formation of SQ-FeS in various mutants of cyt bc1 under oxygenic and anoxygenic conditions. It was found that the highest probability of SQ-FeS formation is seen for the mutants in which HD is arrested or significantly shifted toward the Qo site (+2Ala or +1Ala mutant, respectively). An increase in SQ-FeS formation correlates with a decrease in ROS generation in comparison to native enzyme. However, a mitochondrial mutation (G167P of R. caps. numbering) located on cyt b induces shift of HD out of the Qo site, which completely abolished generation of SQ-FeS. This correlates with strong ROS generation even in the absence of ANT. The results suggest that formation of SQ spin-coupled to FeS is reduces of the probability of SQ reaction with O2 [1].

References: 1. M. Sarewicz, Ł. Bujnowicz, S. Bhaduri, S.K. Singh, W.A. Cramer, A. Osyczka, Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc1/b6f, Proc. Natl. Acad. Sci. U.S.A., 114 (2017) 1323–1328.

P3 a/3 Functional and structural characterization of Alternative Complex III Filipa Calisto1, Joana S. Sousa2, Deryck J. Mills2, Julian D. Langer3, Patrícia N. Refojo1, Miguel Teixeira1, Werner Kühlbrandt2, Janet Vonck2 and Manuela M. Pereira1,4 1Instituto de Tecnologia Química e Biológica – António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal 2Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 3Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 4Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal

The electron transfer through the membrane complexes of the respiratory chain is coupled to the generation of the proton-motive force that drives the synthesis of ATP. Complexes I and IV couple electron transfer with transport of protons across the membrane, while complex III, the bc1 contributes to the proton gradient by a Q-cycle mechanism. Alternative Complex III (ACIII) is a quinol:cytochrome c/HiPIP oxidoreductase membrane complex widespread in the Bacteria domain, and a functional substitute of the bc1 [1,2]. The structure of ACIII from Rhodothermus marinus, recently solved by cryo-electron microscopy at 3.9 Å resolution [3], shows three integral transmembrane subunits and four periplasmic subunits. The periplasmic domain accommodates six hemes and four FeS clusters, which form two divergent electron transfer wires. The two putative proton pathways and the quinol-binding site identified in ACIII structure suggest that ACIII operates by a redox-driven proton translocation mechanism, totally unrelated to the Q-cycle of complex III. We aim to functionally characterize ACIII and its potential electron shuttles, for which we used several complementary biochemical and biophysical approaches including enzymatic assays, fluorescence spectroscopy and isothermal titration calorimetry.

1. M.M. Pereira, J.N. Carita, M. Teixeira, Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: A novel multihemic cytochrome bc, a new complex III, Biochemistry 38 (1999) 1268-1275 2. B.C. Marreiros, F. Calisto, P.J. Castro, A.M. Duarte, F.V. Sena, A.F. Silva, F.M. Sousa, M. Teixeira, P.N. Refojo, M.M. Pereira, Exploring membrane respiratory chains, BBA Bioenerg. 1857 (2016) 1039-1067 3. J.S. Sousa, F. Calisto, J.D. Langer, D.J. Mills, P.N. Refojo, M. Teixeira, W. Kühlbrandt, J. Vonck, M.M. Pereira, Structural basis for energy transduction by respiratory alternative complex III, Nat. Commun. 9 (2018) 1728, doi:10.1038/s41467-018-04141-8

P3 a/4

“Independent motion” model for the Qo site of cytochrome bc1 implicated from the combinatorial mutational studies in Rhodobacter capsulatus Robert Ekiert, Arkadiusz Borek, Artur Osyczka Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

Cytochrome (cyt) bc1 catalyses electron transfer from quinol to cyt c contributing to generation of protonmotive force. A functional connection between the quinol oxidation site (Qo) and cyt c is secured by various mechanistic elements, including the large-scale motion of the [2Fe-2S] cluster-containing head domain of iron-sulfur protein (ISP-HD). In this work we examined enzymatic activities of mutants that combined the impediment in motion of ISP-HD with other effects: alterations of midpoint redox potential of heme c1 and [2Fe-2S] cluster, and structural impediment for the binding of quinol at Qo. The tests included examination of the sensitivity of the mutated enzymes to changes in the ionic strength that, due to its electrostatic influence, effectively manipulates equilibrium between cyt bc1 and cyt c. We observed that certain structural and/or redox potential changes were accommodated by the enzyme, which retained its overall electron transfer capability. In some cases the inhibitory effects of single mutations were compensatory to each other in double mutants, restoring the enzyme functionality. Our results are consistent with the notion that random oscillations of ISP-HD between Qo and cyt c1 help maintaining equilibrium between the cofactors transferring electrons from Qo to cyt c. We propose an independent motion model for

Qo in which the motion of the Qo substrate (to/from Qo and within the site to reach the active position) and the motion of ISP-HD occur independently and the reaction at Qo requires coincidental meeting of quinol and the [2Fe-2S] cluster [1]. We suggest that this independence may be a potential factor limiting the rate of catalysis.

1. A. Borek, R. Ekiert, A. Osyczka. Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b, iron-sulfur protein and cytochrome c1. Biochim Biophys Acta (2018), doi: 10.1016/j.bbabio.2018.04.010

P3 a/5 Prediction of AOX inhibitors from molecular structure Alicia Rosell Hidalgo, Taravat Ghafourian, Luke Young and Anthony L. Moore Department of Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Brighton, United Kingdom

The alternative AOX is a non-protonmotive ubiquinol-oxygen oxidoreductase which belongs to the diiron carboxylate protein family. It is a monotopic protein which is found in all higher plants, some algae, yeasts, protists and a number of parasites including Trypanosoma brucei, the etiological agent of human African sleeping sickess or trypanosomiasis [1]. A number of AOX inhibitors have been discovered over the past years in an attempt to treat trypanosomiasis and for crop protection purposes, but little is still known about the ligand-protein interaction and the chemical characteristics of the inhibitors that are essential for potent inhibition. Furthermore, owing to the rapidly growing resistance to existing drugs, new compounds with improved potency and pharmacokinetic properties are urgently required. We used two computational approaches, ligand-protein docking and Quantitative Structure-Activity Relationships (QSAR), to investigate binding of AOX inhibitors to the enzyme and the molecular characteristics required for potency. Docking studies followed by protein-ligand interaction fingerprint (PLIF) analysis using AOX enzyme and the mutated analogues revealed the importance of the residues Leu 122, Arg118 and Thr 219. QSAR analysis using stepwise regression analysis with experimentally obtained IC50 values as the response variable and the computed molecular descriptors of compounds resulted in a multiple regression model with a good prediction accuracy. We will describe how the QSAR model highlights the importance of the presence of hydrogen bond donor groups on the aromatic ring of ascofuranone derivatives, acidity of the compounds, and a linear (non-bulky) shape of the compounds on the inhibitory effect on AOX.

References [1] L. Young, B. May, T. Shiba, S. Harada, D.K. Inaoka, K. Kita, A.L. Moore, Structure and Mechanism of Action of the Alternative Quinol Oxidases, in: W.A. Cramer, T. Kallas (Eds.) Cytochrome Complexes: Evolution, Structures, Energy Transduction, and Signaling, Springer Netherlands, Dordrecht, 2016, pp. 375-394.

P3 a/6 Molecular effects of human cytochrome b mutation G34S causing exercise intolerance studied in bacterial model Patryk Kuleta, Robert Ekiert, Iwona Mieszczak, Arkadiusz Borek, Artur Osyczka Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

Cytochrome b is one of core subunits of mitochondrial complex III, a key enzyme of the electron transport chain. From several mutations causing mitochondrial diseases found in human cytochrome b, G34S, located between the quinol reduction catalytic Qi site and heme bH was identified in patients suffering from exercise intolerance. We have studied the molecular effects of G34S using the analogous mutation G48S in bacterial counterpart of complex III (cytochrome bc1) in purple bacteria Rhodobacter capsulatus. G48S mutant grows slower than wild type (WT) under cytochrome bc1-dependent photosynthetic conditions and isolated complexes of G48S mutant exhibit lower than WT enzymatic activity (48 s-1 vs 140 s-1, respectively). Kinetic transients of light-induced redox reactions showed that the reverse electron transfer from quinol to heme bH at the Qi site was more prominent in G48S compared to WT. Furthermore, EPR measurements did not reveal presence of semiquinone at the Qi site (SQi) in G48S under typical conditions where SQi generated from reverse reaction can be observed in WT. These results suggest that midpoint redox potential (Em) of heme bH is elevated in G48S, which was confirmed by potentiometric redox titrations. It can be proposed that change in the redox properties of heme bH in G48S and its influence on electron and proton transfers at the Qi site are primary molecular cause of the disease. The identified properties of G48S make this mutant useful for studying the electron and proton reactions at the Qi site.

P3 a/7

Structural and functional characterization of the interaction of yeast cytochrome bc1 complex with recombinant cytochrome c Vishnupriya Pandey1, 2, Friedel Drepper3, Bettina Warscheid3,4, Carola Hunte1,4 1Institute for Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany 2Faculty of Biology, University of Freiburg, Freiburg, Germany 3Faculty of Biology, Institute of Biology II, Biochemistry–Functional Proteomics, University of Freiburg, Freiburg, Germany 4BIOSS Centre for Biological Signalling, University of Freiburg, Freiburg, Germany

Biological energy conversion relies on cofactor-mediated transfer of electrons, which requires precise spatial and temporal control of the distance, orientation and environment of cofactors. Snapshots of a transient electron-transfer complex of the respiratory chain were captured with X-ray structures of Saccharomyces cerevisiae cyt bc1 complex with the mobile carrier cytochrome c (cyt c) bound [1,2]. The interaction is mainly stabilized by non-polar forces provided by a small contact area which surrounds the respective heme clefts. The peripheral electrostatic interactions are in agreement with a steered binding which enforce the distinct orientation of the electron donating and accepting heme cofactors. Yet, it is not known whether physiological electron transfer is accomplished by this single conformation or by multiple productive complexes and why only a single cytochrome c is bound to the dimeric cyt bc1 in the X-ray structures. In order to address the role of single interface residues for structure and function of the electron transfer complex, we produced isoform-1 yeast cyt c in native form. Heterologous production conditions were optimized and the protein quality was assessed.

Quantitative lysine-trimethylation was confirmed by mass-spectrometry. The interaction of recombinant cyt c with yeast cyt bc1 was structurally and functionally characterized. The complex was successfully crystallized and its X-ray structure determined. This opens the way to systematically study structure-function relationships in the electron transfer complex.

[1] Solmaz, SR. and Hunte, C. (2008) Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer. J Biol Chem 283:17542-17549.

[2] Lange, C. and Hunte, C. (2002) Crystal structure of the yeast cytochrome bc1 complex with its bound substrate cytochrome c. Proc Natl Acad Sci USA 99:2800-2805.

P3 a/8

Thermodynamic properties of two magnetically distinct forms of semiquinone in the catalytic Qi site of cytochrome bc1 Sebastian Pintscher, Rafał Pietras, Marcin Sarewicz, Artur Osyczka Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland

Cytochrome bc1 monomer accomodates two quinone-specific catalytic sites (Qi and Qo) which are located on the opposite sides of the membrane and catalyze the opposing redox reactions. In the Qi site a step-wise reduction of ubiquinone to ubiquinol takes place, and this reaction involves formation of a stable intermediate named SQi. While some of the physical and thermodynamic properties of SQi as well as structural details of the SQi binding pocket were described through the extensive EPR investigations, little was known about possible magnetic interaction of SQi with adjacent heme bH. Consequently, significant discrepancies in stoichiometry were reported when equilibrium redox titrations of SQi were compared to reduction level of the heme. In our recent work [1], we revealed the existence of two distinct populations of SQi differing in paramagnetic relaxation. We concluded that the slow-relaxing SQi (SQiS) reflects the previously known form present alongside the reduced (thus diamagnetic) heme bH (the heme adjecent to the Qi site), while the fast- relaxing SQi (SQiF) corresponds to the form magnetically coupled with the oxidized heme bH. Identification of SQiF called for re- investigation of the thermodynamic properties of SQi. In this context, here we show the results of EPR-monitored equilibrium redox titrations of isolated cytochrome bc1 from Rhodobacter capsulatus for the pH range 5 to 9. Our comparisons of the levels for hemes b reduction with the levels of both SQiF and SQiS sheds new light on interactions between the Qi site cofactors and solvs the long-standing puzzle with Qi stoichiometry.

References:

[1] S. Pintscher, R. Pietras, M. Sarewicz, A. Osyczka, Electron sweep across four b-hemes of cytochrome bc1 revealed by unusual paramagnetic properties of the Qi semiquinone intermediate, BBA-Bioenergetics, 1859 (6) (2018), pp. 459-469

P3 a/9 Role of the Q-loop for the function of Escherichia coli cytochrome bd oxidase Alexander Theßeling, Jo Hoeser, Matthias Zumkeller and Thorsten Friedrich Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany

Cytochrome bd oxidase is a terminal oxidase of the respiratory chains in many prokaryotes. It is expressed at low oxygen concentrations and couples the reduction of oxygen to water with the oxidation of a quinol (QH2). In doing so, one positive charge per electron is translocated across the membrane contributing to the proton motive force. Cytochrome bd shows no similarities to other terminal oxidases such as heme-copper oxidases and alternative oxidases [1]. In Escherichia coli, cytochrome bd oxidase consists of three different subunits CydA, CydB and CydX [2,3]. Subunit I contains three heme groups: b558 near the QH2 binding site and the heme groups b595 and d, proposed to form a di-heme center [4]. In subunit I, a hydrophilic loop between transmembrane helices 6 and 7, the so called Q-loop, is proposed to play a central role in quinone binding. It length varies significantly in bd oxidase from various species. We generated E. coli mutants containing a Q-loop of variable length. The protein was homologously produced in E. coli and purified by means of affinity-, and size exclusion-chromatography. The effect of shortening the Q-loop on bd oxidase assembly and stability is described.

References [1] P. A. Cotter, ,V. Chepuri, R. B. Gennis and R. P. Gunsalus, Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product, J. Bacteriol. (1990), 172,6333-6338. [2] R. J. Allen, E. P. Brenner, C. E. VanOrsdel, J. J. Hobson, D. J. Hearn and M. R. Hemm, Conservation analysis of the CydX protein yields insights into small protein identification and evolution, BMC Genomics, (2014), 15:946 [3] J. Hoeser, S. Hong, G. Gehmann, R. B. Gennis, T. Friedrich, Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site, (2014) FEBS Lett., (2014) , 588, 1537-1541. [4] V.B. Borisov, E. Forte, S. A. Siletsky, P. Sarti and A. Giuffrè, Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition, Biochim. Biophys. Acta, (2011), 1807, 1398-1413.

P3 a/10 Cryo-EM structure of the alternative complex III from Rhodothermus marinus Janet Vonck1, Joana S. Sousa1, Filipa Calisto2, Deryck J. Mills1, Julian D. Langer3, Patrícia N. Refojo2, Miguel Teixeira2, Werner Kühlbrandt1, Manuela M. Pereira2,4 1Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 2Instituto de Tecnologia Química e Biológica – António Xavier, Universidade Nova de Lisboa, ITQB NOVA, Oeiras, Portugal 3Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 4Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal

Alternative Complex III (ACIII) is a member of a family of membrane-bound enzymes with quinol:HiPIP/cytochrome c oxidoreductase activity. The protein was first isolated from Rhodothermus marinus [1], and later found to be widespread in the Bacteria domain [2]. ACIII can functionally replace the cytochrome bc1/b6f complexes, but is structurally unrelated. We have determined the structure of the ~270 kDa ACIII complex by single particle cryo-EM at a resolution of 3.9 Å [3]. Six of the seven subunits of the ACIII gene cluster with iron- sulfur clusters and six c-type hemes were traced in the structure. A previously unknown subunit was identified by peptide mass fingerprinting. We identified the quinol-binding site and two putative proton pathways in the transmembrane subunits. A minor fraction of the data contained the previously characterized supercomplex of ACIII with the electron acceptor, caa3 oxygen reductase [4]. The structure introduces ACIII as a redox-driven proton pump and provides insights into its energy transduction mechanism.

1. M.M. Pereira, J.N. Carita, M. Teixeira, Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: A novel multihemic cytochrome bc, a new complex III, Biochemistry 38 (1999) 1268-1275 2. P.N. Refojo, F.L. Sousa, M. Teixeira, M.M. Pereira, The alternative complex III: A different architecture using known building modules, BBA Bioenergetics 1797 (2010) 1869-1876 3. J.S. Sousa, F. Calisto, J.D. Langer, D.J. Mills, P.N. Refojo, M. Teixeira, W. Kühlbrandt, J. Vonck, M.M. Pereira, Structural basis for energy transduction by respiratory alternative complex III, Nature Communications 9 (2018) 1728, doi:10.1038/s41467-018-04141-8 4. P.N. Refojo, M. Teixeira, M.M. Pereira, The alternative complex III of Rhodothermus marinus and its structural and functional association with caa3 oxygen reductase, BBA Bioenergetics 1797 (2010) 1477-1482

P3 a/11 Comparison of the kinetic parameters of alternative oxidases purified from Trypanosoma brucei, Sauromatum guttatum and Arabidopsis thaliana Fei Xu1,2, Alice C. Copsey1, Mario R.O. Barsottini1, Anthony L. Moore1 1Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Falmer, United Kingdom 2Applied Biotechnology Center, Wuhan Institute of Bioengineering, Wuhan, China

The alternative oxidase (AOX) is a cyanide-insensitive terminal oxidase found in plants, fungi and some protozoa. In thermogenic plants (e.g., Sauromatum guttatum), AOX is responsible for heat generation, whilst in non-thermogenic species (e.g., Arabidopsis thaliana), it plays a key role in energy homeostasis and protection against oxidative stress [1]. In protozoa, respiration of the bloodstream forms of Trypanosoma brucei depends solely on AOX (TAO). Generally, the activities of AOXs are varied, although the amino acids surrounding the quinol binding site are extremely well conserved. Therefore, it is important to understand what factors affect the Vmax of AOXs？

Recently, we expressed TAO, SgAOX and AtAOX1a in the E. coli ∆hemA mutant FN102 and the activities, Km and Vmax of AOXs were investigated. Results showed that TAO had the highest Vmax, followed by the AtAOX1a, and then the SgAOX. SgAOX and AtAOX1a exhibited 1.5-fold and 6-fold increase in the activity (O2 consumption) following the addition of 10 mM pyruvate. The activity of AtAOX1a was also improved by 5 mM glyoxylate (6-fold) and 10 mM oxaloacetate (3.5-fold). Data on the Km and Vmax of these AOXs alongside IC50 & Ki’s for a variety of inhibitors following incorporation into proteoliposomes will be presented.

References [1] A.L. Moore, T. Shiba, L. Young, S. Harada, K. Kita, K. Ito, Unraveling the heater: new insights into the structure of the alternative oxidase, Annu Rev Plant Biol, 64 (2013) 637-663.

P3 a/12 Development of novel AOX inhibitors for fungicidal resistance management Luke Young, Mary Albury, Alice Copsey, James Misselbrook, Anthony L. Moore Biochemistry & Biomedicine, School of Life sciences, University of Sussex, Falmer, Brighton, United Kingdom

The alternative oxidase (AOX) is a non-protonmotive quinol oxidase located within the mitochondria of all plants, fungi and a number of pathogens such as Trypanosoma brucei brucei. The primary function for AOX within fungal mitochondria is energy homeostasis, acting as an overflow when the regular ETC is under stress thereby reducing ROS production. Treatment of fungal pathogens with traditional Qo fungicides results in an upregulation of the AOX, allowing the fungus to survive and potentially develop drug resistance [1]. Addition of AOX inhibitors has been demonstrated to potentiate the effectiveness of these fungicides [2], and as such we are developing specific AOX inhibitors to apply alongside traditional compounds to improve their fungicidal efficacy. AOX’s from various sources have been successfully expressed in a heme-deficient E.coli system, ranging from agrochemically important resistant strains such as Septoria tritici and Botryotinia fuckeliana, to AOX’s with human pathogenic implications such as Candida auris, the causative agent of candidiasis, and Ciona intestinalis, an AOX which is currently being used in gene therapy trials for patients with mitochondrial deficiencies. Work will be presented on the characterisation of each of these AOX proteins with respect to molecular activators, kinetic parameters and inhibition, with novel ascofuranone derivatives proving effective inhibitors in low nanomolar concentrations across all samples.

[1] B.N. Ziogas, B.C. Baldwin, J.E. Young, Alternative respiration: A biochemical mechanism of resistance to azoxystrobin (ICIA 5504) in Septoria tritici, Pesticide Science, 50 (1997) 28-34. [2] P.M. Wood, D.W. Hollomon, A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Q(o) site of Complex III, Pest Management Science, 59 (2003) 499-511.

P4 a/1 Exploring the reductive phase of Cytochrome c Oxidase: assignment of heme’s redox states and relative structure changes through potential-resolved FTIR Federico Baserga, Hendrik Mohrmann, Sven T. Stripp, Joachim Heberle Department of Physics, Freie Universität Berlin, Germany

Cytochrome c Oxidase (CcO) is a protein central to cellular respiration. Together with other enzymes, it contributes to the creation of the proton gradient essential to ATP synthesis. It is well known that CcO reduces molecular oxygen to water and pumps protons while undergoing a cycle initiated by electron injection from Cytochrome c. At the beginning of this catalytic cycle, the reduced Cytochrome c transfers electrons to the CuA cofactor of the enzyme. This step is followed, respectively, by the reduction of the metal centers of heme a and heme a3 and triggers a series of changes in the residues neighboring the active center. The mechanistic details of this machinery are not yet completely understood, partially because of the deficiency of time-resolved data providing structural information on the physiological cycle. We combine steady-state attenuated total reflection (ATR) FT-IR spectroscopy with electrochemistry in order to disentangle structural changes coupled to the reduction of single heme cofactors. We can control the redox state of their metal centers by mediated electron injection. After binding carbon monoxide to the active center of CcO and raising the applied potential, we monitor the shift of the C≡O stretching vibration, and ultimately its disappearance due to unbinding. CO is utilized both as a marker for the completely reduced state and as a probe for the Vibrational Stark Effect arising from the electric field of the reduced heme a on the oxidized heme a3. This approach allows us to clearly separate potentials corresponding to the oxidation states of the protein’s cofactors and to correlate them with the protein’s IR difference spectra.

P4 a/2 Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential Markus L. Björck, Peter Brzezinski Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden

Cytochrome c oxidase is the final electron acceptor in the mitochondrial electron-transfer chain where it catalyzes the reduction of oxygen to water. This electrogenic reaction is coupled to proton transfer across the inner mitochondrial membrane, which also contributes to formation of the proton electrochemical potential. Results from earlier studies (reviewed in [1]) have elucidated a detailed reaction mechanism, which involves step-wise intramolecular transfer of protons and electrons to the substrate O2. However, most of these earlier studies were performed with detergent-solubilized enzyme, i.e. without the proton electrochemical gradient that is present under native conditions in the mitochondrial membrane. The effect of a gradient on specific proton and electron transfer events in cytochrome c oxidase is currently unknown. Here, we have studied the effects of an ATP-induced membrane potential on the single- turnover reaction of cytochrome c oxidase with O2 in intact mitochondrial membranes using the flow-flash method. The results indicate that proton translocation, but not electron-transfer, is influenced (slowed) by the membrane potential. The data suggest that, as a result of the membrane potential, a previously-proposed [2,3], but not observed during turnover, reduced ferryl state, Fr, is formed at the catalytic site.

References: 1. Brzezinski, P., Gennis, R.B, Cytochrome c oxidase: exciting progress and remaining mysteries, J. Bioenergetics and Biomembranes 40 (2008) 521-531 2. Zaslavsky, D., Smirnova, I.A., Ädelroth, P., Brzezinski, P., Gennis, R.B, Observation of a novel transient ferryl complex with reduced

CuB in cytochrome c oxidase, Biochemistry 38 (1999) 2307-2311 3. Sharma, V., Wikström, M, The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase, Biochim. Biophys. Acta 1857 (2016) 1111-1115

P4 a/3 Conformational Transitions of Respiratory Cytochrome c Oxidase Marian Fabian2, Katarina Kopcova1, Ludmila Blascakova2, Tibor Kozar2, Daniel Jancura1,2 1Department of Biophysics, P. J. Šafárik University, Košice, Slovakia 2Center for Interdisciplinary Biosciences, Technology and Innovation Park, P. J. Šafárik University, Košice, Slovakia

Membrane bound respiratory oxidases catalyze the four-electron reduction of molecular oxygen to H2O. This redox reaction is associated with the generation of the transmembrane proton gradient. In spite of significant progress the identity of the structural elements involved in the proton translocation have not been fully elucidated [1]. Several alternative models of the redox-linked proton pumping have been suggested. A common feature of these models is a redox dependent conformational cycling between the proton- input state and proton-output state. These two conformations should be present in both oxidized and reduced forms of the participating metal center. Here we explore in more detail the availability of two conformational states for cytochromes a and a3 of bovine heart CcO. Second derivative of the absorption spectra of CcO demonstrated the redox dependent split of the Soret band of cytochrome a and cytochrome a3-ligated with cyanide. The reduction stimulated splitting of the Soret band very likely originates from the appearance of the water near both hemes. Our findings indicate that the Soret band electronic transitions of cytochrome a may discriminate between the open and closed water channel that is presumably participating in the proton pumping [2].

Acknowledgement This work was supported by Slovak Grant Agency (VEGA-1/0464/18) and the Slovak Research and Development Agency (APVV-15-0485).

References [1] S. Yoshikawa, and A. Shimada. Reaction mechanism of cytochrome c oxidase, Chem Rev 115 (2015) 1936-1989 [2] S. Yoshikawa, K. Muramoto, and K. Shinzawa-Itoh, Proton-pumping mechanism of cytochrome c oxidase, Annu Rev Biophys 40 (2011) 205-223

P4 a/4 EPR detection of radical(s) in cytochrome c oxidase Daniel Jancura1,2, Marian Fabian2 1Department of Biophysics, Faculty of Science, Safarik University, Kosice, Slovakia, 2Center for Interdisciplinary Biosciences, Technology and Innovation Park, Safarik University, Kosice, Slovakia

Catalytic mechanism of cytochrome c oxidase (CcO) involves formation of ferryl intermediates P and F. The production of these intermediates is accompanied by a formation of protein-based radical(s). The reaction of oxidized CcO with hydrogen peroxide also leads to the formation of P and F and corresponding radical species. However, the application of electron paramagnetic spectroscopy (EPR) to detect such radical has resulted only in the observation of low amounts relative to the concentration of the P intermediate. A possible reason for this fact is a coupling of the unpaired electron of radical with the paramagnetic metal center(s) within the catalytic site of CcO. We have developed a new approach, a moderate destabilization of the enzyme structure by protein denaturant, guanidinium chloride (Gnd.Cl), to detect stoichiometric amount of the radical in CcO. In this situation, a coupling between protein-based radical and a metal center(s) is broken. As our results show, the yield of the EPR observed radical in P is significantly increased in the presence of Gnd.Cl relative to that in the absence of denaturant. In a sample with 2 M Gnd.Cl, the yield of the detected radical reached ~50% of the P population. The origin of EPR detected radical(s) and their possible roles in the catalytic cycle of CcO is discussed.

Acknowledgement: This work was supported by Slovak Grant Agency (VEGA-1/0464/18) and the Slovak Research and Development Agency(APVV-15-0485).

P4 a/5 A novel setup for time-resolved IR spectroscopy on Cytochrome c Oxidase Pit Langner, Federico Baserga, Hendrik Mohrmann, Joachim Heberle Experimental Molecular Biophysics, Department of Physics, Freie Universität Berlin, Berlin, Germany

Cytochrome c Oxidase (CcO) is the fourth and terminal complex in the mitochondrial respiratory chain. Physiologically, it uses four electrons provided by cytochrome c to pump four protons into the intermembrane space, while catalytically reducing oxygen to water. The atomic details of the sequential steps that go along with this redox-driven proton translocation are still a matter of debate. We use CcO from Rhodobacter sphaeroides to study the correlation between the active center's redox state and the protonation events in the enzyme's catalytic cycle. Time-resolved infrared spectroscopy is a well-established method to investigate transient protonation changes but applying it to CcO poses a plethora of challenges. Our goal is to use a quantum cascade laser (QCL) setup and slow-flowing, COpoised CcO solution in a microfluidics channel transparent to mid-infrared radiation to eventually study oxygen binding on the reduced enzyme after CO flash-off. We report on a novel setup designed for this purpose.

P4 a/6 Definition of the Electron Transfer Pathway between Cytochrome c and Cytochrome Oxidase Francis Millett1, Martha Scharlau1, Lois Geren1, Eugene Y Zhen2, Ling Ma3, Ray Rajagukguk4, Bill Durham1, Shelagh Ferguson-Miller5 1Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA 2Ely Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA 3Rocky Mountain Cancer Center, Lakewood, CO, USA 4Department of Pharmacy, Eisenhower Health, Rancho Mirage, CA, USA 5Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA

A new crystal structure for the cytochrome c – cytochrome oxidase (Cc – CcO) complex has recently been determined [1] which is quite different from one previously determined by computational docking analysis [2]. A new pathway for electron transfer from the Cc heme to CcO CuA was proposed: heme c → Cc-C14 → Cc-K13 → CcO-Y105 → CcO-M207 → CuA [1]. To test this new pathway, we have carried out mutagenesis and kinetic studies of the reaction of ruthenium-labeled horse Cc (Ru-39-Cc) with bovine and R. sphaeroides CcO. Laser flash photolysis of a 1:1 complex between Ru-39-Cc and bovine CcO at low ionic strength results in electron transfer from -1 Ru-39-Cc to CuA in CcO with an intracomplex rate constant of ka = 80,000 s . The Ru-39-Cc K13A, K72A, K86A, and K87A mutants each increased the dissociation constant KD of the complex with bovine CcO significantly, but did not change the value of ka. The Rs.

CcO Y144S mutant (bovine Y105) only caused a 3.8-fold decrease in ka. In contrast, the Rs. CcO W143F mutant (bovine W104) caused a 450-fold decrease in ka, but did not lead to a significant change in KD or the redox potential of CuA. These results are not consistent with the pathway proposed in [1]. It is proposed that electron transfer occurs in the conformation [2] in which W143 (bovine W104) is in

Van der Waals contact with the Cc heme and the CcO CuA ligand M263 (M207) and mediates electron transfer from heme c to CuA. Supported by NIH grants GM20488, 8P30GM103450. References [1] S. Shimada, K. Shinzawa-Itoh, J. Baba, S. Aoe, A. Shimada, E. Yamashita, J. Kang, M. Tateno, S. Yoshikawa, T. Tsukihara, Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode, EMBO J. 36 (2017) 291-300. [2] V. A. Roberts, M. E. Pique, Definition of the interaction domain for cytochrome c on cytochrome c oxidase. III. Prediction of the docked complex by a complete, systematic search, J. Biol. Chem. 274 (1999) 38051-60.

P4 a/7 Role of subunit COX8A in biogenesis of cytochrome c oxidase in human fibroblasts Daria Rotko1, Bogusz Kulawiak1, Wolfram S. Kunz2 and Adam Szewczyk1 1Nencki Institute of Experimental Biology, Warsaw, Poland 2Institute of Experimental Epileptology and Cognition Research, and Department of Epileptology and Life & Brain Center, University of Bonn, Bonn, Germany

Biogenesis of cytochrome c oxidase (COX) protein complex is a tightly regulated process, which is executed under dual genetic control. According to the current paradigm, it proceeds in the modular fashion, where preassembled complexes of subunits and chaperones constitute discrete entities in the sequential COX assembly line. While COX deficiencies are some of the most frequent causes of respiratory chain disorders in humans, mutations in the nuclear genes of structural subunits of COX are very rare and few clinical cases have been reported to date. One of them, manifested in the patient with the Leigh-like syndrome, was ascribed to a mutation in COX8A leading to aberrant splicing, decreased transcript amounts and the loss of wild type protein. COX8A is the smallest nuclear-encoded structural subunit of COX with a poorly known function, implicated to have a role in maintaining the stability of COX. To study how COX8A deficiency affects organization of cytochrome c oxidase in the patient’s fibroblasts, we analyzed the pattern of assembly of COX into supramolecular complexes by blue native polyacrylamide gel electrophoresis (PAGE). The amount of COX was largely decreased and the residual COX was stabilized in the respirasomes together with complexes I and III. Further assessment of steady-state protein levels of individual COX subunits was done by SDS PAGE and Western blot, and followed by characterization of the transcription profile by quantitative reverse-transcription polymerase chain reaction. Our data suggest a differential effect of COX8A deficiency on the COX subunits within the same assembly modules, highlighting complexity of the dynamics between COX assembly and degradation. This project was supported by the Marie Sklodowska-Curie COFUND grant No. 665735 and Polish National Science Center grant No. 2015/18/E/NZ1/00737.

P4 a/8

Towards time-resolved structural studies of ba3-type cytochrome c oxidase Cecilia Safari, Swagatha Ghosh, Rebecka Andersson, Gisela Brändén University of Gothenburg, Department of Chemistry and Molecular Biology, Sweden

Cellular respiration is the process where energy in the form of electrons are used to produce storable chemical energy in the form of ATP. This process demands a series of electron transfers through the respiratory chain protein complexes, where the last protein complex is cytochrome c oxidase (CcO). CcO reduces di-oxygen to two water molecules at the same time as it functions as a proton pump. The free energy from the reduction-oxidation reactions is stored as an electrochemical proton gradient, and there is yet no clear picture of how proton pumping actually works in the CcOs. We have so far developed a convenient method for batch-crystallization in lipidic cubic phase (LCP) which has generated the first room- temperature Serial Femtosecond Crystallography (SFX) structure of ba3 CcO, free from radiation damage [1]. We have also performed time-resolved carbon monoxide (CO)-binding studies of ba3 CcO using both SFX and X-ray solution scattering. The purpose of the CO- binding studies has been to mimic O2-binding to the active site without starting the electron transfer reactions, as a first step to study the structural changes that occur. The long-term goal with our structural studies is to elucidate the fundamental proton pumping mechanism, which is considered to be a conserved mechanism among all oxidases.

1. R. Andersson, C. Safari, R. Dods, E. NangoR. Tanaka, A. Yamashita, T. Nakane, Kensuke Tono, Y. Joti, P. Båth, E. Dunevall, R. Bosman, O. Nureki, S. Iwata, R. Neutze, G. Brändén, Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature, Scientific Reports 7 (2017)

P4 a/9 Novel insights in to lipid-protein interactions from large scale atomistic molecular dynamics simulations of cytochrome c oxidase Vivek Sharma1,2, Aapo Malkamäki1 1Department of Physics, University of Helsinki, Helsinki, Finland 2Institute of Biotechnology, University of Helsinki, Helsinki, Finland

The terminal step of mitochondrial electron transport chain is oxygen reduction to water, which is catalyzed by complex IV or cytochrome c oxidase. Decades of biochemical and biophysical research, combined with structural data, have provided detailed insights into the relatively well-understood mechanism of redox-coupled proton pumping in cytochrome c oxidase [1]. Currently, theoretical and computational approaches are being used to decipher the subtle mechanistic questions [2,3], which are otherwise difficult to solve with latest experimental technology. In this work, we have performed fully atomistic classical molecular dynamics simulations of the dimeric form of cytochrome c oxidase. The data from microseconds long simulations allow us to characterize the crystallographic cardiolipin binding sites in unprecedented dynamic framework and provide novel insights into conserved lipid-protein interactions. We discuss functional implications of these interactions in cytochrome c oxidase mechanism and regulation.

References: 1. M. Wikström, K. Krab, V. Sharma, Oxygen activation and energy conservation by cytochrome c oxidase, Chem. Rev. 118 (2018) 2469-2490. 2. V. Sharma, P. Jambrina, M. Kaukonen, E. Rosta, P. Rich, Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations, Proc. Natl. Acad. Sci. USA. 114 (2017) E10339-E10348. 3. M. Wikström, V. Sharma, Proton pumping by cytochrome c oxidase – A 40 year anniversary, Biochim. Biophys. Acta. In press.

P4 a/10 Extraction and Reconstitution of S. cerevisiae Cytochrome c Oxidase in Liposomes without the Use of Detergent Irina A. Smirnova, Pia Ädelroth, Peter Brzezinski Department of Biochemistry and Biophysics, the Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden E-mail: [email protected]

Functional studies of proton translocation by individual respiratory chain enzymes require reconstitution in phospholipid vesicles that also provide a near-native lipid environment. However, the currently used methods to reconstitute individual complexes involve the use of detergents for isolation of the enzyme of interest, which may result in removal of native lipids that are important for maintaining structure and function. A recently-developed method based on the use of styrene maleic acid co-polymer (SMA) allows isolation of membrane proteins in nanodiscs, surrounded by a disc of native lipids [1]. In an earlier study we used SMA to isolate pure and active cytochrome c oxidase (CytcO) from S. cerevisiae mitochondria [2]. Here we describe the direct incorporation of SMA-CytcO nanodiscs into small and large liposomes without the use of detergent. The efficiency of the incorporation was about 90%. The CytcO molecules were oriented with the cytochrome-c binding site facing to the outside (85 % of the CytcOs). Turnover of CytcO resulted in formation of the membrane potential (∆ψ) as monitored using permeant cations, the fluorescent dye (TMRE) and tetraphenylphosphonium chloride (TPP). The new method thus allows purification of active membrane proteins together with native lipids and reconstitution in vesicles of well-defined lipid composition without the use of detergent.

1. J.M. Dörr, S. Scheidelaar, M.C. Koorengevel, J.J. Dominguez, M. Schäfer, C.A. van Walree, J.A. Killian, The styrene–maleic acid copolymer: a versatile tool in membrane research, European Biophysics Journal 45 (2016) 3-21. 2. I.A. Smirnova, D. Sjöstrand, F. Li, M. Björck, J. Schäfer, C. von Ballmoos, G. Lander, P. Ädelroth, P. Brzezinski, Isolation of pure yeast Complex IV in native lipid nanodiscs, Biochim. Biophys. Acta 1858 (2016) 2984-2992.

P4 a/11 Computational Investigation of the Hydration of the H Channel of Bovine Heart Cytochrome c Oxidase Bo Thomsen, Yuji Sugita Theoretical Molecular Science Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan) Email: [email protected]

Cytochrome c Oxidase (CcO) is known for its role in producing the electrochemical potential across the inner mitochondrial membrane through the reduction of oxygen and redox coupled proton pumping. In structural studies of Bovine Heart CcO (BHCcO) the proton pumping has been suggested to proceed through the H channel [1], a channel not conserved in the structures of CcOs from other organisms. A recent computational report [2] using conventional molecular dynamics (MD) to study the H channel of BHCcO identifies the channel as a dipole well, since no water molecules were found in critical regions of the H channel in multiple conventional MD trajectories, thereby making proton transport through the H channel unlikely. The difference in hydration between crystal structure and in vitro has been used in previous computational studies to explain the connection between proton pumping channel and loading site in CcO structures for other organisms [3]. The free energy barriers for this increased hydration from the crystal structure to in vitro structure might however prohibit increased hydration, unless one performs untraceably long conventional MD simulations. We therefore seek to investigate the hydration of the H channel of BHCcO using steered MD to increase the hydration by pulling water into the channel, thereby exploring the hydration of the H channel more efficiently and extensively than conventional MD, to elucidate the function of the H channel of BHCcO.

References: 1. S. Yoshikawa, A. Shimada, Reaction Mechanism of Cytochrome c Oxidase, Chem. Rev. 115 (2015) 1936-1989 2. V. Sharma, P. G. Jambrina, M. Kaukonen, E. Rosta, P. R. Rich, Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations, Proc. Natl. Acad. Sci. U.S.A. 114 (2017) E10339-E10348 3. M. Wikström, M. I. Verkhovsky, G. Hummer, Water-gated mechanism of proton translocated by cytochrome c oxidase, Biochim. Biophys. Acta Bioenerg. 1604 (2003) 61-65

P4 a/12 The stochastic modelling of proton pumping: the contribution of the K-channel Victoria Titova1, Stanislav Boronovskiy1, Jean-Pierre Mazat2, Yaroslav Nartsissov1, Stephane Ransac2 1Institute of cytochemistry and molecular pharmacology, Moscow, Russian Federation 2Laboratoire de métabolisme énergétique cellulaire IBGC – CNRS, Bordeaux, France

Cytochrome c oxidase (CcO) is a terminal enzyme complex of the mitochondrial respiratory chain. It catalyzes oxygen reduction to water alongside with cytochrome c oxidation. These reactions are coupled with proton pumping through the membrane from the N-side to the P-side and therefore the membrane potential is generated. In the present study CcO activity was estimated via the computer algorithm based on stochastic approach where the catalytic cycle of enzyme is reported as a set of consecutive transitions between distinct states with the various numbers of metabolites. The present work is dealing with CcO activity under different pH. The investigated schemes of functioning of CcO included four different schemes. The first pair of schemes assumed the pumping of consumed only via the D-channel protons, and the pumping occurred when either the binuclear center was fullfilled with protons or it has bound three or four protons. The second pair of schemes assumed the pumping of consumed both via the D- and the K-channel protons, and the pumping also occurred when the binuclear center was fulfilled with protons or not. K-proton flow increased from 8% of total proton flow to 96% under pH increase from 4 to 10, besides equal contributions to total proton flow were observed under pH 8.5. This fact shows that one can observe a change of channel modes under the alkalization of the enzyme environment: the D-channel activity decreases significantly in favor of the K-channel. K-proton flow increased under raising pH, then the peak under pH from 8.4 to 8.6 and further decrease were observed in the model scheme, assuming K-protons transmembrane transfer. The peak of the pumping efficiency was also obtained when pumping of K-protons occurs under the binuclear center full of protons. However the pumping efficiency monotonically increased when three or four protons were present in the binuclear center.

P4 a/13 Protonation state and conformational changes at the K-channel entry of cytochrome c oxidase Alexander Wolf1, Jovan Dragelj2, Ernst-Walter Knapp2, Ulrike Alexiev1 1Department of Physics, Freie Universität Berlin, Berlin, Germany 2Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin

Cytochrome c oxidase (CcO) transports protons across the membrane through the K- and D-pathway. Only active in the reductive phase of the enzyme, the K-channel delivers two chemical protons to the binuclear center. While differences in the protonation state of specific residues and water organization around the channel have been implicated [1], the exact mechanism of K-channel gating and the conformational changes accompanying it are still not fully understood. By employing site-directed labeling of CcO from Paracoccus denitrificans with environmentally sensitive fluorophores, we have shown a significant pKa and polarity shift [2] of K-channel surface-bound reporter groups upon reduction of the enzyme, indicating long-range interactions between the binuclear center and surface. Fluorescence Correlation Spectroscopy of these CcO-fluorophore conjugates revealed only slightly increased protonation rates in the oxidized state [4]. By accompanying fluorescence spectroscopic techniques with molecular dynamics (MD) simulations, we show that the redox-state of the enzyme drives protonation and local conformational changes at the K-channel surface. [1] J. Liu, C. Hiser S. Ferguson-Miller, Role of conformational change and K-path ligands in controlling cytochrome c oxidase acitivity, Biochem. Soc. Trans. 45 (2017) 1087-1095 [2] K. Kirchberg, H. Michel, U. Alexiev, Exploring the entrance of proton pathways in cytochrome c oxidase from Paracoccus denitrificans: Surface charge, buffer capacity and redox-dependent polarity changes at the internal surface, Biochim. Biophys. Acta 1827 (2013) 276-284 [3] A. Wolf, C. Schneider, T.Y. Kim, K. Kirchberg, P. Volz, U. Alexiev, Fluorescence correlation spectroscopy as a tool to investigate the protonation dynamics of cytochrome c oxidase Phys. Chem. Chem. Phys. 18 (2016) 12877-12885 [4] K. Kirchberg, H. Michel, U. Alexiev, Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase, J. Biol. Chem. 287 (2012) 8187-8193

P4 a/14 Physiological and structural analysis of cytochrome c oxidase activating protein Higd1a Yuya Nishida, Yasunori Shintani, Seiji Takashima Medical Biochemistry, Osaka University Graduate School of Medicine, Osaka (Japan) E-mail: [email protected]

The respiratory chain (RC) transport electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a directly interact with cytochrome c oxidase (CcO) and increased its activity, thereby leading to increased ATP synthesis [1]. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]mito) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP]mito under hypoxia and preserved cardiac function in zebrafish. Next, to gain an insight molecular mechanism by which Higd1a increased CcO activity, we aimed to perform structural analysis of the complex between CcO and Higd1a by cryo-EM. First, we successfully obtained the electron density map of the Higd1a-free CcO at ~4 angstrom resolution. Then, we examined the Higd1a-CcO complex stabilized condition, and confirmed that the complex was highly stabilized in a Higd1a-destabilized condition. These results hold promise for successfully determining the Higd1a-CcO complex structure by cryo-EM.

1. T. Hayashi et al., Higd1a is a positive regulator of cytochrome c oxidase, Proc. Natl. Acad. Sci. U.S.A. 112 (2015) 1553-1558

P4 a/15 Proton kinetics in ba3 oxidase Federica Poiana1, Christoph von Ballmoos2, Pia Ädelroth1 and Peter Brzezinski1 1Stockholm university, 2Bern University [email protected]

Ba3 cytochrome c oxidase (CcO) is the terminal oxidase of thermophilic bacterium Thermus thermophilus; it reduces oxygen to water and pumps protons across the membrane, similarly to A-type oxidases found in mitochondria and other bacteria. Interestingly, despite maintaining the overall folding structure of A-type oxidases, ba3 CcO harbors many significant differences, both in sequence and functioning mechanism [1]. For example, ba3 has only one proton input pathway, which is located in the same position as the K-pathway of aa3 CcO but has no conserved residues with the latter [2], in particular the key residue E286 responsible for proton gating in proximity of the active site is lacking in B-type CcO. Furthrmore, it has been shown that ba3 CcO pumps protons to a reduced yeald, 0.5H+/e- instead of 1H+/e- of aa3 CcO [3]. These observations led to the conclusion that ba3 CcO has developed a different functioning mechanism with respect to A-type CcOs. Recent studies have investigated how mutations of selected residues in the K-pathway analogue influence proton uptake and O2 reduction [4,5]. Our research has then extended those findings by analyzing O2 catalysis in two additional variants of the K-pathway analogue to answer the key question: which residues are fundamental for proton uptake? According to our results, we can identify E15(II) as the only crucial residue in the pathway; since, upon mutation to a glutamine, O2 reduction is not achieved past the A->P transition. Furthermore we focused on proton release in single turnover. According to previous experiments, a possible O2 reduction mechanism has been proposed where three protons are taken up and one is released. Proton uptake kinetics showed net proton uptake of only two protons, hinting that the last uptake and the first release could be simultaneous. By reconstituting ba3 CcO into liposomes, we were able to measure the proton release only and relate it to the F->O intermediate, supporting therefore the suggested mechanism. References

[1] I.A. Smirnova, D. Zaslavsky, J.A. Fee et al. Electron and proton transfer in the ba 3 oxidase from Thermus thermophilus, J Bioenerg Biomembr (2008) 40: 281 [2] T. Tiefenbrunn, W. Liu, Y. Chen, V. Katritch, C.D. Stout, Fee JA, et al. (2011) High Resolution Structure of the ba3 Cytochrome c Oxidase from Thermus thermophilus in a Lipidic Environment. PLoS ONE 6(7) [3] C. von Ballmoos, P. Ädelroth, R. B. Gennis, P. Brzezinski, Proton transfer in ba3 cytochrome c oxidase from Thermus thermophilus, Biochimica et Biophysica Acta (BBA) – Bioenergetics, (2012), 650-657, [4] C. von Ballmoos, N. Gonska, P. Lachmann, R. B. Gennis, P. Ädelroth, and P. Brzezinski, Mutation of a single residue in the ba3oxidase specifically impairs protonation of the pump site Proc Natl Acad Sci, (2015), 3397-3402 [5] I. Smirnova, J. Reimann, C. von Ballmoos, H.-Y. Chang, R. B. Gennis, J. A. Fee, P. Brzezinski, and P. Ädelroth, Functional Role of Thr-312 and Thr-315 in the Proton-Transfer Pathway in ba3 Cytochrome c Oxidase from Thermus thermophilus, Biochemistry (2010), 7033-7039

P4 b/1 Creatine Transporter (CrT) Deficiency Impairs Brain Energetics Under Stress Hong-Ru Chen1,2, Xiaohui Zhang-Brotzge2, Ton J. DeGrauw2, Diana M. Lindquist3, Siming Wang4, Chia-Yi Kuan1,2 1Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, USA 2Department of Pediatrics, Emory University School of Medicine, Atlanta, USA 3Imaging Research Center, Department of Radiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA 4Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, USA

Creatine (Cr) and phosphocreatine (PCr) provide immediate replenishment of ATP to fuel diverse cellular functions. Our daily need for Cr/PCr comes from the diet and de-novo biosynthesis through Cr synthesis enzymes, but creatine transporter (CrT) is needed to maintain the brain Cr level. In CrT deficiency, a disease that was first reported in 2001 and constitutes the second highest cause of X- linked mental retardation, the brain Cr/PCr peaks are almost absent on magnetic resonance spectroscopy. Importantly, the CrT-deficient patients do not respond to oral Cr supplement treatment, in contrast to patients with Cr synthesis enzyme mutations. Thus, there is an unmet need for better understanding the functions of CrT to develop the therapy of CrT deficiency. To this end, we have generated CrT mutant mice and used them to examine the impacts of Cr/PCr-deficiency on brain energetics and stress responses. We found that adult CrT-/y mice manifest greatly reduced Cr/PCr levels and dendritic spine density in the brain. Further, CrT-/y mice have elevated susceptibility to starvation-induced autophagy, neonatal hypoxia-ischemia, and ischemic brain injury, which are all correlated with increased pAMPK and reduced mTOR signaling activities. Moreover, we showed that using a novel Cr supplement method, one can administer Cr to CrT-deficient mice after cerebral ischemia to significantly reduce brain damage. Together, these results suggest an important role by CrT to maintain the brain energetics under stress and a promising treatment of CrT deficiency.

P4 b/2 Hexokinase is the main modulator of H2O2 production and calcium handling during post-natal brain development in mitochondria Eduardo S Ferreira, Antonio Galina Department of Medical Biochemistry Leopoldo de Meis, IBqMLM-Federal University of Rio de Janeiro, Brazil

Brain has a high demand for glucose and oxygen but weight only 2% of the body, which could lead to oxidative imbalance due to the great capacity of mitochondria to produce Reactive Oxygen Species (ROS) and its low antioxidant capacity. About 90% of the brain’s Hexokinase 1 (mHK-I) is bound to mitochondrial outer membrane and its activity modulates the Mitochondrial Membrane Potential

(ΔΨM)-dependent ROS production. After birth, glucose usage rises as energy source. Mitochondrial content and oxygen consumption also increase and superoxide dismutase is implemented, regulating ROS. In fact, hydrogen peroxide (H2O2), and calcium, are necessary signaling molecules to correctly drive brain cell differentiation, migration division and neuroplasticity after birth. Mitochondria was shown to generate ROS and uptake calcium and mHK modulates both mechanisms. This way, we sought to determine mtHK roles during post- natal development in: 1) mitochondrial H2O2 generation, comparing the antioxidant enzymes, and 2) mitochondrial calcium uptake; to understand the control of its signaling molecules. We performed high resolution respirometry, H2O2 sensitive AmplexRed, enzyme activities and CaGreen assays. We show that oxygen consumption development is closely followed by ROS production and mHK activity, the latter being highly correlated with de ROS production (r2 = 0,79), in contrast with the mitochondrial antioxidant enzymes such as Glutathione Reductase (r2 = 0,029), Glutathione Peroxidase (r2 = 0,008) and Thioredoxin Reductase (r2 = -0,6). Despite being low after birth, since post-natal day 7, the mHK activity is able to reduce as much as 90% of total ROS production during succinate oxidation. Finally, newborn mHK is unable to increase mitochondrial calcium uptake, effect accomplished by P60 mHK. We conclude that, although low postnatally, mHK activity can developmentally regulate mitochondrial H2O2 and calcium uptake, may contributing to ROS and calcium signaling after birth.

P4 b/3 Perinatal Asphyxia and Brain Development: Mitochondrial Damage Without Anatomical or Cellular Losses Lima JPM, Rayêe D, Silva-Rodrigues T, Pereira PRP, Mendonca APM, Rodrigues-Ferreira C, Szczupak D, Fonseca A, Oliveira MF, Lima FRS, Lent R, Galina A, Uziel D Institute of Medical Biochemistry Leopoldo de Meis

Perinatal asphyxia remains a significant cause of neonatal mortality and is associated with long-term neurodegenerative disorders. In the present study, we evaluated cellular and subcellular damages to brain development in a model of mild perinatal asphyxia. Survival rate in the experimental group was 67%. One hour after the insult, intraperitoneally injected Evans blue could be detected in the fetuses' brains, indicating disruption of the blood-brain barrier. Although brain mass and absolute cell numbers (neurons and non-neurons) were not reduced after perinatal asphyxia immediately and in late brain development, subcellular alterations were detected. Cortical oxygen consumption increased immediately after asphyxia, and remained high up to 7 days, returning to normal levels after 14 days. We observed an increased resistance to mitochondrial membrane permeability transition, and calcium buffering capacity in asphyxiated animals from birth to 14 days after the insult. In contrast to ex vivo data, mitochondrial oxygen consumption in primary cell cultures of neurons and astrocytes was not altered after 1% hypoxia. Taken together, our results demonstrate that although newborns were viable and apparently healthy, brain development is subcellularly altered by perinatal asphyxia. Our findings place the neonate brain mitochondria as a potential target for therapeutic protective interventions.

P4 b/4 Novel pathogenic heteroplasmic substitution m.14597 A>G in the MT-ND6 gene in female with Leber’s hereditary optic neuropathy Tatiana D Krylova1, Natalia L Sheremet2, Vyacheslav Yu Tabakov1, Polina G Tsygankova1, Yulia S Itkis1, Vitalii V Kadyshev1, Ekaterina Yu Zakharova1 1Research Centre For Medical Genetics, Moscow, Russia 2Research Institute of Eye Diseases, Moscow, Russia

Leber’s hereditary optic neuropathy (LHON, OMIM 535000) is one of the most common mitochondrial disease and characterized by a subacute bilateral loss of vision. LHON is caused by mutations in mitochondrial DNA (mtDNA). Now about 19 LHON pathogenic mutations in mtDNA have been published (according to www.mitomap.org). 35 y.o. woman with clinical presentation of LHON was negative for 19 common mutations (test performed with MLPA (Multiplex Ligase-dependent Probe Amplification) and automatic direct sequencing). We applied the whole mtDNA sequencing by NGS (Next-Generation Sequencing) on IonTorrent S5 and identified probably pathogenic variant m.14597 A>G (MT-ND6 gene) in heteroplasmic state in the patient’s DNA extracted from blood, fibroblasts and urine sediment (24%, 76%, 86% mutant load respectively). The results were confirmed by direct sequencing. Previously this variant also in heteroplasmy was associated with dysarthria and dystonia [1]. High-resolution respirometry on patient’s fibroblasts was applied to reveal OXPHOS and ETC capacity defects. Almost two times difference for the FCR R/E, netR/E was detected in intact cells between patient’s and control’s samples; in patient’s permeabilized cells we observed increased CII/E, Rot/E, and decreased R/CII, CI/CII ratios compared to control cells what indicates complex I dysfunction and complex II compensatory effect. Established data allows to suggest high pathogenicity of m.14597 A>G substitution.

1. Y. Yang et al, Clinical whole-exome sequencing for the diagnosis of mendelian disorders, N Engl J Med. 369(16) (2013) 1502-11.

P4 b/5 Characterization of Reversible Loss of Flavin from Mitochondrial Complex I in Ischemic Brain Injury Anna Stepanova1, Sergei Sosunov1, Anna Kahl2, Vadim Ten1, Alexander Galkin1,2 1, Department of Pediatrics, Columbia University, , NY, USA 2, Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA

Mitochondrial dysfunction plays a central role in ischemia/reperfusion (I/R) brain injury. Accumulation of succinate in ischemia may favor the process of reverse electron transfer (RET) when a fraction of succinate-derived electrons is directed upstream to mitochondrial Complex I (C-I) and reactive oxygen species (ROS) are formed. C-I is also known to be affected first by brain ischemia but the mechanism of damage is not fully understood. Brain I/R was induced using hypoxia/ischemia model in neonatal mice or middle cerebral artery occlusion model of stroke in adult mice. We assessed C-I activity and flavin content in mitochondrial membranes ex vivo. For in vitro studies of intact brain mitochondrial, we tested the effect of RET on H2O2 release, C-I activity, and flavin content under different conditions, e.g., varying oxygen concentration

([O2]). We demonstrated post-ischemic C-I activity decrease and loss of the enzyme’s flavin in both in vivo models of I/R. We found RET- induced inactivation of C-I in mitochondria in vitro, which resulted in a progressive decline in the H2O2 release rate. C-I inactivation was caused by redox-dependent dissociation of flavin from the enzyme under conditions of RET. Addition of exogenous flavin to the in vitro assay partially recovered the activity of C-I, indicating the reversibility of the inactivation. Mitochondrial H2O2 release rate is linearly dependent on oxygen level [1] while the dynamics of C-I flavin loss were the same at different [O2]. Therefore, RET-induced inactivation of C-I was not dependent on the level of ROS. We propose that this previously undescribed process of flavin dissociation from C-I may underlie early mitochondrial impairment in cerebral ischemia.

1. A. Stepanova, A. Kahl, C. Konrad, V. Ten, A. Starkov, A. Galkin, Reverse electron transfer results in a loss of flavin from mitochondrial complex I: potential mechanism for brain ischemia reperfusion injury, J Cereb Blood Flow Metab. 37 (2017) 3649–3658.

P4 b/6 Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer’s disease neurons Niamh M. C. Connolly2, Pierre Theurey1, Ilaria Fortunati3, Susette Lauwen2, Camilla Ferrante3, Paola Pizzo1,4, Jochen H. M. Prehn2 ¹Dept. of Biomedical Sciences, University of Padua, Padua, Italy ²Dept. of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland 3Dept. of Chemical Sciences, University of Padua, Padua, Italy 4Neuroscience Institute - Italian National Research Council (CNR), Padua, Italy

Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer’s disease (AD). We combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain, but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the respiratory chain. To validate these predictions, we utilised fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, non-intuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity.

P5 a/1 ATP synthase purification and subunit composition analysis in the models of mammalian Complex V deficiencies Efimova Iuliia, Nůsková Hana, Tauchmanová Kateřina, Vrbacký Marek, Kovalčíková Jana, Pecina Petr, Ho Hien, Houštěk Josef, Mráček Tomáš Department of Bioenergetics, Institute of Physiology, The Czech Academy of Sciences, Prague

Mitochondrial F1Fo ATP synthase produces as much as 90% of cellular ATP. Biogenesis of this multi-subunit enzyme with two-genome origin is sophisticated - modular model has been proposed, however the detailed information about the latter stages of the enzyme assembly is incomplete. In our laboratory, we have recently established number of transgenic models deficient for the subunits of ATP synthase: a, A6L, ε, γ, δ, DAPIT, MLQ and also for a TMEM70 assembly factor. The aim of this project is to analyze the subunit composition and characterize the individual assembly intermediates of ATP synthase in these cellular and animal models. Preliminary results revealed presence of non-canonical intermediates of ATP synthase biogenesis on various models of complex V deficiencies under different electrophoretic conditions. Subunit composition of the observed intermediates in TMEM70 and DAPIT knockout models remains unknown. Our subunit composition data are only based on 2D-SDS PAGE analysis of subcomplex bands cut out form native gels. At present, we are working on adaptation of the technique of affinity purification through IF1 inhibitor protein [1, 2] to isolate individual F1 domain containing subassemblies and study their subunit composition using mass spectrometry and electrophoretic techniques. Obtained results should improve our understanding of complex V maintenance and assembly and its supramolecular organization. Supported by the Czech Ministry of Health (16-33018A).

[1] J. V Bason, M. J. Runswick, I. M. Fearnley, and J. E. Walker, Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase., J. Mol. Biol., 406 (2011) 443–53. [2] M. J. Runswick, J. V Bason, M. G. Montgomery, G. C. Robinson, I. M. Fearnley, and J. E. Walker, The affinity purification and characterization of ATP synthase complexes from mitochondria., Open Biol., 3 (2013) 120-160.

P5 a/2 Bioinformatic analysis of prokaryotic F-type ATP synthase operons Boris A. Feniouk1,2, Tatiana D. Kholina1, Daria V. Dibrova1,2 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia 2A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

FOF1-ATP synthase is a multisubunit membrane enzyme that interconverts two main “energy currencies” of a living cell – ATP and protonmotive force. We investigated the variety of ATP synthases in prokaryotes by analyzing 711 genomes that represent the diversity of eubacteria and archaea [1]. The analysis confirmed that F-type ATP synthase is an eubacterial enzyme and is very rarely found in archaea. It was also revealed that F-type ATP synthase is absent in some eubacteria, including Spirochaetaceae, Synergistales, Acholeplasmatales, Thermales, Deinococcales. On the other hand, we found many species having two or even three different F-type ATP synthases. In most cases the additional enzymes were sodium-translocating N-type ATPases. The operon structure of F-type ATP synthase varied between different bacteria. A common case is a single operon containing genes of all subunits and of UncI protein that is involved in assembly of the FO part of the enzyme. But in some species, there are two or more (up to six!) operons coding ATP synthase subunits. We also found ATP synthase operons that contain genes coding other proteins with known function (e.g. uracil-xanthine permease, threonine efflux protein, ribonuclease R) or hypothetical proteins. One of the latter hypothetical proteins is found in several species of Flavobacteriia and Cytophagia. It is a hydrophobic protein with four transmembrane helices predicted. The corresponding gene is located between genes coding subunit a and UncI protein. We speculate this hypothetical protein, like UncI, might be involved in assembly of FO.

This work was supported by the Russian Science Foundation research project №14‐14‐00128.

[1] M.Y. Galperin, K.S. Makarova, Y.I. Wolf, E.V. Koonin, Expanded microbial genome coverage and improved protein family annotation in the COG database, Nucleic Acids Res. 43 (2015) D261–9.

P5 a/3 Single particle analysis of V1-ATPase from thermophilic bacterium Aya Furuta1, Nao Takeuchi1, Atsuko Nakanishi1, Jun-ichi Kishikawa1, Kaoru Mitsuoka2, Ken Yokoyama1 1Department of Molecular Biosciences Kyoto Sangyo University, Kyoto, Japan 2Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan

VoV1 from Thermus thermophilus (T.thVoV1) is a rotary molecular motor that consisted of hydrophilic ATP-driven motor, V1, and integral membrane motor, Vo, driven by proton motive force. The V1 is composed of a rotary axis of DF subunits and an A3B3 stator ring. To clarify the molecular mechanism of V1, we have attempted to determine structure of V1 by single particle analysis using cryogenic electron microscopy (cryo-EM). The purified V1 was applied to carbon grids with or without thin carbon backing (carbon coated grids).

The vitrified grid was observed by Titan Krios (FEI). The single particle images of V1 were extracted from the cryo-EM images and subjected to 2D classification using RELION 2.1. When the cryo grid without thin carbon backing was used, orientation of most particle images of V1 were up or bottom; very few side orientation images were found in cryo-EM images. The same case happened when using a carbon coated grid without any surface modification. As a result, this strong preferred orientation of particle images of V1 impeded the

3D reconstitution of V1 at high resolution. In this study, we attempted to overcome the preferred orientation by modification of thin coated carbon and sample preparation to obtain high resolution map of the V1. Firstly, we modified the thin carbon membrane on the grid with poly-lysine. It was expected that the interactions between positive charge of poly-lysine and negative charge of V1 change the distribution of V1 orientations. Secondly, V1 with a detergent, LMNG (Lauryl Maltose-Neopentyl Glycol) was used for preparation of cryo-grids. These modified methods changed the distribution of orientation of particle images; the preferred orientation of V1 was partially overcome. We selected particles from good 2D classes from a dataset, and combined these particles for 3D classification.

We finally obtained 3D reconstruction of V1 at 5.8 resolution.

Å

P5 a/4 Structure, mechanism, and regulation of the chloroplast ATP synthase Alexander Hahn1, Janet Vonck1, Deryck Mills1, Thomas Meier1,2, Werner Kühlbrandt1 1Department of Structural Biology, Max-Planck-Institute of Biophysics, Frankfurt, Germany 2Department of Life Sciences, , United Kingdom

Plant and cyanobacteria use light energy to produce an electrochemical proton gradient across the photosynthetic membrane to drives the synthesis of ATP by the chloroplast ATP synthase (cF1Fo). The catalytic α3β3 head (cF1) performs sequential ATP synthesis which is powered by the cFo rotary motor in the membrane. cFo contains a rotor ring of 14 c-subunits and subunit a that conducts the protons to and from the c-ring protonation sites. The central stalk of subunits γ and ε transmits the torque from the Fo motor to the catalytic cF1 head, resulting in the synthesis of three ATP per revolution. The peripheral stalk subunits b, b' and δ act as a stator to prevent unproductive rotation of cF1 with cFo. cF1Fo has a unique redox sensor that couples ATPase activity to the light sensitive redox potential of the stroma. We reconstituted the ATP synthase from spinach chloroplasts into lipid nanodiscs and determined its structure by electron cryo- microscopy (cryo-EM) at a resolution of 2.9 Å (cF1) to 3.4 Å (cFo). In the cF1 ATPase head, we observe nucleotides with their coordinating Mg ions and water molecules, allowing assignment to the three well-characterized functional states involved in rotary ATP synthesis. Subunit δ on top of the ATPase head binds to all three α subunits, ensuring that only one peripheral stalk can attach. The loosely entwined, long α-helices of the peripheral stalk subunits b and b' clamp the integral membrane subunit a to the c-ring and function as elastic spring that absorbs and redistributes energy between tree, surprisingly, unequal rotation steps. Protons are translocated through a hydrophilic channel on the lumenal surface to the glutamate residues on the c-ring rotor which carry protons for an almost full rotation before releasing them into the stroma through a second hydrophilic channel. A strictly conserved arginine separates the access and exit channels, preventing leakage of protons through the membrane. Subunit γ has an L-shaped double hairpin that functions as redox sensor and blocks rotation to avoid wasteful ATP hydrolysis at night.

References A. Hahn, J. Vonck, D.J. Mills, T. Meier, W. Kühlbrandt, Structure, mechanism, and regulation of the chloroplast ATP synthase, Science 360, (2018) 620

P5 a/5 Water molecules in proton half-channels of F0F1-ATP synthase: prediction and implications Leonid A. Ivontsin, Elena V. Mashkovtseva, Yaroslav R. Nartsissov Institute of Cytochemistry and Molecular Pharmacology, Moscow, Russia

Over the past decades, it has been clearly shown that water plays an important role in proteins functions. It is also the case for F0F1- ATP synthase, which is a universal molecular motor that provides synthesis of ATP due to the energy of the proton gradient. Regardless recent structural studies have led to the determination of proton half-channels location, many issues remain unanswered. The key unresolved problem is the proton transfer coupling with the ATP synthesis. Water molecules can penetrate inside the proteins, becoming an integral part of the enzymes. They bind to each other and solvate charges, thus determining the movement of ions within the enzymes. Simulation of possible distributions of water molecules in the F0F1- ATP synthase input half-channel was carried out. By means of cluster analysis on the obtained general set of fixed water molecules locations, we defined a series of areas where water molecules are most likely to be found. In each such area a water molecule could be observed with a probability higher than the threshold. Based on the locations of the enzyme amino acid residues and the resulting distributions of water molecules, profiles of the electrostatic potential were calculated. Probability of proton transfer from one charged center to another was determined by quantum-mechanical model of one-dimensional motion that allowed to consider the purely quantum effects inherent in microparticles. The problem of proton movement through the sequence of charged centers in the half-channel is solved using a stochastic approach. While investigating of the system and its parameters under different conditions and modifications, a range of proton transport times was obtained, which possible corresponds to different enzyme activity. The observed set of proton paths through the input half-channel showed several modes of transitions through the enzyme, which occurred to be very sensitive to the water molecules distribution inside.

P5 a/6 Role of ATPase Inhibitory Factor 1 (IF1) in metabolic regulations of insulin secreting INS-1E cells Anežka Kahancová, Filip Sklenář, Petr Ježek and Andrea Dlasková Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic

ATPase Inhibitory Factor 1 (IF1) is a small (10 kDa) nuclear-encoded protein, which is involved in regulation of mitochondrial ATP synthase activity. Well established function of IF1 is to inhibit hydrolysis of ATP by the ATP synthase under the conditions, of dissipated proton gradient across the mitochondrial inner membrane, such as during starvation or hypoxia/ischemia. On the other hand, the previously reported ability of IF1 to inhibit the ATP synthesis [1] is still a matter of discussion and further studies are required. We have previously demonstrated that IF1 silencing in model pancreatic β-cells (INS-1E) upregulates ATP cellular levels and leads to concomitant increase in insulin secretion [2]. To examine if also IF1 overexpression will influence ATP levels and insulin secretion, we have established INS-1E cells stably overexpressing IF1 protein. Consistently with previous data, these cells produced less ATP and insulin and their oxygen consumption rate was lower. Altogether, our results point to IF1 as an important regulator of cellular ATP levels in pancreatic β-cells, which expression has to be tightly regulated to ensure adequate insulin secretion. Furthermore, we have studied submitochondrial localization of IF1 using super-resolution microscopy dSTORM (direct stochastic optical reconstruction microscopy). Obtained 3D images have revealed that IF1 is not freely diffused within the mitochondrial tubule but instead localizes in clusters. We have employed advanced image analysis tools to characterize these clusters and compare them with clusters formed by the ATP synthase.

1. J. García-Bermúdez, J.M. Cuezva, The ATPase Inhibitory Factor 1 (IF1): A master regulator of energy metabolism and of cell survival, BBA 1857(8) (2016) 1167-1182 2. A. Kahancová, F. Sklenář, P. Ježek, A. Dlasková, Regulation of glucose-stimulated insulin secretion by ATPase Inhibitory Factor 1 (IF1). FEBS Lett. 592(6) (2018) 999-1009

Supported by GACR grant No. 17-08565S

P5 a/7

Regulatory C-terminal domain of the ε subunit in FoF1 ATP synthase is important to maintain cellular membrane potential by activating ATP-dependent H+ pumping in Bacillus subtilis Yasuyuki Kato-Yamada1, Genki Akanuma1, Tomoaki Tagana1, Maho Sawada1, Shota Suzuki1, Tomohiro Shimada2, Kan Tanaka2, Fujio Kawamura1 1Department of Life Science, College of Science, Rikkyo University, Tokyo, Japan 2Laboratory for Chemistry and Life Science, Institute of Innovative Science, Tokyo Institute of Technology, Yokohama, Japan

The C-terminal domain (CTD) of the ε subunit in FoF1-ATPase/synthases (FoF1) plays an important role in the regulation of FoF1 activities. Although the regulatory mechanisms of F1 or FoF1 activities mediated by the ε subunit in vitro have been clarified by various approaches, the physiological significance of the regulation of FoF1 by the ε subunit remains elusive. To address this issue, we constructed and characterized Bacillus subtilis mutant, harboring a deletion in the regulatory CTD of the ε subunit (εΔC). Previous observations with Bacillus subtilis F1-ATPase (BF1) showed that, unlike other F1-ATPases, the ε subunit has an activating effect on

ATPase activity of BF1 [1]. Measurements with the inverted membrane vesicles from the mutant strain showed that the regulatory CTD of the ε subunit has an activating effect on the H+-pumping activity through the activation of ATPase activity, while the ATP synthesis activity was not altered very much. Although the growth under the standard conditions was essentially the same as the wild type, cells harboring the εΔC mutation were overwhelmed by the wild-type cells when co-cultured. To emphasize the effect of the mutation, we introduced the εΔC mutation into the Δrrn8 strain harboring only two of ten rrn (rRNA) operons, whose translational activity is low [2]. The growth of the Δrrn8 εΔC mutant stalled at late-exponential phase. The significant reduction of cellular membrane potential and changes in the ATP concentration were observed during growth stalling of the Δrrn8 εΔC mutant. These findings suggest that, in Bacillus subtilis, the CTD of the ε subunit is important to keep the cellular membrane potential through activating the ATP-dependent H+-pumping activity of

FoF1.

References

1. J. Mizumoto, Y. Kikuchi, Y.H. Nakanishi, N. Mouri, A. Cai, T. Ohta, T. Haruyama, Y. Kato-Yamada, ε Subunit of Bacillus subtilis F1- ATPase relieves MgADP inhibition, PLoS ONE 8 (2013) e73888 2. K. Yano, T. Wada, S. Suzuki, K. Tagami, T. Matsumoto, Y. Shiwa, T. Ishige, Y. Kawaguchi, K. Masuda, G. Akanuma, H. Nanamiya, H. Niki, H. Yoshikawa, F. Kawamura, Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis, Microbiology (United Kingdom) 159 (2013) 2225–2236

P5 a/8 De novo designed axis works as a rotor of rotary motor Jun-ichi Kishikawa1, Mihori Baba1, Atsuko Nakanishi1, Kaoru Mitsuoka2, Ken Yokoyama1 1Department of Molecular Biosciences, Kyoto Sangyo University, Kyoto (Japan) 2Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka (Japan)

V1-ATPase (V1) is an ATP-driven rotary motor which rotates a central rotor protein DF against the surrounding stator A3B3 ring. The rotary motion of the rotor is coupled to the conformational changes of the stator associated with ATP hydrolysis. This coupling mechanism between the rotor and the stator is a key for the efficient torque generation. To clarify the molecular mechanism, we generated artificial molecular motor system composed of the A3B3 ring and an exogenous rod protein FliJ from flagella motor, which has no sequence similarity with V1 subunit D. As a result, we have demonstrated that the exogenous FliJ protein functions as a rotor of molecular motor [1]. This results strongly suggest that any residue-specific interactions between the stator ring and the rotor are not need for the torque generation. To confirm this proposal, in this study, we do novo designed artificial parallel coiled-coil composed of simple repeating sequence and examined whether the designed coiled-coil functions as a rotor. Single particle analysis using cryo-EM revealed that the designed coiled-coil forms a complex with A3B3 ring by penetrating through the center cavity of the ring. In addition, the

ATPase activity of the complex was much higher than that of A3B3 ring only. We also indicated that the complex showed unidirectional and continuous rotation by experiments of single molecular observation. The rotational velocity and estimated torque of the complex were ~1/20 and ~1/6 of that of wild-type V1 respectively. These results indicate that the designed coiled-coil functions as the rotor in the stator ring of molecular motor. References 1. M. Baba, K. Iwamoto, R. Iino, H. Ueno, M. Hara, A. Nakanishi, J. Kishikawa, H. Noji, K. Yokoyama, Rotation of artificial rotor axles in rotary molecular motors. Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 11214-11219

P5 a/9 Proton translocation, force generation and dimerization in the F-type ATP synthase of Polytomella sp Niklas Klusch1, Bonnie J. Murphy1, Julian D. Langer2, Deryck J. Mills1, Özkan Yildiz1, Werner Kühlbrandt1 1Department of Structural Biology; Frankfurt, Germany 2Department of Molecular Membrane Biology | Max Planck Institute of Biophysics, Frankfurt am Main, Germany

Mitochondrial F-type ATP synthases generate ATP, the universal energy currency of the cell, by rotatory catalysis. They are located in the inner mitochondrial membrane, where they form V-shaped homodimers. ATP synthesis is driven by the electrochemical proton gradient across the membrane. We determined the structure of the complete mitochondrial ATP synthase dimer from the unicellular green alga Polytomella sp. at near-atomic resolution by single-particle electron cryo-microscopy. Two proton half channels that enable rotary catalysis were traced at the interface between subunit a and the c-ring rotor in the membrane. The high-resolution cryo-EM map enabled us to build a complete model of the dimer, including the unique ATP synthase-associated (ASA) subunits that form the dimer interface and the massive peripheral stalk. The structure of the peripheral stalk revealed a previously unknown ASA subunit in the membrane, bringing the total up to 10. The new ASA10 subunit is involved in dimer formation and thus plays a crucial role for the integrity of the nanomachine in the inner mitochondrial membrane.

P5 a/10 Single-molecule analysis of bovine mitochondrial F1-ATPase for direct assignment of crystal structures and rotational pausing states Ryohei Kobayashi, Hiroshi Ueno, Hiroyuki Noji Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo, Japan

F1-ATPase (F1) is the ATP-driven molecular motor, which has been extensively studied by both single-molecule studies and structural analysis [1,2]. However, the model F1’s for both studies have been different; F1 from thermophilic bacteria (TF1) for single-molecule studies while bovine mitochondrial F1 (bMF1) for structural analysis. This does not allow us to conclusively determine of the correlation between crystal structures and rotational pausing states found in the single-molecule studies. In order to directly assign the crystal structures to rotational pausing states, we have conducted single-molecule rotation assay of bMF1. So far, to determine the angular position of ATP hydrolysis, we have adopted 3 systems; ATPγS, AMPPNP, and catalytic-site mutant (βE188D). At [ATPγS] near or below Km, we found two major pauses during 120° rotation, indicating that they might correspond to the pauses waiting for ATP binding and ATP hydrolysis. The angular distance of ATP hydrolysis pause from the ATP binding pause was about 80°. Similar tendency was found in ATP-driven rotation of βE188D. In addition, we investigated the pause positions inhibited by AMPPNP which is expected to stop rotation at the angle of ATP hydrolysis. After confirming the binding pauses at low [ATP] (100 nM), AMPPNP (500 nM) was introduced into the reaction mixture. The rotations were stopped within 2 minutes. The angular distance of the pause stalled by AMPPNP from the ATP binding pause was about 80°. These observations all suggested that ATP hydrolysis reaction occurs at ~80° after ATP binding pause, which is consistent with the crystal structures of bMF1. Assignment of Pi-release waiting angle is undergoing.

[1] R. Watanabe, R. Iino, H. Noji, Phosphate release in F1-ATPase catalytic cycle follows ADP release, Nat. Chem. Biol. 6 (2010) 814- 820 [2] J.P. Abrahams, A.G.W. Leslie, R. Lutter, J.E. Walker, Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria, Nature. 370 (1994) 621-628

P5 a/11 A disease-causing hydrogen bond in ATP synthase of mitochondrial genetic origin Roza Kucharczyk1, Natalia Skoczeń1,2, Alain Dautant2,3, Marine Bouhier2,3, Deborah Tribouillard-Tanvier2,3, Jean-Paul di Rago2,3 1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland 2CNRS, Institut de Biochimie et Génétique Cellulaires, Bordeaux, France 3Université de Bordeaux, IBGC, Bordeaux, France

Neuro-muscular disorders have been associated to mutations in the mitochondrial F1Fo ATP synthase. The ATP synthase is described in terms of an integral membrane proton-translocating domain (FO) and a peripheral ATP-generating catalytic domain (F1). The subunit a (6), together with a ring of subunits c, is involved in the transfer of protons from one side of the membrane to the other and is encoded in humans by a mitochondrial gene, MT-ATP6. We recently identified a mutation in this gene (m.8969G>A), leading to replacement of a highly conserved serine residue into asparagine at amino acid position 148, in a 14-year-old Chinese female who initially developed an isolated nephropathy followed by a complex clinical presentation with brain and muscle problems [1]. An equivalent of this mutation in yeast (atp6-S175N) was shown to prevent FO-mediated proton transfer and weaken FO to F1 association. Herein we identified four first- site intragenic suppressors (aN175D, aN175K, aN175I, and aN175T), which, in light of a recently published atomic structure of yeast FO, provides strong indication that the detrimental consequences of the original mutation result from the establishment of a hydrogen bound between aN175 and a nearby glutamate residue (aE172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS175N mutation can be suppressed by second-site suppressors

(aP12S, aI171F, aI171N, aI239F, and aI200M), of which some are very distantly located (by 20-30 Å) from the original mutation. The possibility to disrupt a pathogenic hydrogen bond through long-range effects is an interesting observation that holds promise for the development of therapeutic molecules.

This work was supported by grants from National Science Center of Poland (2016/23/B/NZ3/02098) to R.K 1. S. Wen, K. Niedzwiecka, W. Zhao, S. Xu, S. Liang, X. Zhu, H. Xie, D. Tribouillard-Tanvier, M.F. Giraud, C. Zeng, A. Dautant, R. Kucharczyk, Z. Liu, J.P. di Rago, H. Chen, Identification of G8969>A in mitochondrial ATP6 gene that severely compromises ATP synthase function in a patient with IgA nephropathy, Scientific reports, 6 (2016) 36313.

P5 a/12 Phonon assisted proton ATPase - electron tunnelling model Elena Lacatus Polytechnic University of Bucharest, Romania

ATPase activities in living cells are carried out though the protein conformational dynamics and governed by quantum field theory (QFT) (van der Waals force field, London) [1]. Proteins boast quantum coherence [2], and the donor-acceptor distance fluctuation generates the phonon tunnelling effect during proton-coupled electron transport (PCET). Bose-Einstein condensation was used in Molecular Dynamics modelling research meant to describe the quantum superposition and quantum entanglement of proton/ phonon pumping mechanism. Thus, the signalling mechanism occurs as a collective effect during coherent ATPase proton/phonon pumping and electron tunnelling in protein. Phonon tunnelling explains the energy transport in cell membrane, and the electron transport model describes electron tunnelling of a single protein molecule during ATPase (the proton pump involved in a wide variety of physiological processes) that periodically generates energy in cells (20-30Å length range within 10fs timeframe) [1] A functional cell membrane model [3] has to properly describe ion channel gating pores [4], voltage-gated pores and the ATPase mechanism to create reliable nano-bio-info interfaces for the integrated modular-design concept of proactive biosensors

References 1. A.A. Stuchebrukhov, Long-Distance Electron Tunneling in Proteins, Laser Phys. 20:125, (2010); doi.org/10.1134/S1054660X09170186 2. A. W. Chin, J. Prior, R. Rosenbach, et al., The role of non-equilibrium vibrational structures in electronic coherence and recoherence in pigment–protein complexes, Nature Physics volume 9, pages 113–118 (2013); doi.org/10.1038/nphys2515 3. E. Roberts, A. Magis, et al., Noise Contributions in an Inducible Genetic Switch: A Whole-Cell Simulation Study, PLoS Comput Biol 7(3): e1002010. (2011); doi.org/10.1371/journal.pcbi.1002010 4. E. Lacatus, Ion channel path of cellular transduction, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1837 (2014); doi.org/10.1016/j.bbabio.2014.05.266

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Biochemical properties of Escherichia coli FOF1-ATP synthase with Leu249Gln substitution in beta subunit Anna S. Lapashina1,2, Boris A. Feniouk1,2 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia 2A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

Noncompetitive inhibition of ATPase activity by MgADP is a common regulatory feature of all FOF1-ATP synthases studied so far. When MgADP is bound at a catalytic site without phosphate, a conformational transition may occur leading to inactive state with ADP trapped in a catalytic site. The trapped ADP can be expelled upon membrane energization and the enzyme re-activates. In chloroplasts, mitochondria, and some bacteria phosphate counteracts this inhibition and increases ATPase activity of FOF1. On the other hand, in Escherichia coli enzyme, phosphate inhibits ATPase activity and seems to enhance MgADP-inhibition. In our previous work, we've described a single mutation, Leu249Gln, in beta subunit of E. coli ATP synthase, which reversed the effect of phosphate on ATPase activity of inverted membrane vesicles. In this study, we demonstrate that purified E. coli F1 and FOF1 complexes containing this mutation are indeed activated or, at least, not inhibited by inorganic phosphate. Sulfite, which is known to relieve MgADP inhibition, had almost no effect on the wild-type enzyme, but stimulated ATP hydrolysis by the mutant complexes severalfold. The ATPase activity of the mutant complexes is also more susceptible to azide inhibition. We suggest that betaLeu249Gln

FOF1 is inhibited by MgADP more pronouncedly than the wild-type complex.

The relationship between MgADP, pmf and phosphate in regard to E. coli FOF1-ATPase is also discussed.

This work was supported by the Russian Science Foundation research project №14‐14‐00128.

P5 a/14

2+ 2+ ADP/ Mg inhibition of F1 ATPases is tuned using Mg affinity governed by species-specific γ subunit contacts to α and β subunits Duncan G. G. McMillan1, Kirsten C. C. Knobel1, Karolina Šubrtová2, Stefan R. Marsden1, Gregory M. Cook3, Hiroyuki Noji4, Peter-leon Hagedoorn1, Achim Schnaufer2 1Department of Biotechnology, Delft University of Technology, The Netherlands 2Research and Centre for Immunity, Infection, and Evolution, University of Edinburgh, United Kingdom 3Department of Microbiology and Immunology, Otago University, New Zealand 4Department of Applied Chemistry, The University of Tokyo, Japan

Adenosine diphosphate together with magnesium, are commonly regarded as an inhibitor of ATP hydrolysis, but species variability of this effect, and mechanism of this variability, has remained elusive. Yet the influence of this ‘product inhibition’ has been widely noted across all described F1Fo ATP synthases.

The Caldalkalibacillus thermarum TA2.A1 F1 domain of the F1Fo ATP synthase is latent in ATP hydrolysis [1] with the highest stepping torque described to date [2]. Here we show that monodirectionality and ATP hydrolysis is governed by affinity for ADP/Mg, and the amount of ADP/Mg the enzyme co-purifies with dictates as-isolated hydrolysis activity. We show that both ADP molecules need to be removed before single molecule rotation is observed from a locked state. In spite of this, we observe that high Mg concentration is capable of eliminating ATP hydrolysis. High Mg concentrations inhibit several F1 ATPases to varying degrees, suggesting this is a common mechanism. In contrast the Trypanosoma brucei F1 ATPase is insensitive to high Mg concentration. Examination of current crystallographic data of C. thermarum F1 [3,4] shows that the N-terminus of the γ subunit contacts the α and β subunits to form a tripartite hydrogen bond network. We reveal that the loss of this network causes a partial loss in Mg-mediated ATP hydrolysis inhibition, suggesting that these bonds govern magnesium affinity. In contrast, the Trypanosoma brucei F1 ATPase structure [5] lacks these hydrogen bonds, and they are variable in other ATP synthases. Isothermal calorimetry reveals that loss of this hydrogen bonding network in C. thermarum F1 allows the release of 5.6 kcal/mol more energy per ATP hydrolyzed – a number in very close agreement with molecular models of the activation energy of 3 hydrogen bonds between 2 alpha-helical peptides [6]. These studies reveal that the

N-terminus of the gamma subunit controls F1Fo ATP synthase hydrolysis activity by controlling magnesium affinity.

References

1. D.G.G. McMillan, S. Keis, P. Dimroth, G. M. Cook, A Specific Adaptation in the a subunit of Thermoalkaliphilic F1Fo-ATP Synthase Enables Oxidative Phosphorylation at High pH but Not at Neutral pH Values, The Journal of Biological Chemistry 282, (2007) 17395- 17404 2. D.G.G. McMillan, R. Watanabe, H. Ueno, G.M. Cook, H. Noji, Biophysical characterization of a thermoalkaliphilic molecular motor with a high stepping-torque gives insight into evolutionary ATP synthase adaptation. The Journal of Biological Chemistry 291 (2016) 23965-23977

3. A. Stocker, S. Keis, J. Vonck, G.M. Cook, P. Dimroth, The Structural Basis for Unidirectional Rotation of Thermoalkaliphilic F1- ATPase, Structure 15, (2007) 904–914

4. S.A. Ferguson, G.M. Cook, M.G. Montgomery, A.G.W. Leslie, J.E. Walker, Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum, Proceedings of the National Academy of Sciences 113 (2016) 10860-10865 5. M.G. Montgomery, O. Gahura, A.G.W. Leslie, A. Zíková J.E. Walker, ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites, Proceedings of the National Academy of Sciences 115 (2017), 2102-2107 6. S-Yi. Sheu, D-Y. Yang, H.L. Selzle, E.W. Schlag, Energetics of Hydrogen bonds in peptides, Proceedings of the National Academy of Sciences 100 (2003), 12683–12687

P5 a/15 Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus Atsuko Nakanishi1, Jun-ichi Kishikawa1, Masatada Tamakoshi2, Kaoru Mitsuoka3, Ken Yokoyama1 1Department of Molecular Biosciences, Kyoto Sangyo University, Kyoto, Japan 2Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan 3Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan

Proton translocating rotary ATPases couple ATP hydrolysis/synthesis in the soluble domain with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Structures of complete rotary ATPases have been revealed at a secondary structural level by using cryogenic electron microscopy (cryo-EM). Nevertheless, the limited (6~7 Å) resolution of the whole complex structure limited understanding of the molecular mechanism of rotary ATPases. Here, we present the structure of intact V/A type H+-rotary ATPase from the bacterium Thermus thermophiles by single particle analysis using cryo-EM enabling the identification of three rotational states based on the orientation of the rotor subunit [1]. The three different structures provide insights into the movement of each subunit during rotation. Furthermore, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains by using masked refinement and classification with signal subtractions. These reconstructions are of higher resolution than any EM map of intact rotary ATPase obtained previously, providing a clear proton translocation path from both sides of the membrane. In addition, our structure revealed the detailed insight into the contact surface between rotor subunits, and molecular basis of structural robustness of the peripheral stator apparatus. These results show the close integration of the different domains of rotary ATPase allowing tight energy coupling between the soluble V1 and the membrane Vo moieties.

References 1. A. Nakanishi, J. Kishikawa, M. Tamakoshi, K. Mitsuoka, K. Yokoyama, Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus. Nat. Commun. (2018) 9(1):89. doi: 10.1038/s41467-017-02553-6

P5 a/16 Subunit a of ATP synthase – essential for ATP synthase dimerization in Saccharomyces cerevisiae Katarzyna Niedzwiecka1, Emilia Baranowska1, Chiranjit Panja1, Roza Kucharczyk 1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

Mitochondrial ATP synthase, an enzyme catalyzing ATP from ADP and inorganic phosphate, exists in mitochondria in form of dimers, oligomers or in complexes with the respiratory chain complexes. Mutations in the mitochondrial MT-ATP6 gene that encodes subunit a, have been associated to neuromuscular disorders but also were identified in cancer samples. We have exploited budding yeast S. cerevisiae as a model to study the effects of five tumor-associated mutations in this gene on the energy metabolism [1]. We have shown that two of these mutations (atp6-P163S and atp6-K90E, equivalent to those found in prostate and thyroid cancer samples), in OM45- GFP background, affect ROS and calcium homeostasis. Moreover the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium treatment was delayed in the double mutants [2]. Herein we present a genetic evidence that subunit e of ATP synthase, necessary for dimerization of the enzyme, is essential for yeast atp6-P163S OM45- GFP mutant. The results let us to hypothesize that structural changes of ATP synthase, dependent on subunits a and e, are important for yPTP induction and that this channel functioning is essential for yeast cell fitness.

This work was supported by grants from National Science Center of Poland (2013/11/B/NZ1/02102) to R.K [1] K. Niedzwiecka, A.M. Kabala, J.P. Lasserre, D. Tribouillard-Tanvier, P. Golik, A. Dautant, J.P. di Rago, R. Kucharczyk, Yeast models of mutations in the mitochondrial ATP6 gene found in human cancer cells, Mitochondrion, 29 (2016) 7-17. [2] K. Niedzwiecka, R. Tisi, S. Penna, M. Lichocka, D. Plochocka, R. Kucharczyk, Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast, Biochim. Biophys. Acta, 1865 (2018) 117-131.

P5 a/17 The Influence of Vacuolar Proton-Translocating ATPase (V-ATPase) in Health and Disease Through the Lens of Glycolysis Karlett J. Parra, Summer R. Hayek Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA

The ability of cells to adapt to fluxuations in glucose availability is crucial for their survival and involves the vacuolar proton-translocating

ATPase (V-ATPase), a multi-domain proton pump found in all eukaryotes. V-ATPase hydrolyzes ATP via its V1 domain and uses the energy released to transport protons across membranes via its Vo domain. This activity is critical for pH homeostasis and generation of a membrane potential that drives cellular metabolism. We will present new mechanistic models to explain how the V-ATPase-glycolysis axis is regulated and coordinated in both fungi and humans. The effect of glucose flux on V-ATPase activity has been best characterized in the Saccharomyces cerevisiae (S. cerevisiae) model fungus. In yeast cells exposed to glucose starvation, the V1 and Vo domains disassemble from one another, thereby inhibiting V-ATPase and protecting against energy depletion. Reactivation of glycolysis reverses this process. We have established that PFK-1 and the glycolytic flow regulate V-ATPase at steady state and under disassembly and reassembly conditions. These results linked PFK-1 α-subunit to V1Vo catalytic coupling; α-subunit may act as a negative regulator of V1Vo coupling. Additional studies in human cells have shown reciprocal regulation of this pathway, with alterations in V-ATPase activity influencing downstream glycolysis. Inhibition of V-ATPase activity increased the protein level and nuclear localization of the alpha subunit of the transcription factor HIF-1 (HIF-1α) via decreased hydroxylation and degradation of HIF-1 α. These results linked V-ATPase activity to HIF-1 α-dependent up-regulation of glycolysis. This suggests that the connection between V- ATPase and glycolysis is ubiquitous but that the specific pathways and regulatory mechanisms may differ between systems.

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+ The effect of point mutations in Escherichia coli H -FOF1 ATP synthase on the MgADP-inhibition of ATPase activity Tatiana E. Shugaeva1, Anna S. Lapashina1,2, Boris A. Feniouk1,2 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia 2A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

+ The ATPase activity of H -FOF1-ATP synthase can be regulated in several ways, the most common of which is MgADP-dependent inhibition. When proton electrochemical gradient across the membrane is low, MgADP binding to the catalytic site in the absence of phosphate may lead to ATP synthase inactivation. The degree of this inhibition varies greatly between different organisms: ATP synthase in chloroplasts, mitochondria and some bacteria (Bacillus sp. PS3, Bacillus subtilis) has strong MgADP-inhibition, whereas it is much weaker in Escherichia coli enzyme. Also, it was shown that phosphate addition stimulates ATP hydrolysis in Bacillus sp. PS3 ATP synthase but inhibits it in E. coli enzyme. Multiple alignment of ATP synthase beta subunit sequences revealed a few amino acid residues, including positions corresponding to E. coli beta189 (Phe in E. coli and Leu in Bacillus sp. PS3) and beta249 (Leu in E. coli and Gln in Bacillus sp. PS3) that might be involved in the regulation of the enzyme. We have previously shown that a point mutation L249Q in beta subunit of E.coli ATP synthase reversed the effect of phosphate on ATPase activity and enhanced MgADP-inhibition in E. coli enzyme. In this work we report that mutation F189L also modified the effect of phosphate and that the double mutation βL249Q/F189L further enhanced the MgADP-inhibition compared to single mutation βL249Q. ATP synthesis activity, however, was only marginally decreased in the double mutant.

This work was supported by the Russian Science Foundation research project №14‐14‐00128.

P5 a/19 Membrane potential driven ATP synthesis in single reconstituted F-ATP synthase Hendrik Sielaff, Michael Börsch Single Molecule Microscopy Group, Jena University Hospital, Jena, Germany

F-ATP synthases consists of two elastically coupled nanomotors that rotate against each other to hydrolyze ATP or synthesize ATP at the expanse of a proton motive force (pmf) [1]. The mechanism of ATP hydrolysis in the F1 portion has been studied in detail by single molecule fluorescence microscopy [2], and is supported by high-resolution structural data. However, a comprehensive mechanism of

ATP synthesis by the membrane-coupled FOF1 holoenzyme, driven by a pmf, is still lacking, because of the experimental challenges (1) of long-term studies on single molecules [3], and (2) to crystalize membrane enzymes. Recently, advances have been made by capturing the enzyme in an anti-Brownian electrokinetic trap [4], and obtaining a high-resolution cryo-EM structure. Here, we are aiming to establish a new approach for long-term studying of single molecules. By reconstitution of the FOF1 complex in a lipid bilayer system we can maintain a constant electrochemical potential that drives ATP synthesis, while at the same time an optical setup allows us to observe movements of fluorescently labeled single molecules. Combining electrophysiological with optical measurements enables us to get a deeper insight into the mechanism of ATP synthesis.

References 1. H. Sielaff, H. Rennekamp, A. Wächter, H. Xie, F. Hilbers, K. Feldbauer, S.D. Dunn, S. Engelbrecht, W. Junge, Domain compliance and elastic power transmission in rotary FOF1-ATPase, Proc. Natl. Acad. Sci. USA 105 (2008) 17760-17765 2. H. Sielaff, J. Martin, D. Singh, G. Biuković, G. Grüber, W.D. Frasch, Power Stroke Angular Velocity Profiles of Archaeal A-ATP Synthase Versus Thermophilic and Mesophilic F-ATP Synthase Molecular Motors, J. Biol. Chem. 291 (2016) 25351-25363

3. H. Sielaff, M. Börsch, Twisting and subunit rotation in single FOF1-ATP synthase, Philos. Trans. R. Soc. Lond. B Biol. Sci. 368 (2013) 20120024 4. M. Dienerowitz, F. Dienerowitz, M. Börsch, Measuring nanoparticle diffusion in an ABELtrap, J. Opt. 20 (2018) 034006

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Chemo-mechanical Coupling Mechanism of Rotation of Mammalian F1-ATPase by Static and Dynamic X-ray Crystallographic Studies Toshiharu Suzuki1,2,3, Eiki Yamashita4, Seiki Baba5, Kunio Hirata6, Naoya Iida7, Takashi Kumasaka6, Toru Hisabori2, Toshiya Endo3, Masasuke Yoshida3, Hiroyuki Noji1 1Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo, Japan 2Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan 3Department of Molecular Bioscience, Kyoto-Sangyo University, Kyoto, Japan 4Institute of Protein Research, Osaka University, Osaka, Japan 5Japan Synchrotron Radiation Research Institute (JASRI), Hyogo, Japan 6SPring8-center, Riken, Hyogo, Japan 7Department of Physics, Waseda University, Tokyo, Japan

FoF1-ATP synthase is a highly-efficient rotary motor achieving energy conversion between chemical potential of ATP and + electrochemical potential of H established across biological membranes via rotation [1]. F1-ATPase is an ATPase-domain of FoF1-ATP synthase and functions as a rotary molecular motor driven by ATP hydrolysis. The approximately 50kDa rotor shaft rotates relative to the surrounding ~330kDa stator ring. To know the operation mechanism of the efficient chemo-mechanically-coupled rotation, we have established analytical systems for recombinant human F1 [2] and bovine F1 [3]. Microscopic single-molecule analysis of human F1 revealed a scheme of ATPase-driven rotation [2]. X-ray crystallographic studies of bovine F1 provided several molecular structures at up to 1.66 Å resolution, which included eight rotation interim snapshots for the release of product phosphate (Pi) or ADP. The former interim structures identified stepwise rotation of rotor shaft and widening of the Pi-binding pocket during the Pi-release. The stepwise displacements in Pi-mimicking water molecules, arginine finger, and p-loop Lys residues in the pocket induced a global rearrangement of subunits, which led to driving stepwise rotation of the rotor shaft (~20-deg). The sequence of the structural transition was further confirmed by our dynamic time-divided X-ray crystallographic study. In addition, the ADP-releasing intermediate structures unveiled a conformational rearrangement in the catalytic site (especially in p-loop) during ADP-releasing. All of the rotation intermediate structures supported the previously proposed rotation scheme of human F1. These results provide the power-generating chemo-mechanical coupling mechanism of mammalian F1 that elastic force accumulated in the protein molecule, originally provided by ATP’s binding to the enzyme, is released step-by-step by the trigger of the Pi-release and converted into the physical power (torque) to drive rotation of the rotor shaft.

1. N. Soga, K. Kimura, K. Kinosita Jr., M. Yoshida, S. Suzuki, Perfect chemo-mechanical coupling of FoF1-ATP synthase, Proc Natl Acad Sci USA, 114(2017) 4960-4965 2. T. Suzuki, K. Tanaka, C. Wakabayashi, E. Saita, M. Yoshida, Chemo-mechanical coupling of human mitochondrial F1-ATPase motor, Nature Chem Biol 10(2014) 930-936

3. T. Suzuki, N. Iida, J. Suzuki, Y. Watanabe, T. Endo, T. Hisabori, M. Yoshida, Expression of mammalian mitochondrial F1-ATPase in Escherichia coli depends on two chaperone factors, AF1 and AF2, FEBS Open Bio 6(2016) 1267-1272

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Rotation of the engineered F1-ATPase with non-catalytic α-type P-loops Hiroshi Ueno1, Rie Koga2, Tomoko Masaike3, Nobuyasu Koga2, Hiroyuki Noji1 1Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan 2Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Japan 3Department of Applied Biological Science, Graduate School of Engineering, Noda, Japan

Rotary ATPases are unique rotary molecular motors that function as energy conversion machines. Among all known rotary ATPases,

F1-ATPase is the best characterized rotary molecular motor. There are many high-resolution crystal structures and the rotation dynamics have been investigated in detail by extensive single-molecule studies [1]. However, its design principle, e.g., the origin of conformational change of F1, remains elusive. To reveal it, we focused on the P-loops located at the nucleotide binding sites of α and β subunits. ATP is bound to α subunit without catalysis for a regulatory role whereas β subunit hydrolyzes ATP for torque generation.

Crystal structures of F1 suggest large conformational changes of P-loop in β subunit during catalysis in contrast to the counterpart in α subunit [2]. Interestingly, the P-loops of α and β subunits have the consensus sequence (GxxxxGKT) but are distinctly different at x residues. Therefore, we substituted x residues of β subunit with that of α subunit and analyzed the rotation of engineered F1s.

Surprisingly, the engineered F1 with all α-type P-loops showed ~1,000 times slower rotation. We report the details of how such β-α conversion influences the rotation of F1.

References

1. H. Noji, H. Ueno, D.G.G. McMillan, Catalytic robustness and torque generation of the F1-ATPase, Biophys. Rev. 25 (2017) 103-118

2. J.P. Abrahams, A.G. Leslie, R. Lutter, J.E. Walker, Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria, Nature 370 (1994) 621-628

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Electrophysiological properties of channel formed by bovine FOF1 ATP synthase in planar lipid bilayer Andrea Urbani1, Valentina Giorgio1,2, Chimari Jiko3, Janna F. M. Bogers4, Duncan G. G. McMillan4, Andrea Carrer1, Christoph Gerle5, Ildikò Szabò2,6, and Paolo Bernardi1,2 1Department of Biomedical Sciences, University of Padova, Padova, Italy 2Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Italy 3Kyoto University, Kyoto, Japan 4Department of Biotechnology, Delft University of Technology, The Netherlands 5Laboratory of Protein Crystallography, Institute for Protein Research, Osaka University, Osaka, Japan 6Department of Biology, University of Padova, Padova, Italy

Permeability transition (PT) in mitochondria is triggered by Ca2+ and specific activators and finally leads to increased permeability to ions and solutes of the inner mitochondrial membrane (IMM). In the 1970s the idea was advanced that PT is mediated by a Ca2+-regulated pore, named the Permeability Transition Pore (PTP). By means of patch clamp on mitoplasts the PTP was identified as a high- conductance channel, named Mitochondrial Mega Channel (MMC). Despite many years of study and the key role of PTP opening in cell death and several diseases, the molecular nature of the pore remains unclear. In 2013 Giorgio et al. provided evidence that dimers of mammalian FOF1 ATP synthase purified from native gels can form the PTP/MMC [1] but the exact molecular mechanism is still unclear.

The present work aimed at further understanding whether mammalian FOF1 ATP synthase generates channel in single channel recordings in planar lipid bilayer (PLB). Highly pure FOF1 ATP synthase was prepared from bovine hearts using the very mild, lipid-like detergent LMNG [2]. The resulting preparation was analyzed by clear native PAGE, SDS-PAGE, mass spectrometry, and negative stain electron microscopy and was shown: (i) to be intact and inclusive of all subunits; and (ii) to be active and highly sensitive to oligomycin (>95%). After incorporation into the PLB channel activity was assessed in the presence of different concentrations of Ca2+ and Bz-423, a 2+ 2+ compound that is able to sensitize the PTP to Ca . Here we provide evidence of high conductance, Ca -dependent mammalian FOF1 ATP synthase channel activity resembling the MMC-PTP key features, including inhibition by PTP specific compounds.

[1] V. Giorgio et al., Dimers of mitochondrial ATP synthase form the permeability transition pore, PNAS 110(15) (2013) 5887-9 [2] S. Maeda et al., Two-dimensional crystallization of intact F-ATP synthase isolated from bovine heart mitochondria, Acta Crystallographica Section F, Structural Biology and Crystallization Communications 69 (2013) 1368–1370

P5 a/23 Vacuolar-ATPase E subunit somatic missense mutations identified in the COSMIC database result in increased enzyme activity Bradleigh Whitton1, Matthew Rose-Zerilli1, Simon J. Crabb1, Graham Packham1, Haruko Okamoto2,3 1Faculty of Medicine, Cancer Sciences Unit, University of Southampton, Southampton General Hospital Tremona Road Southampton SO16 6YD, United Kingdom 2Biological Sciences, University of Southampton, Southampton, University Road, Highfield campus, United Kingdom 3Institute of Life Sciences, University of Southampton, Southampton, United Kingdom

V-ATPases are multi-protein complexes that catalyse the ATP-dependent transport of protons across intracellular and plasma membranes. Mammalian V-ATPase comprises 13 distinct subunits forming a cytosolic V1 (A, B, C, D, E, F, G and H) and a membrane- integral VO (a, c, c”, d and e) domain. Overexpression of V-ATPase subunits has been linked to cancer cell migration, invasion and metastasis. Mutations in V-ATPase subunits have been identified in various cancers, however the functional impact of these mutations is unknown. Previous work by us and others has shown that a substitution in several key amino acid residues in the N-terminal alpha helix of the yeast V1E subunit affects V-ATPase enzyme kinetics. Therefore, we hypothesised that cancer associated V1E subunit mutations may impact upon mammalian V-ATPase activity. On this basis, three somatic missense mutations, glutamine (Q), valine (V) and lysine (K) at glutamate (E) 61 in the human E2 isoform were identified in lung and skin tumour samples in the COSMIC database (v80). Since the E61 is conserved between subunit isoforms E1 and E2 across the mammalian species, we also introduced these substitutions into human E1 isoform. These mutations were introduced into human E subunit isoforms by a PCR-based cloning method. A V-ATPase ΔE null mutant yeast model was then complemented with the human E2 or E1 subunits with or without these substitutions to see whether the mutations had a cancer specific effect. V-ATPase activity in isolated vacuolar membranes was assessed by measuring inorganic phosphate production in the presence or absence of the specific V-ATPase inhibitor bafilomycin-A1. Our results indicated that the ATPase activity was significantly increased in the V-ATPases with E61 to Q and V substitutions in both human E1 and E2 subunits compared to the wild-type subunit. These results show for the first time that cancer-associated V-ATPase subunit mutations alter enzyme activity.

P5 a/24 Mechanism of Light Regulation on Chloroplast ATP Synthase Revealed by Single-Particle cryo-EM Jay-How Yang1, Petra Fromme1,2, Po-Lin Chiu1,2 1Center for Applied Structural Discovery (CASD), Biodesign Institute, Arizona State University, Tempe, AZ, USA 2School of Molecular Sciences, Arizona State University, Tempe, AZ, USA

The activities of chloroplast ATP (cATP) synthase are tightly coupled to photosynthetic electron transport chain, which generates an electrochemical membrane potential and drives the rotation of motors to produce ATP molecules upon illumination. To understand this regulation mechanism, we have determined two structures of the chloroplast ATP synthase of Spinacia oleracea using single-particle cryo-EM. Our cryo-EM c-ATP-synthase structures reveal the structural details of the dark-activated (oxidized) and light-activated (reduced) state of the chloroplast ATP synthase, respectively. The oxidized disulfide bridge was identified on the γ subunit, leading to a conformation that fosters an interaction with catalytic β subunit thereby prohibiting the rotation of the γ-c14 rotor. In the reduced state, γ subunit releases from binding to β subunit and allows the γ-c14 rotor free to rotate. The mechanism of this redox switch, together with the hypothesized proton translocation pathway, gives us a mechanistic view of energy production through light regulation on cATP synthase. Moreover, our final cryo-EM reconstructions show a complete view of the cATP-synthase complex, which is composed of 3β3

δ γεabb’c14 subunits and present an architecture of F-type ATP synthase the structures provide structural details of the mechanism⍺ of the light regulation of the ATP-synthase. Furthermore, the structure of the oxidized dark state allowed us to detect a structural asymmetry in the c-ring, where the outer helices of the c subunits facing the proton conducting a-subunit are rotated thereby enabling direct proton transfer from the conserved aArg189 of the a subunit to the cGlu61 of the c ring, thereby driving ATP-synthesis.

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The Chemomechanical Coupling of F1 ATPase of Paracoccus denitrificans Mariel Zarco-Zavala1, Duncan G.G. McMillan2, Toshiharu Suzuki1, Hiroshi Ueno1, Rikiya Watanabe1, Francisco Mendoza-Hoffmann3, José J. García-Trejo3, Hiroyuki Noji1 1Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan 2Department of Biotechnology, Delft University of Technology, Delft, The Netherlands 3Department of Biology, Chemistry Faculty, National Autonomous University of Mexico, Mexico City, México

Biophysical studies of the bacterial and eukaryotic F1-ATPases have shown differences in their chemomechanical coupling schemes [1]. However, the mechanical basis of that divergence remains unclear. To address this question, we examined the rotatory dynamics of

Paracoccus denitrificans (Pd) F1-ATPase. Pd is an α-proteobacterium phylogenetically related to the ancestors of mitochondria. The

PdF1 has a canonical bacterial type composition, but harbor a unique α-proteobacteria inhibitor, the ‘ζ subunit’, whose inhibitory domain resembles the mitochondrial IF1 [2].

Using single-molecule microscopy, we visualized a typical stepwise ATPase-driven rotation. However, in contrast to the other F1-

ATPases, the rotational sub-steps usually observed around Km were not detected in the PdF1. To confirm this finding, we compared the dwell positions of single PdF1 molecules at low [ATP] and those at high [ATP]; additionally, we compared the ATP binding dwell angles with the angular position of the catalytic dwell identified by the use of chemical inhibitors. The results revealed that the binding and cleavage of ATP occur at the same angle in the PdF1, contrary to the bacterial and eukaryotic F1-ATPase. Finally, we determined that the ζ subunit blocks the rotation of PdF1 in the same angular position of the catalytic dwell, verifying its functional similarity with mitochondrial IF1. Overall, these data could provide insight into the tune of the chemomechanical mechanism of the ATPase during evolution.

References [1] T. Suzuki, K. Tanaka, C. Wakabayashi, E.-i. Saita, M. Yoshida, Chemomechanical coupling of human mitochondrial F1-ATPase motor, Nature chemical biology, 10 (2014) 930-936. [2] J.J. García-Trejo, M. Zarco-Zavala, F. Mendoza-Hoffmann, E. Hernández-Luna, R. Ortega, G. Mendoza-Hernández, The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria, The Journal of biological chemistry, 291 (2016) 538-546.

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The High-Resolution Structure and Inhibition of Trypanosoma brucei F1-ATPase Alena Zíková1, Ondřej Gahura1, Martin G. Montgomery2, Andrew G. W. Leslie2, John E. Walker2 1Biology Centre ASCR, Institute of Parasitology, Ceske Budejovice, Czech Republic 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom

The sophisticated rotational mechanism of ATP synthesis coupled to the proton translocation across biological membranes imposes significant constraint to the evolutionary diversification of F-type ATP synthases. The compositional and structural variability of these enzymes is restricted to the regions not engaged in the proton pumping, torque transmission, or ATP generation. The catalytic subcomplex, F1-ATPase, has long been considered entirely invariant across eukaryotes. However, the F1-ATPase purified from Trypanosoma brucei differs from the previously described enzymes by the presence of an essential p18 subunit. Using X-ray crystallography, we generated an atomic model of the F1-ATPase and showed that p18 is associated with the external surface of each of the three alpha-subunits, thereby elaborating the F1-domain.

Interestingly, T. brucei FoF1-ATP synthase functions as an ATPase in the infectious stage of the parasite as it hydrolyzes ATP to maintain the mitochondrial membrane potential in the absence of the canonical electron transport chain. We demonstrated that this activity can be blocked in vitro by a protein inhibitor TbIF1, which acts in vivo to prevent the depletion of ATP under chemical hypoxia. In contrast, the genetically induced expression of TbIF1is lethal for the infectious form. We have also determined that the developmental regulation of TbIF1 expression is tightly controlled at the post-transcriptional level by two RNA-binding proteins Rbp10 and Rbp6 that bind to 3’UTR specific motifs of the TbIF1 mRNA, thus targeting the transcript for degradation. Since TbIF1 is a natural inhibitor of the

FoF1-ATP synthase complex, we are actively pursuing the molecular details of their interactions to provide us with the grounds for the structure-based design of new trypanocidal drugs.

P5 a/27 Mechanistic features of subunit ε of the Mycobacterial F-ATP synthase Nebojša Bogdanović, Shashi Bhushan, Gerhard Grüber School of Biological Sciences, Nanyang Technological University, Singapore

Bacterial F-ATP synthases are composed of 8 different subunits in the proposed stoichiometry of α3:β3:γ:δ:ε:a1:b2:c9-15, where the soluble domain F1 has a hexameric head made up of α3:β3. Subunits γ and ε form the central stalk, and a homodimer of subunits b the peripheral stalk. The membrane-embedded integral FO domain contains the subunits a and c, which are responsible for ion- translocation. A modified gene that encodes mycobacteria-specific, C-terminal domain of subunit Mtα was used together with α3:β3:γ chi chi and Mtε genes to generate the heterologous α 3:β3:γ and α 3:β3:γ:Mtε complexes of the chemically-driven motor F-ATP synthase. Highly purified and monodisperse proteins were produced for the electron microscopy to obtain the 2D-classification and 3D- chi reconstructions of negatively stained EM-images. The 3D-reconstruction of α 3:β3:γ:Mtε complex enabled the assignment of the subunits in the complex. ATPase activity of the purified proteins was measured and mutational studies have been employed to map the critical epitopes in the coupling subunit Mtε.

P5 b/1 Drug development for human African trypanosomiasis targeting its cyanide-insensitive respiration Yasutoshi KIDO1,2,3, Daniel Ken INAOKA3, Tomoo SHIBA4, Shigeharu HARADA4, Hiroyuki SAIMOTO5, Naoto UEMURA2, Kiyoshi KITA3 1Dept. Parasitology, Grad. Sch. Med., Osaka City Univ., Japan 2Dept. Clin. Pharm. and Therap., Oita Univ., Japan 3Trop. Med. Global Health, NAGASAKI Univ., Japan 4Applied Biology, Kyoto Inst. Tech., Japan 5Fuc. Engineering, Tottori Univ., Japan

The Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei threats global health. It eventually leads to lethal condition if untreated. It is estimated that 13 million people live in areas at moderate to high risk and approximately 5,000 people are newly infected annually. Only two drugs are available for stage 2 of the disease, but those exhibit significant toxicities, and the efficacy is limited. We have found that ascofuranone (AF), produced by a filamentous fungus, to be a potent lead compound which specifically eradicates the trypanosomes at very low concentrations. This lead compound inhibits the mitochondrial cyanide-insensitive alternative oxidase, which is essential for the parasites survival but not for human. Therefore, the target of this research is to develop novel anti- HAT drugs, which should also be useful for the livestock trypanosomiasis known as Nagana. We have elucidated the three-dimensional structure of drug target and identified pharmacophores of the lead compound by the structure activity relationship study using more than 200 derivatives. We have designed several derivatives to cure T. brucei infected mice model. In addition to pharmacodynamics test, we are conducting lead optimization based on pharmacokinetic, pharmacodynamic, and toxicity studies (Absorption, Distribution, Metabolism, Excretion, and Toxicity; ADMET).

P6 a/1 Proton Translocations in Channelrhodopsin-1 from Chlamydomonas augustae Ramona Schlesinger1, Maria Walter1, Vera Muders1, Kirsten Hoffmann1, Victor Lorenz-Fonfria2, Joachim Heberle2 1Freie Universität Berlin, Department of Physics, Genetic Biophysics, Berlin, Germany 2Freie Universität Berlin, Department of Physics, Experimental Molecular Biophysics, Berlin, Germany

CaChR1 is a red shifted channelrhodopsin-1 from the eyespot of Chlamydomonas augustae, which translocates cations upon light excitation and thus might be an interesting candidate in different kinds of optogenetic applications. We will present protonation/deprotonation events taking place within the protein during the photocycle. To understand which amino acids are involved, we created different variants of CaChR1 and analyzed them with time-resolved UV-Vis spectroscopy and FT-IR difference spectroscopy. In the latter case, we could assign several bands to individual amino acids in the carboxylic region as well as S-H region. A special focus was to figure out the involvement of cysteines to the mechanism of the channel as seven out of fourteen cysteines in total are localized in a belt like formation in the core of the protein flanking the chromophore retinal. We can show that two cysteines influence assigned signals in the carboxylic region indicating a direct or indirect interaction with carboxylates.

P6 b/1 Disease-segregating Aifm1 mutation causes myopathy in knockin mice Daniele Bano Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE, Bonn)

Apoptosis-inducing factor (AIF) is a NADH-dependent Flavoprotein localized in the mitochondrial intermembrane space. AIF interacts with the oxidoreductase CHCHD4/Mia40 and, consequently, contributes to the maintenance and stability of the oxidative phosphorylation (OXPHOS) system. Pathogenic mutations in the AIFM1 gene result in recessive X-linked forms of mitochondrial diseases, leading to severe clinical manifestations such as childhood-onset myopathies and hereditary peripheral neuropathies. The direct consequence of any disease-segregating AIFM1 mutation has not been studied in experimentally tractable model organisms yet. Here, we provide a line of evidence that the disease-associated mutation Aifm1 (R200 del) causes pathology in knockin mice. Compared to hypomorphic Harlequin mutant mice, Aifm1 (R200 del) knockout animals are less variable in terms of phenotypes, as they show a more consistent body weight reduction, hind limb clasping and kyphosis across individual animals. Importantly, Aifm1 (R200 del) knockout mice exhibit early-onset myopathy associated with nemaline-like inclusions and cytochrome c oxidase-negative muscle fibers. Over time, Aifm1 (R200 del) knockin mice develop peripheral neuropathy, however they do not show signs of neurodegenerative and inflammatory processes in the cerebellum, as instead observed in Hq mutant animals. Mechanistically, mutant AIF protein alters mitochondrial bioenergetics in a tissue-specific and time-dependent manner. As a result of mitochondrial deficiency, sustained Akt/mTOR signaling induces aberrant folate-driven 1C metabolism and other molecular signatures common of mitochondrial diseases. Taken together, our findings broaden our understanding of the pathophysiological mechanisms link to AIF deficiency. As an added value, our novel knockin mouse model may become a useful tool in the preclinical assessment of treatments for mitochondrial diseases.

P6 b/2 Repeated exposure to hyperbaric hyperoxia affects mitochondrial functions of the lung fibroblasts Jiri Dejmek1,2, Michaela Kripnerova2,4, Michaela Markova2,3, Miroslava Cedikova2,3, Vaclav Babuska5, Lukas Bolek1,2, Jitka Kuncova2,3 1Institute of Biophysics, Czech Republic 2Biomedical Center, 3Institute of Physiology, 4Institute of Biology, 5Institute of Medical Chemistry and Biochemistry, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic

Hyperbaric oxygen (HBO) therapy, i.e. breathing pure oxygen under increased environmental pressures serves as a treatment for diverse medical conditions. However, elevated oxygen concentration can be detrimental to central nervous system or lungs. Our study aimed to evaluate the effects of repeated exposure to HBO on mitochondrial respiration on human lung fibroblasts HFL1 using assessed by high-resolution respirometry (HRR), cell viability estimated by PrestoBlue® reaction, morphology analyzed by routine phase contrast and fluorescent microscopy, and superoxide dismutase (SOD) and citrate synthase (CS) activities. The cells were exposed to HBO for 2 hours per day for 5 consecutive days. 24 hours after the last exposure, HBO cells displayed significantly smaller area and perimeter, compromised viability and elevated SOD activity. No changes were detected in CS activity or quality of mitochondrial network. Respiratory activity was determined using two titration protocols: 1. digitonin, malate, palmitoyl carnitine, ADP, cytochrome c, glutamate, pyruvate, succinate, FCCP, rotenone, antimycin A and TMPD / ascorbate (Dig, M, Pcar, G, P, S, u, Rot, AmaA, TM), 2. Dig, M, G, D, c, P, S, oligomycin, u, Rot, AmA and TM. HRR revealed impaired mitochondrial oxygen consumption manifested by increased leak respiration, decreased activity of complex II and compromised ATP-related oxygen consumption when fatty acids were oxidized. Our findings document that in conditions mimicking chronic intermittent exposure to HBO, lung fibroblasts suffer from compromised mitochondrial respiration linked to complex II and impaired cellular growth in spite of increased antioxidant defense. Underlying mechanism of this HBO-induced mitochondrial dysfunction should be further explored.

Support: NPU I – Nr. LO1503, CHU Research Fund (Progres Q39), Specific Student Research Project Nr. 260394/2017.

P6 b/3 Assessing the role of mitochondrial glycerol 3-phosphate dehydrogenase in the antidiabetic effect of metformin - a mass spectrometry imaging study Ove Eriksson2, Giulio Calza1, Elisabeth Nyberg1,2, Matias Mäkinen2, Rabah Soliymani1, Annunziata Cascone2, Dan Lindholm2,3, Emanuele Barborini4, Marc Baumann1, Maciej Lalowski1 1Meilahti Clinical Proteomics Core facility, HiLIFE, Faculty of medicine, Biomedicum Helsinki 1, University of Helsinki, Finland 2Biochemistry/Developmental Biology, Faculty of medicine, Biomedicum Helsinki 1, University of Helsinki. 3Minerva Foundation Institute for Medical Research, Biomedicum Helsinki 2, Helsinki, Finland 4Tethis S.p.A. Via Russoli 3, Milano, Italy

Metformin is the first line drug for type 2 diabetes but its mechanisms at molecular level remain unclear. Several biochemical effects of metformin have been studied comprising an inhibition of complex I of the mitochondrial respiratory chain, an activation of AMP- dependent protein kinase, an inhibition of PKA, and an inhibition of mitochondrial glycerol 3-phosphate dehydrogenase. Here, we have studied the acute effect of a therapeutically relevant intrahepatic concentration of metformin on glucose production from lactate [1]. We employed the perfused rat liver as experimental system since it enables full control of drug dosage. We used MALDI mass spectrometry imaging to estimate the concentration of metformin in the livers and we measured the concentration of glucose in the effluent medium. MALDI mass spectra of freeze-clamped rat liver perfused with metformin showed a peak at m/z 130.16 which was assigned to metformin. The detection limit was at a tissue concentration of 250 nM, and uptake of metformin to the liver occurred with a Km of 0.44 mM. At a liver concentration of 30 microM, metformin did not induce any suppression of the basal or lactate-induced glucose release. These results show that MALDI imaging can be used to estimate the tissue concentration of metformin in a micromolar concentration range. Our findings challenge the view that metformin attenuates liver glucose release through an inhibition of mitochondrial glycerol 3- phosphate dehydrogenase (EC 1.1.5.3) [2].

1. G. Calza, E. Nyberg, M. Mäkinen, R. Soliymani, A. Cascone, D. Lindholm, E. et al. Lactate-induced glucose output is unchanged by metformin at a therapeutic concentration - a mass spectrometry imaging study of the perfused rat liver. Front Pharm 9 (2018) 141. 2. A.K. Madiraju, D.M. Erion, Y. Rahimi, X.-M. Zhang, D.T. Braddock, R. A. Albright, et al. Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase. Nature 510 (2014) 542–546.

P6 b/4 Cardioprotective effect of dehydrosilybin preconditioning in isolated rat heart Eva Gabrielova1, Lenka Bartosikova2, Jiri Necas2, Martin Modriansky1 1Department of Medical Chemistry and Biochemistry, Olomouc, Czech Republic 2Department of Physiology, Faculty of Medicine and Dentistry, Palacký University in Olomouc, Olomouc, Czech Republic

Cardiovascular disease, particularly ischemic heart disease, is a worldwide health problem. A major goal in the management of myocardial infarction is to reduce postmyocardial infarction complications and mortality by reversing myocardial ischemia and limiting the infarct size. Silymarin is a phytochemical isolated from the milk thistle plant Silybum marianum. The main activity of silymarin is an antioxidant effect of its flavonolignans and other polyphenolic constituents, which is attributable to radical scavenging of both free radicals and reactive oxygen species. A minor flavonolignan component of silymarin is 2,3-dehydrosilybin (DHS). The aim of the current study was to examine the preconditioning effect of DHS on isolated rat heart after reperfusion by the Langendorff technique and to investigate the possible involvement of signalosomes in cardioprotection induced by DHS. The rats (male Wistar, 300 g, 10 weeks) were anesthetized with 2% Rometar + 1% Narkamon. After the i.p. heparine injection the hearts were perfused with a modified Krebs-Henseleit solution (pH 7.4). Schedule of preconditioning was following: stabilization/compound perfusion/global ischemia/reperfusion proceeded at intervals of 5/5/30/60 min. Left ventricle pressure (LVP), heart rate, blood flow and contractility (dP/dtmax) were measured. We tested LDH activity in perfusate, the heart was cut into slices and stained by 1% TTC, or signalosomes were isolated and eNOS and PKCε were detected by western blot. We used quercetin (500 nM) and bradykinin (100 nM) to compare their effect with DHS. DHS (100 nM) caused a decrease of LDH release. A high viability of the heart tissue was maintained after DHS pretreatment. An increase of eNOS was observed in isolated singalosomes. Our data showed that an inhibitor of src kinase family, PP2, decreased the effect of DHS. Our data suggest that DHS is cardioprotective lessening ischemia/reperfusion damage partly via eNOS and src kinase signalling.

This work was supported by grants NPU LO1304 and Czech Science Foundation 16-16667S.

P6 b/5 Altered mitochondrial dynamics due to DNM2 mutations Anikó Gal1,2, Shamim Naghdi1, David Weaver1, Andras Gezsi2, Maria Judit Molnar2, György Hajnóczky1 1MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA 2Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary

The Dynamin2 protein (DNM2) has diverse roles in cell functions, including in clathrin mediated endocytosis at the plasma membrane and together with DRP1, in mitochondrial division [1,2]. DNM2 depletion blocks mitochondrial division and results in an elongated, hyper-fused mitochondrial network [2]. In this study, we tested the effect of human DNM2 mutations (R369W and R465W) on mitochondrial dynamics in patient derivated primary fibroblasts. Both patients have centronuclear myopathy, axonal neuropathy and ophthalmoplegia externa. Their myopathological results showed mitochondrial dysfunctions and in their muscle biopsy sample mtDNA, multiple deletions were found. In addition, the patient with R369W has repetitive discharges and serious cardiomyopaty as well [3]. The patient with R465W beside the common sympthoms has hypoacusis and glycogen accumlation and paracristallin inclusion in her muscle biopsy sample. The mitochondrial fusion activity, network continuity and colocalizations were measured by confocal laser scanning microscopy. The gene expression study was performed using TaqMan gene expression array plates. In patient fibroblasts, mitochondria display fragmentation, aggregation, and decreased network continuity and fusion activity, which changes can be reversed by genetic rescue of DNM2. The mitochondrial interactions with tubulin or ER seemed to be unaltered. The gene expression analysis targeted to mitochondrial dynamics genes showed an increased expression of RAC1, CDC42 and TOMM7 genes. Our findings indicate that the R369W and R465W DNM2 mutations could result abnormal mitochondrial morphogenesis and quality control via inhibition of mitochondrial fusion mechanisms and involving dysregulation of RAC1, CDC42 and TOMM7 expression.

1. M. Bitoun, A.C. Durieux, B. Prudhon, J.A. Bevilacqua, A. Herledan, V. Sakanyan, A. Urtizberea, L. Cartier, NB. Romero, P. Guicheney, Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis. Hum Mutat. 30 (2009) 1419-1427. 2. J. E. Lee, L. M. Westrate, H. Wu, C. Page, G. K. Voeltz, Multiple dynamin family members collaborate to drive mitochondrial division. Nature 540 (2016), 139-143. 3. A. Gal, G. Inczedy-Farkas, E. Pal, V. Remenyi, B. Bereznai, L. Geller, Z. Szelid, B. Merkely, M.J. Molnar, The coexistence of dynamin 2 mutation and multiple mitochondrial DNA (mtDNA) deletions in the background of severe cardiomyopathy and centronuclear myopathy. Clin Neuropathol. 34 (2015): 89-95.

P6 b/6 Mitochondrial bioenergetics in skeletal muscle of premanifest and early manifest transgenic minipig model for Huntington’s disease Hana Hansikova1, Marie Rodinova1, Jana Krizova1, Hana Stufkova1, Bozena Bohuslavova2, Georgina Askeland3, Zaneta Dosoudilova1, Stefan Juhas2, Zdenka Ellederova2, Jiri Zeman1, Lars Eide3, Jan Motlik2 1Laboratory for Study of Mitochondrial Disorders, Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic 2Laboratory of Cell Regeneration and Cell Plasticity, Institute of Animal Physiology and Genetics AS CR, v.v.i. Libechov , Czech Republic 3Department of Medical Biochemistry, Institute of Clinical Medicine, University of Oslo, Norway

Skeletal muscle wasting and atrophy is one of the severe clinical impairment connected with progression of Huntington’s disease (HD). Mitochondrial dysfunction may play significant role in aetiology of the HD but exact condition of mitochondria during development of the HD in muscle has not yet been carefully investigated. We analysed muscle (q. femoris) from 24, 36, 48 and 66-month-old transgenic minipigs expressing the N-terminal part of human mutated huntingtin (TgHD) and age-matched wild-type (WT) siblings (6 TgHD + 6 WT in each age). Respiratory chain complexes (RCC), citrate synthase (CS), pyruvate dehydrogenase (PDH) activity and levels were analyzed by spectrophotometric, radiolabelled and immunoelectrophoretic methods. Respiration was measured by polarography. Mitochondrial energy generating system capacity was characterized by oxidation rate of labelled substrates. Ultrastructure was analyzed by transmission electron microscopy. Genome integrity was assessed by q-PCR. The effect of HD, gender and aging were statistically analyzed. Ultrastructural analyses in 48-month-old TgHD revealed local disorganization of myotubules, dilatation of sarcoplasmic reticulum, increased content of glycogen, higher density of mitochondria and incipient cristae disarrangement in comparison with WT. Activity of CS and RCC complexes IV and I+III and ratio II+III/CS and II/CS were significantly decreased in TgHD. Oxygen consumption showed significantly decreased ratio CII/CIV in TgHD contrary to WT. Oxidation rates characterizing function of ATPase and ANT translocator were found to be gender specific. Protein analyses proved lower content of OPA1 from 48 month in TgHD. Genotype specific effect on mitochondrial DNA (mtDNA) damage but not on mtDNA copy number or nuclear DNA damage in TgHD was observed in the age of 66 month. Our results showed that mitochondrial function in muscle decreases slowly during premanifest stage of HD and biochemical phenotype appears at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression of skeletal muscle in HD and are in concordance with mobility problems observed in this model.

Supported by: Czech-Norwegian Research Programme 7F14308 (MSMT), NPU1609 (MSMT), MZ CR AZV 16-31932A

P6 b/7 Creating patient-specific stem cell models and characterizing metabolic changes in Leigh’s Syndrome Shilpa Iyer1, Ajibola Bakare1, Olivia Kolenc2, Kelly Scheulin3, Harrison E. Grace3, Raj R. Rao2, Edward J. Lesnefsky4, Kyle P. Quinn2, Franklin West3 1Dept of Biological Sciences, Fulbright College of Arts and Sciences, Univ of Arkansas, Fayetteville, Arkansas, USA 2Dept of Biomedical Engineering, College of Engineering, Univ of Arkansas, Fayetteville, Arkansas, USA 3Regenerative Bioscience Center, University of Georgia, Athens, GA, USA 4Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, VA, USA

Leigh’s Syndrome (LS), a classic mitochondrial (mt) disease has no current cure and no adequate cellular model for understanding the rapid fatality associated with the disease. Fatality results from excess accumulation of mutant mtDNA leading to failure of mt- bioenergetics. Other symptoms include developmental, neural, cardiac and muscle impairments. To generate cell models and to assess the mt-functional changes associated with LS, 5 patient-derived fibroblast cell lines carrying Complex I (CI- T10158C; T12706C) and Complex V (CV- T8993G; T9185C) mutations were sequenced, and used in this study. Label-free multiphoton microscopy was used to non-invasively evaluate redox potential in each cell line based on an optical redox ratio of FAD and NADH auto-fluorescence. A significantly higher optical redox ratio was associated with both CV and CI mutant cells compared to normal fibroblasts (FB). Mt- respiration was measured using XF96 flux Analyzer. CV mutant cells showed significantly higher maximal and spare respiratory capacity rate compared to FB. Compensatory Glycolytic and Post 2-DG Acidification rate and non-mt respiration rate was also significantly higher in CV mutant cells relative to FB. No significant difference was observed in respiration or glycolytic measurements in CI mutant cells, relative to FB. These results indicate that CV mutant cells were consuming oxygen at a faster rate than CI mutant cells to compensate for the severe defect in ATP synthase. mRNA reprogramming technology was used to reprogram LS fibroblasts into clinical grade human induced pluripotent stem cells (hiPSCs). LS-hiPSCs exhibited the hallmarks of a pluripotent cell and were able to differentiate into multiple lineages. Next-generation sequencing is being used to quantify the mutant mtDNA burden in LS FB, and LS- hiPSCs over time. Overall, these studies will lead to unique model systems for understanding the pathophysiology of LS and other mt disorders.

P6 b/8 Aerobic metabolism adaptations of human endothelial EA.hy926 cells related to chronic hypoxia Agnieszka Koziel, Wieslawa Jarmuszkiewicz Department of Bioenergetics, Adam Mickiewicz University, Poznan (Poland)

In this study we analyzed the influence of chronic exposure to hypoxia on aerobic metabolism of human umbilical vein endothelial cells (ECs). The impact of hypoxia on mitochondrial respiratory function in ECs and isolated mitochondria of these cells were assessed in

EA.hy926 cell line cultured for 6 days at 1% (hypoxia-treated ECs) and 20% (control ECs) O2 concentration.

Chronic exposure to hypoxia lead to various changes in aerobic metabolism of ECs. In ECs, exposure to 1% O2 for 6 days triggered a shift from aerobic toward anaerobic catabolic metabolism. Growth of ECs under chronic hypoxia conditions did not affect ECs viability and mitochondrial biogenesis. Nevertheless, compounded fermentation were noticed in ECs exposed to chronic hypoxia. Under chronic hypoxia conditions, mitochondrial respiration during oxidation of carbohydrates, fatty acids and glucogenic amino acids were declined. Contrarily, ketogenic amino acid oxidation were elevated in hypoxic ECs. Significantly elevated reactive oxygen species (ROS) production (intracellular and mitochondrial) were observed in ECs exposed to chronic hypoxia. Nevertheless, antioxidant defence (superoxide dismutases SOD1 and SOD2, and uncoupling protein 2, UCP2) were not escalated. Moreover, UCP2 activity and expression were diminished in hypoxic ECs. Considerably augmented expression and activity of complex II, and declined expression and activity of complex I were observed in mitochondria of hypoxic ECs. The augmented activity of complex II resulted in elevation in succinate-sustained mitochondrial ROS production, mainly through increased reverse electron transport. Our results point out a crucial role of mitochondria in metabolic adaptation of ECs exposed to chronic hypoxia conditions. We present an important role of succinate, complex II, and reverse electron transport in hypoxia adaptation of ECs. We speculate that mitochondrial ROS could play a significant signalling role in ECs under hypoxic conditions.

Acknowledgments This work was supported by the National Science Center Grants, Poland (2012/07/N/NZ3/00495 and 2016/21/B/NZ3/00333) and KNOW Poznan RNA Centre (01/KNOW2/2014).

P6 b/9 Impact of sepsis on mitochondrial respiration of the porcine kidney Jitka Kuncova1,2, Michaela Markova1,2, Jiri Chvojka3, Lenka Valesova3, Jan Benes4, Zdenek Tuma2, Martina Grundmanova1,2, Martin Matejovic2,3 1Institute of Physiology, Faculty of Medicine in Plzeň, Charles University, Pilsen, Czech Republic 2Biomedical Center 3Department of Internal Medicine I, 4Department of Anesthesiology and Intensive Care Medicine, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic

Sepsis, life-threatening organ dysfunction caused by a dysregulated host response to infection, represents a fundamental medical problem due to their high incidence and mortality rate. Sepsis-induced acute kidney injury (S-AKI) is a common complication of sepsis in patients admitted to intensive care units. S-AKI is thought to be linked to mitochondrial dysfunction, however, little evidence has been provided to date. In this study, we evaluated mitochondrial respiration in kidney biopsies of pigs subjected to non-severe sepsis (NS) or septic shock (SS) in relation to renal hemodynamic parameters. In 15 anaesthetized ventilated pigs of both sexes, NS or SS was induced by fecal peritonitis after baseline hemodynamic parameters were determined and first biopsy specimen was taken from kidney cortex. Systemic and renal hemodynamic parameters were monitored throughout the experiment (cardiac output, mean arterial pressure, renal blood flow, vascular resistance, renal venous pressure, medullary and cortical tissue perfusion rate and oxygen partial pressures). Further tissue biopsies were taken 24 and 48 hours after sepsis induction. Tissue biopsies were processed by high-resolution respirometry (oxygraph OROBOROS, Austria) to determine states LEAK, OXPHOS I, I+II, ETS I+II, II, and ROX. NS led to an increase in Complex II related states (OXPHOS I+II, ETS I+II, II) without substantial change in the state LEAK. In SS, there were compromised Complex I activity and increased LEAK state. In conclusion, sepsis leads to alterations in mitochondrial respiration in the porcine kidney cortex. In NS, changes point to a better efficiency of oxygen utilization, suggesting some degree of adaptation to metabolic challenge. In contrast, SS is associated with impaired mitochondrial function, possibly resulting in compromised energy production in the proximal tubules.

Support: NPU I – Nr. LO1503, CHU Research Fund (Progres Q39), Specific Student Research Project Nr. 260394/2017.

P6 b/10 Mitochondrial ATP production is required for Wnt signaling modulation Luigi Leanza1, Roberto Costa1, Roberta Peruzzo1, Magdalena Bachmann1, Giulia Santinon2, Mattia Vicario3, Giulia Dalla Montà1, Andrea Mattarei4, Ruben Quintana Cabrera1, Enrico Moro2, Luca Scorrano1, Massimo Zeviani5, Mario Zoratti3,6, Cristina Paradisi7, Francesco Argenton1, Marisa Brini1, Tito Calì3, Sirio Dupont2, Ildikò Szabò1,6 1Department of Biology, University of Padova, Italy 2Department of Molecular Medicine, University of Padova, Italy 3Department of Biomedical Sciences, University of Padova, Italy 4Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy 5MRC Mitochondrial Biology Unit, Cambridge, UK 6CNR Institute of Neuroscience, Padova, Italy 7Department of Chemical Sciences, University of Padova, Italy

Mitochondria are central organelles for cellular metabolism and are involved controlling both cell proliferation and death. The way(s) how mitochondrial function/dysfunction affects downstream signaling pathways in the context of proliferation is still poorly defined. While Wnt signaling, crucial for cellular proliferation and differentiation and often upregulated in cancer, is known to influence mitochondrial function, the possibility that mitochondrial function affects Wnt signaling has not been explored so far. Here we show that sub-lethal concentrations of different pharmacological compounds all able to decrease mitochondrial ATP production down-regulate canonical Wnt signaling in different cancer cell lines. The same pharmacological treatments led to reduced Wnt signaling in vivo, in Danio rerio (zebrafish) reporter lines, while leaving other important signaling pathways such as Sonic hedgehog (Shh) unaffected, indicating specificity of the mitochondria-Wnt signaling axis. In accordance, fibroblasts from patients harboring a genetic mutation leading to impaired function of the respiratory chain complex III, displayed reduced Wnt signaling with respect to healthy cells. The mechanism linking mitochondrial function to the regulation of Wnt signaling has also been investigated. The new paradigm proposed here further underlines the importance of mitochondrial fitness and suggests that chemotherapeutics causing mitochondrial dysfunction may have an additional benefit.

P6 b/11 Investigating the role of the mitochondria shaping protein Opa1 in controlling adipocyte size Marta Medaglia1, Camilla Bean1, Luca Scorrano1,2 1University of Padova, Department of Biology, Padova, Italy 2Venetian Institute of Molecular Medicine, Padova, Italy

White adipose tissue is specialized in the storage and release of fat, the balance of which is critical to maintain healthy energy homeostasis. In addition to its lipid-storing capacity, WAT has been described as an important endocrine organ controlling the systemic handling of energy substrates. Excessive lipid load causes adipocyte stress, which in turn accounts for many adverse effects of obesity; also conditions of absence or scarcity of WAT such as lipodystrophy and cachexia are associated with severe metabolic complications. Mitochondrial dysfunction has been first associated to impaired glucose tolerance 40 years ago, and it’s still under debate whether mitochondrial dysfunction is the cause or a consequence of insulin resistance and type 2 diabetes. In particular, the role of the fusogenic protein Opa1 remains to be elucidated. Controlled Opa1 overexpression favors adipocyte plasticity, resulting in overall improved glucose metabolism and insulin sensitivity. Adipocyte-specific deletion of Opa1 triggers a lipodystrophic phenotype, with adipocytes unable to adapt to metabolic challenges to increase lipid storage. Moreover, RNAseq analysis performed on Wt and Opa1tg pre-adipocytes and mature adipocytes reveals changes in pathways associated with control of cell size. We therefore hypothesize that Opa1 impacts on signaling pathays that are responsible for regulating cell size.

P6 b/12 The role of Mitochondrial Superoxide Release in Acute and Chronic Hypoxia Sensing and Signaling of the Pulmonary vasculature Oleg Pak1, M. Hüttemann2, S. Scheibe 1, M. Gierhardt1, A. Sydykov1, K. Schäfer1, H. A. Ghofrani1, R. T. Schermuly1, M. P. Murphy3, L. I. Grossman2, N. Weissmann1, N. Sommer1 1Excellence Cluster Cardiopulmonary System, University of Giessen and Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany 2Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 540 E. Canfield, Detroit, Michigan 48201, USA 3MRC Mitochondrial Biology Unit, CB2 0XY Cambridge, United Kingdom

Introduction: Acute alveolar hypoxia induces reversible hypoxic pulmonary vasoconstriction (HPV) of precapillary pulmonary arteries to redirect blood from poorly to better ventilated areas of the lung to optimize gas exchange. In contrast, chronic hypoxia triggers the development of pulmonary hypertension (PH). The exact role of mitochondrial superoxide release in HPV and PH remains controversial. We hypothesized that a pulmonary specific regulatory subunit 4 of the mitochondrial cytochrome c oxidase (Cox4i2) regulates the release of mitochondrial superoxide which may underlie hypoxic signaling. Methods: We investigated the release of different reactive oxygen species (ROS) as well as the effect of ROS inhibition and Cox4i2 deletion (Cox4i2-/-) on acute and chronic hypoxic signaling in pulmonary smooth muscle cells (PASMCs) in vitro, in isolated mouse lungs and in mice in vivo. ROS release was inhibited by mitochondria-targeted antioxidant MitoQ and S3QEL2, which inhibits superoxide release from complex III. Result: Intracellular and mitochondrial superoxide concentration, as well as hydrogen peroxide levels were increased in PASMCs after 5 min, but decreased after 5 days of hypoxia. MitoQ and S3QEL2 significantly inhibited HPV in isolated lungs. HPV was absent in isolated lungs of Cox4i2-/- mice. Cox4i2-/- abolished the acute hypoxia-induced rise of superoxide level in PASMCs, as well as downstream hypoxic signaling. Cox4i2-/- PASMCs lacked hypoxia-induced mitochondrial hyperpolarization, but showed similar oxygen-dependent respiration as wild-type PASMCs. In contrast to acute HPV, MitoQ application and Cox4i2-/- did not affect the development of chronic hypoxia-induced PH. Conclusion: An increase of mitochondrial superoxide levels mediates acute, but not chronic hypoxic signaling of the pulmonary vasculature. Cox4i2 is essential for HPV, possibly by a mechanism involving mitochondrial hyperpolarization, which may promote superoxide release at complex III.

P6 b/13 Bioenergetics of the Costello syndrome Rossignol Rodrigue1,2,6, Dard Laetitia1,2, Hubert Christophe1,2, Amoedo Dias Nivea1,2, Elodie Dumon1,2, Claverol Stéphane2,3, Bonneu Marc2,3,4, Bellance Nadège1,2, Lacombe Didier1,2,5 1INSERM U1211, Bordeaux Bordeaux, France 2Bordeaux University, Bordeaux, France 3Functional Genomics Center (CGFB), Proteomics Facility, Bordeaux, France 4Bordeaux-INP Avenue des Facultés 33405 Talence Cedex, France 5CHU Bordeaux, Haut-Lévèque Hospital, Pathology department, Bordeaux 6CELLOMET, Functional Genomics Center (CGFB), Bordeaux, France

The RAS pathway is a highly conserved cascade of protein-protein interactions and phosphorylation that is at the heart of signalling networks that govern proliferation, differentiation and cell survival. Recent findings indicate that the RAS pathway plays a role in the regulation of energy metabolism via the control of mitochondrial form and function but little is known on the participation of this effect in RAS-related rare human genetic diseases. Germline mutations that hyperactivate the RAS pathway have been discovered and linked to human developmental disorders that are known as RASopathies. Individuals with RASopathies, which are estimated to affect approximately 1/1000 human birth, share many overlapping characteristics, including cardiac malformations, short stature, neurocognitive impairment, craniofacial dysmorphy, cutaneous, musculoskeletal, and ocular abnormalities, hypotonia and a predisposition to developing cancer. Here, we analysed the bioenergetics of the Costello syndrome (CS) using a mice model expressing HRASG12S, patients-derived fibroblasts and fibroblasts expressing lentiviral constructs carrying HRASG12S/V/A mutations. Our findings revealed a consistent alteration of mitochondrial physiology in the heart and the skeletal muscle of the Costello mice that was also found in the cell models of the disease. The underpinning mechanisms involved a molecular control of the AMPK signalling pathway by mutant forms of HRAS.

P6 b/14 Mitochondrial DNA copy number and Complex I activity in pediatric acute lymphoblastic leukemia Archna Singh1, Ayushi Jain1, Sameer Bakhshi2, Jayanth Kumar1 1Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India 2Department of Medical Oncology, B.R.A.I.R.C.H, All India Institute of Medical Sciences, New Delhi, India

Studies in pediatric acute lymphoblastic leukemias (ALL) show alterations in mitochondrial DNA(mtDNA) copy number. Our study aims to investigate copy number changes, effects on metabolism and mitochondrial electron transport chain (ETC) complexes’ enzyme activity and plausible underlying mechanisms. Bone marrow aspirates from 120 ALL cases and peripheral blood samples from healthy controls (n=26) were analyzed for mtDNA copy number and gene expression of regulators of mitochondrial replication and transcription (TFAM, PolG) by real-time PCR. The patient mtDNA copy numbers were significantly higher than that in controls (p-value<0.0001) as were TFAM and PolG gene expressions (p-value<0.0001 for both genes). ETC Complex I enzyme activities were compared in patients (n=67) and controls (n=25) {p value=0.29, median(range)=7.59 (0.5798–111) and 6.69 (0.03467–18.01) µmol/min/µg respectively} using an ELISA kit. Next-Generation sequencing (Illumina HiSeq2500 platform) was done for whole mitochondrial genome sequencing on patient (n=12) and control (n=5) samples. Variants having allele frequency from 5%-95% were identified as Heteroplasmic variants while variants having allele frequency less than 5% and greater than 95% were identified as homoplastic variants. Heteroplasmy was seen in 10 out of 12 ALL patient samples and in 1 out of 5 healthy controls. Heteroplasmic variations were present in the ALL patient samples in the regulatory region (D-loop) and various mitochondrial genes that encode for ETC complexes: ND2, ND4, ND5 of Complex I; COX2 and COX3 of Complex IV and ATPs6 of Complex V. The positions 464, 10935, 13762 were the most frequent sites for heteroplasmy. Homoplasmic variations were present in all samples and all throughout the mtDNA. Further analysis of interactions between mitochondrial DNA mutations, enzyme activities and copy number changes may provide insights into mechanisms modulating mitochondrial function in ALL.

P6 b/15 Evaluating the Bioenergetics of Myoblast Migration on Aligned Nanofiber Scaffolds Kalyn S. Specht1, Abinash Padhi2, Amrinder S. Nain2, David A. Brown1 1Virginia Tech, Department of Human Nutrition, Foods, and Exercise and the Virginia Tech Center for Drug Discovery, Blacksburg, Virginia 2Virginia Tech, Department of Mechanical Engineering, Blacksburg, Virginia

Cell migration is influential in many physiological processes such as morphogenesis and wound healing, as well as in disease states such as cancer metastasis. Native cellular environments are composed of fibrous proteins of varying geometric features (diameter, orientation, spacing and hierarchical assembly). We engineered fibronectin-coated fiber networks of controlled diameter (curvature) and alignment, then interrogated the bioenergetic pathways utilized in single cell migration. To do so, we introduced two mitochondrial inhibitors, antimycin-a (AMA) and oligomycin and a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). Despite clear effects on mitochondrial respiration, cell speed was not markedly altered after treatment with 2µM AMA (pre: 34.5±2.3 µm/hr and post: 30.1±2.5 µm/hr, n=21; P = NS). Rates of cell migration remained unchanged for the next six hours. Next, we treated cells with 2.5µM oligomycin, and again found cell speed to remain unaffected (pre: 30.1±3.8 µm/hr and post: 28.7±2.8 µm/hr, n=11; P = NS). Unexpectedly, moderate inhibition of glycolytic metabolism with 2-DG [12mM] decreased cell migration velocity from 32.9±3.8 µm/hr to 23.9 ±1.4 µm/hr (n=25, P<0.05) within one hour and remained decreased for the subsequent 6-hour treatment. Interestingly, inhibition with a higher concentration of 2- DG (50mM) abruptly halted cell movement (pre: 31.2±1.6 µm/hr and post: 9.1±0.66 µm/hr (n=20, P <0.05). Co-treatment with 2DG and AMA further decreased cell velocity from 35.4 ±2.1 µm/hr to 14.9±1.3 µm/hr (n=11, P <0.05). Altogether our data suggest that myoblast migration is heavily reliant on glycolysis and contrary to current understanding suggests a diminished role of mitochondrial bioenergetics in cell-ECM interactions during cell development, repair, and pathology.

P6 b/16 X-ray crystallographic structure analysis of seven disease-causing mutants of human lipoamide dehydrogenase Eszter Szabo1, Piotr Wilk2, Reka Mizsei1, Agnes Hubert1, Zsofia Zambo1, David Bui1, Beata Torocsik1, Manfred S. Weiss2, Vera Adam-Vizi1, Attila Ambrus1 1Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, Hungary 2Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany

(Dihydro)lipoamide dehydrogenase (LADH, E3) is a homodimeric flavin-disulfide oxidoreductase that catalyzes the oxidation of dihydrolipoamide using NAD+ as a co-substrate. As a common third subunit of the mitochondrial alpha-ketoacid dehydrogenase complexes and part of the glycine cleavage system the human (h) E3 plays pivotal roles in metabolism. Pathogenic hE3 variants affect several central metabolic pathways simultaneously and lead to E3 deficiency. Clinical manifestations of E3 deficiency are versatile, generally very severe, and not correlating well with the loss in E3 activity. This implies that other auxiliary biochemical mechanisms, presumably the elevated reactive oxygen species (ROS) generating activities of certain pathogenic variants and/or impaired interactions among the subunits of the relevant multienzyme complexes, might also contribute to the pathogenesis [1]. We determined the high-resolution crystal structures of the wild type hE3 and seven of its disease-causing mutants at the resolution range of 1.5 to 2.3 Å. The pathogenic amino acid substitutions are located in either the dimer interface (G426E, D444V, I445M, R447G, and R460G), the active site (P453L), or the cofactor binding region (G194C). Based on the analyses of the crystal structures individual molecular pathomechanisms, of the compromised catalytic activities and altered capacities for ROS generation, are proposed for the respective disease-causing hE3 mutants.

1. A. Ambrus, V. Adam-Vizi, Human dihydrolipoamide dehydrogenase (E3) deficiency: Novel insights into the structural basis and molecular pathomechanism, Neurochem. Int., (2018) [Online early access] DOI: 10.1016/j.neuroint.2017.05.018.

P6 b/17 Alterations in energy transfer pathways in Wfs1 deficient mice Kersti Tepp, Marju Puurand, Aleksandr Klepinin, Indrek Reile, Tuuli Kaambre Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia

Mutations in the wolframin (Wfs1) gene cause Wolfram syndrome, an autosomal recessive neuro-degenerative disorder characterized by early juvenile diabetes mellitus, progressive optic nerve atrophy, diabetes insipidus and deafness. Wfs1 deficient (Wfs1KO) mice develop endoplasmic reticulum stress and unfolded protein responses in cells, but the pathophysiology at organism level is poorly understood. Also, link between Wfs1 deficiency and mitochondrial dysfunction have shown. Phosphotransfer reactions are governed by the metabolic status of the cell. Moreover, phosphotransfer pathways and their rates may correlate with changes in the organization of the mitochondrial energy production system. The creatine kinase (CK), adenylate kinase (AK) and other energy transport pathways create an opportunity for facilitated energy transport [1]. First results that characterize energy transfer pathways in the heart and skeletal muscles of Wfs1KO mice demonstrate a shift in the energy pathway preferences. In the heart muscle of Wfs1KO mice the AK pathway is more active, while the creatine activated respiration is lower than in control animals. On the contrary, in the glycolytic m. rectus femoris the activity of AK pathway shows a slight decrease in comparison to the control. The coupling of hexokinase to OXPHOS is also altered in muscles of Wfs1KO mice. The glycolytic m. gastrocnemius white of Wfs1KO mice display a lower glucose induced respiration than in control animals; while in the oxidative heart muscle of Wfs1KO mice this respiration rate is higher. These changes indicate to compensatory mechanism in response to metabolic alterations.

1. V. Saks, U. Schlattner, M. Tokarska-Schlattner, T. Wallimann, R. Bagur, S. Zorman, M. Pelosse, P.D. Santos, F. Boucher, T. Kaambre, R. Guzun, Systems Level Regulation of Cardiac Energy Fluxes Via Metabolic Cycles: Role of Creatine, Phosphotransfer Pathways, and AMPK Signaling, Springer Ser Biophys, 16 (2014) 261-320.

P6 b/18 ATP synthase assembly defect and liver damage are major pathology hallmarks of inducible Tmem70 knockout in adult mice Marek Vrbacky1, Jana Kovalcikova1, Hana Nuskova1, Katerina Tauchmannova1, Inken Beck2, Otto Kucera3, Zuzana Cervinkova3, Radislav Sedlacek2, Tomas Mracek1, Josef Houstek1 1Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic 2Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic 3Faculty of Medicine, Charles University, Hradec Kralove, Czech Republic

TMEM70 is a transmembrane protein localized in the inner mitochondrial membrane and involved in the biogenesis of the eukaryotic ATP synthase. In patients, TMEM70 mutations cause isolated deficiency of ATP synthase resulting in neonatal mitochondrial encephalocardiomyopathy. Recently we confirmed the essential role of TMEM70 in a mouse model, where homozygous Tmem70-/- deletion led to embryonal lethality, disturbed ATP synthase biogenesis and disordered mitochondrial ultrastructure. To explore the molecular mechanism of TMEM70 action in adult mice, we generated tamoxifen inducible whole body conditional knockout. Mice survived for up to 8 weeks after the induction of gene excision while progressively losing the body weight. We observed pronounced decrease of the fully assembled F1Fo-ATP synthase with F1 subcomplex accumulation in majority of examined tissues, except in heart that was likely protected by the remaining un-excised Tmem70 gene. Histopathology revealed focal necrosis in liver, further corroborated by increased markers of oxidative stress and apoptosis, by blood hyperammonemia, and by increased circulating levels of alanine and aspartate aminotransferases. OXPHOS in Tmem70-/- liver mitochondria exhibited higher membrane potential and poor inhibition of state 3 respiration by oligomycin. More detailed analysis of ATP synthase subcomplexes in Tmem70-/- liver mitochondria by clear native electrophoretic techniques detected formation of large and labile vestigial, subunit c deficient, ATP synthase complex that dissociated into the free F1 subcomplex upon Coomassie blue dye addition. This suggests the role of TMEM70 in c-ring incorporation into the ATP synthase holoenzyme. In conclusion, induction of Tmem70 knockout in adult mice impairs primarily liver function, and it resembles symptoms present during metabolic crises in patients. This contrasts with the mainly cardiologic presentation at disease onset in humans. Supported by grants 14-36804G and 16-33018A.

P7 a/1 Epoxycyclohexenedione-type Compounds Make Up a New Class of Inhibitors of the Bovine Mitochondrial ADP/ATP Carrier Ayaki Aoyama, Masatoshi Murai, Hideto Miyoshi Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Mitochondrial ADP/ATP carrier (AAC) forms a key link between the mitochondrial matrix and cytosol compartments. In contrast to a wide variety of selective inhibitors of the respiratory enzymes, only two classes of inhibitors (atractylosides and bongkrekic acids (BKAs)) have been identified for AAC to date. Through the extensive screening of our chemical library, we discovered epoxycyclohexenedione (ECHD)-type compounds as unique inhibitors of the bovine heart mitochondrial AAC. In the present study, we investigated the mechanism of the inhibition of AAC by ECHDs using bovine submitochondrial particles (SMPs). Careful proteomic analyses of ECHD-bound AAC as well as biochemical characterization using different SH-reagents demonstrated that ECHDs inhibit the function of AAC by covalently binding primarily to Cys57 and secondarily to Cys160. Interestingly, AAC remarkably aggregated in SMPs when incubated with high concentrations of ECHDs for a long period of time. This aggregation was observed under both oxidative and reductive conditions of the SDS-PAGE analysis of SMP proteins, indicating that the aggregation is not caused by intermolecular S-S linkages. When all solvent accessible cysteines (Cys57, Cys160, and Cys257) were modified by N-ethylmaleimide in advance, the aggregation of AAC was completely suppressed. In contrast, when Cys57 or Cys160 is selectively modified by a SH- reagent, the covalent binding of ECHDs to a residual free residue of the two cysteines is sufficient to induce aggregation. Based on these results, we conclude that ECHDs are the first chemicals, to the best of our knowledge, to induce prominent structural alteration of AAC without forming intermolecular S-S linkages [1].

1. A. Aoyama, M. Murai, N. Ichimaru, S. Aburaya, W. Aoki, H. Miyoshi, Epoxycyclohexenedione-type compounds make up a new class of inhibitors of the bovine mitochondrial ADP/ATP carrier, Biochemistry 57 (2018) 1031-1044.

P7 a/2 Biophysical and pharmacological characterization of a channel-forming mitochondrial protein complex Vanessa Checchetto1, Angela Paggio2, Diego De Stefani2, Rosario Rizzuto2, Ildikò Szabò1 1Department of Biology, University of Padova, Italy 2Department of Biomedical Sciences, University of Padova, Italy

Mitochondrial ion channels are of great importance to ensure the proper function of this bioenergetic organelle and to regulate cell fate. However, in many cases our knowledge concerning their molecular identity and regulation is still limited. Recently, we characterized a novel protein complex located in the inner mitochondrial membrane that displays all known pharmacological and electrophysiological properties of the long-sought mitochondrial KATP (mitoKATP) channel. An important role has been assigned to mitoKATP, especially in the heart, in protection from I/R injury, pre-conditioning and post-conditioning but its composition, properties, regulation and function constitute the most controversial topic in the field of mitochondrial channels.

P7 a/3 The putative role of human VDAC1 in Huntington disease pathogenesis Daria Grobys, Andonis Karachitos, Wojciech Grabiński, Martyna Baranek, Hanna Kmita Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland

Huntington disease (HD) is a neurodegenerative disorder caused by CAG trinucleotide repeat expansion in exon 1 of HTT gene encoding huntingtin (Htt). The repeat number higher than 35 results in its mutant form (mHtt) regarded as a triggering factor of neurodegeneration resulting in progressive motor dysfunction, cognitive decline and other behavioral symptoms. The mechanism(s) underlying the neurodegeneration is (are) not completely elucidated. At cellular level the presence of mHtt causes neuron dysfunction and death through a number of phenomena involving disturbances of mitochondrial functions. Importantly, the voltage-dependent anion channel (VDAC) serves as a mitochondrial gatekeeper, controlling the metabolic cross-talk between mitochondria and the rest of the cell as well as expression of mitochondrial proteins. Accordingly, the aim of this study was to check whether VDAC protein is important for Htt and/or mHtt effects on cell viability and mitochondrial coupling status in intact cells, which in turn would indicate VDAC participation in the early stages of HD pathogenesis. For the purpose we applied the yeast Saccharomyces cerevisiae model representing different variants of Htt and mHtt induced expression in the presence or absence of the yeast functional VDAC and isoforms of the human protein. The obtained results indicate that temperature and expression of human VDAC isoforms are important for mHtt cytotoxic effect. Moreover, expression of human VDAC isoforms evokes differences in mHtt-triggered changes in mitochondria coupling status. This allows us to suggest that among human VDAC isoforms hVDAC1 is most probably involved in HD pathogenesis.

Acknowledgments: YIp351Q25, YIp351Q103 constructs were provided by prof. A. Barrientos, US pYX212 hVDACs were provided by prof. V. De Pinto, IT The work was supported by the grant of National Science Centre, Poland, NCN 2011/01/B/NZ3/00359, GDWB-06/2015 and the KNOW RNA Research Centre in Poznan No. 01/KNOW2/2014.

P7 a/4 The LAT1 amino acid transporter: substrate binding site and drug discovery Cesare Indiveri, Mariafrancesca Scalise, Michele Galluccio, Lara Console Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy

LAT1 (SLC7A5) is a plasma membrane transporter mainly localized in basolateral membranes of polarized epithelia and in Blood Brain Barrier; it forms a heterodimer with the glycoprotein CD98 (SLC3A2) through a conserved disulfide between two Cys residues. LAT1 mediates an antiport of neutral and branched amino acids Na+ and pH independent. The interest in deepening the study of LAT1 relies on its well documented over-expression in many tumors suggesting this protein as novel pharmacological target. We conducted the studies on LAT1/CD98 using a combined approach of bioinformatics, proteoliposome model and cell systems following the [3H]His transport in exchange with other amino acids. In particular, we firstly demonstrated that LAT1 is the sole transport competent unit of the heterodimer by using recombinant hLAT1 and hCD98, native proteins extracted from cells or endogenous proteins in intact cells. The transport mechanism of LAT1 mediated antiport is random simultaneous suggesting the existence of a functional dimer. Homology modeling of LAT1, built on the basis of bacterial AdiC, predicted four residues as crucial for substrate binding and translocation: site directed mutagenesis demonstrated that F252 is a gate element, S342 and C335 are responsible of substrate docking and C407 interacts with the intracellular substrate. The presence of these two Cys residues has been exploited for designing inhibitors, based on dithiazole structure, able to specifically interact with -SH groups of LAT1. Thus, a screening has been performed in proteoliposomes and eight compounds have been identified as potent inhibitors with IC50 in the micromolar range. The interaction occurs at the level of C407, forming a mixed disulfide. The C407A mutant showed decreased reactivity towards dithiazoles. Interestingly, the most potent inhibitors are also able to impair viability of cancer cells expressing LAT1.

P7 a/5 Control of brown adipose thermogenesis by a cold-inducible, circadian mitochondrial transporter Iuliia Karavaeva1, Tao Ma1, Elahu G. Sustarsic1, Matthew J. Emmett3, Homa Majd4, Mark P. Jedrychowski5, Susanne Keipert6, Andreas Nygaard Madsen1, Astrid Linde Basse2, Christian Theil Have1, Torben Hansen1, Jacob Bo Hansen2, Birgitte Holst1, Thue W. Schwartz1, Martin Jastroch6, Steven P. Gygi5, Edmund R. S. Kunji4, Mitchell A. Lazar3, Zachary Gerhart-Hines1 1Section for Metabolic Genetics at the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark 2Department of Biology, University of Copenhagen, Copenhagen, Denmark 3Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Department of Genetics, The Institute for Diabetes, Obesity, and Metabolism Perelman School of Medicine at the University of Pennsylvania, Philadelphia, USA 4Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, United Kingdom 5Department of Cell Biology, Harvard Medical School, Boston, USA 6Institute for Diabetes and Obesity, Helmholtz Zentrum München, German Research Center for Environmental Health, Garching, DE

The remarkable energy-dissipating capacity of thermogenic brown adipose tissue (BAT) makes it a promising target with which to combat obesity and associated diseases. BAT thermogenesis is tightly controlled by environmental cold temperature and the intrinsic circadian clock. Yet the key mechanisms underlying both these methods of BAT control remain unknown. Here we report an orphan mitochondrial transporter and its close homolog with novel functions in circadian and cold-induced BAT metabolism. Expression of the transporter mRNA and protein is highly cold-inducible and undergoes circadian oscillation in BAT that is positively correlated with thermogenesis. Interestingly, cold-induced expression of the transporter is not stimulated by adrenergic signaling – the classical BAT activator – but rather by the circadian regulator, REV-ERBα. Knock down of the transporter in murine brown adipocytes causes a dramatic decrease in basal and uncoupled respiration. Double knock down of the transporter and its homolog partially rescues the phenotype of the single knock down of the transporter. These observations imply an important role of the transporters in alternative regulation of BAT metabolism. Identification of the substrate/substrates of the transporters and investigation of their roles in BAT thermogenesis will advance the fundamental understanding of BAT metabolism and potentially contribute to development of novel treatment strategies for metabolic disease.

P7 a/6 The influence of flavonoids on oxygen consumption and membrane potential of isolated endothelial mitochondria via modulation of mitoBKCa channel activity Anna Kicinska1, Piotr Bednarczyk2, Rafał Kampa2,3, Adam Szewczyk3, Wieslawa Jarmuszkiewicz1 1Department of Bioenergetics, Adam Mickiewicz University, Poznan, Poland 2Department of Biophysics, Warsaw University of Life Sciences (SGGW), Warsaw, Poland 3Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Warsaw, Poland

Changes in K+ permeability of the inner mitochondrial membrane are important for induction of protective mechanisms diminishing tissue injuries caused by ischaemia/reperfusion or oxidative stress. Numerous observations suggest that the key role in these cytoprotection phenomena is played by the mitochondrial potassium channels. Previously, the mitochondrial large-conductance calcium- activated potassium channel (mitoBKCa) from EA.hy926 endothelial cells has been characterized [1]. In this study, in a search for new pharmacological modulators of mitochondrial potassium channels, we have examined the influence of cardioprotective flavonoids on endothelial cell mitochondria. We have found that some of these substances influence the bioenergetics of endothelial mitochondria. In isolated mitochondria, the rate of resting non-phosphorylating respiration was increased in response to naringenin and quercetin, during succinate-sustained respiration. This effect was partially reversed by a potassium channel blocker, iberiotoxin. Both flavonoids also caused a slight (~ 1mV) but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP+-specific electrode). The resulting steady state value of membrane potential was partially restored after treatment with iberiotoxin. The influence 2+ of Ca on the flavonoid effect was also examined. The direct influence of studied flavonoids on mitoBKCa channels was further confirmed using patch-clamp measurements. Thus, we show that flavonoids modulate oxygen consumption and membrane potential of isolated endothelial mitochondria via their influence on the mitoBKCa channel activity

1. P. Bednarczyk, A. Koziel, W. Jarmuszkiewicz, A. Szewczyk, Large-conductance Ca² -activated potassium channel in mitochondria of

endothelial EA.hy926 cells, Am J Physiol Heart Circ Physiol 304 (2013) H1415-H1427.⁺

This study was supported by a grant 2016/21/B/NZ1/02769 from the National Science Centre, Poland and partially by KNOW Poznan RNA Centre (01/KNOW2/2014)

P7 a/7 Recording channel activity of the ROMK1/2 protein produced in E. coli Piotr Koprowski, Milena Krajewska, Adam Szewczyk Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

ROMK2 is a mitochondrial isoform of Kir1.1, which also forms plasmalemmal inwardly rectifying potassium channel inhibited by ATP [1]. ROMK2 is believed to be a part of mitoK(ATP) channel, however, the data directly linking the protein to this channel activity is limited [2]. The small amount of protein and infrequently observed activity of ROMK2 in mitochondria of H9c2 cell line prompted us to express the ROMK1/2 protein in E. coli cells. The bacterial expressed ROMK1/2 gives opportunity to directly patch-clamp E. coli protoplast, and reconstitute purified channel in BLM membranes, for functional studies. We observed differences in biophysical properties between these preparations, and the activity of ROMK1/2 reported previously [3]. This discrepancy could be attributed to differences in buffer ionic composition, lipid content of membranes and existence of potential protein partners. This work was supported by Polish National Science Center, grant no. 2015/19/B/NZ1/02794.

1. H. Hibino, A. Inanobe, K. Furutani, S. Murakami, I. Findlay, Y. Kurachi, Inwardly rectifying potassium channels: their structure, function, and physiological roles, Physiol. Rev. 90 (2010) 291-366 2. D.B. Foster, A.S. Ho, J. Rucker, A.O. Garlid, L. Chen, A. Sidor, K.D. Garlid, B. O'Rourke, Mitochondrial ROMK channel is a molecular component of mitoK(ATP), Circ. Res. 111(2012) 446-454 3. C.M. McNicholas, W.B. Guggino, E.M. Schwiebert, S.C. Hebert, G. Giebisch, M.E. Egan, Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator. Proc. Natl. Acad. Sci. 93(1996) 8083-8088

P7 a/8 SK channel activation induces neuroprotection by metabolic reprogramming Inge E. Krabbendam1, Birgit Honrath1,2, Barbara Bakker3, Carsten Culmsee2, Martina Schmidt1, Amalia M. Dolga1 1Dept. of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands 2Dept. of Pharmacology and Clinical Pharmacy, University of Marburg, Marburg, Germany 3Dept. of Pediatrics, Biology, University of Groningen, Groningen, The Netherlands

Activation of small-conductance Ca2+-activated K+ (SK) channels contribute to neuroprotection in conditions of oxidative stress by 2+ attenuating mitochondrial calcium ([Ca ]m), mitochondrial reactive oxygen species (ROS) generation and mitochondrial respiration in neuronal HT22 cells [1]. In this study, we aim to clarify how SK channels mediate effects at mitochondrial complex activity, and whether metabolic shifts are playing a role in their neuroprotective effects. The impact of SK channel activation by CyPPA on mitochondrial function was studied with extracellular flux analysis, lactate measurements, high-resolution respirometry and flow cytometry. Our data show that SK channel opening result in a fast induction of glycolysis followed by a slight reduction in oxidative phosphorylation. Interestingly, opening of SK channels alone resulted in decreased mitochondrial complex I and II activity, a slight increase in mitochondrial ROS production and depolarization of the mitochondrial membrane, suggesting preconditioning mechanisms to be also involved in the neuroprotection. The neuroprotection mediated by CyPPA in situations of oxidative stress was reduced in cells lacking the possibility to undergo glycolysis and also in cells where mitochondrial ROS production was attenuated by specific mitochondrial oxidant scavengers. Overall, these findings indicate a potential therapeutic value of SK channels in diseases associated with mitochondrial oxidative stress.

References: [1] B. Honrath, L. Matschke, T. Meyer, L. Magerhans, F. Perocchi, G.K. Ganjam, H. Zischka, C. Krasel, A. Gerding, B.M. Bakker, M. Bünemann, S. Strack, N. Decher, C. Culmsee, A.M. Dolga, SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake, Cell Death Differ. (2017) 1–13. doi:10.1038/cdd.2017.2.

P7 a/9 Production of ROMK1/2 protein in E. coli for functional studies Milena Krajewska, Piotr Koprowski, Adam Szewczyk Laboratory of Intracellular Ion Channels, Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

ROMK1 (also known as Kir1.1a, a product of the KCNJ1 gene), a member of the Kir channel family, is a plasmalemmal inwardly rectifying potassium channel inhibited by ATP [1]. ROMK2 is a mitochondrial isoform lacking first 19 amino acids and it is believed to be a part of mitoK(ATP) channel. ROMK1/2 proteins form homotetramers. Each subunit is characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment [2]. To obtain purified ROMK1/2 channels for further studies, we used bacterial expression in Escherichia coli cells. Wild type or codon- optimized ORFs coding for ROMK1/2, or chimera between cytoplasmic N- and C-Termini of ROMK1/2 and transmebrane part of KirBac1.3, were fused to sequences of several expression tags, which could assist membrane insertion and folding of eukaryotic proteins in bacteria (SUMO [3], pOmpF [4] and MISTIC [5]). In addition, fusion proteins contained affinity tags for purification (N- or C- terminal 6-His tag or Strep-tagII). The construct with C-terminal affinity tag exhibiting low degradation and highest membrane expression level was chosen for further studies. Here several detergents were screened and, based on solubilization efficiency and tetramer stability, n-Dodecyl β-D-maltoside was used for protein purification. Large amount of E. coli expressed ROMK1/2 protein give opportunity to reconstitute this channel in membranes and membrane nanodiscs for functional studies.

This work was supported by Polish National Science Center, grant no. 2015/19/B/NZ1/02794

1. H. Hibino, A. Inanobe, K. Furutani, S. Murakami, I. Findlay, Y. Kurachi, Inwardly rectifying potassium channels: their structure, function, and physiological roles, Physiological Reviews 90 (2010) 291-366. 2. J.C. Koster, K.A. Bentle, C.G. Nichols, K. Ho, Assembly of ROMK1 (Kir 1.1a) inward rectifier K+ channel subunits involves multiple interaction sites, Biophysical Journal 74 (1998) 1821–1829 3. X. Zuo, S. Li, J. Hall, M.R. Mattern, H. Tran, J. Shoo, R. Tan, S.R. Weiss, T.R. Butt, Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli, Journal of Structural and Functional Genomics 6 (2005) 103-111 4. P.C. Su, W. Si, D.L. Baker, B.W. Berger, High-yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion, Protein Science 22 (2013) 434-443 5. N.S. Alves, S.A. Astrinidis, N. Eisenhardt, C. Sieverding, J. Redolfi, M. Lorenz, M. Weberruss, D. Moreno-Andrés, W. Antonin, MISTIC-fusion proteins as antigens for high quality membrane protein antibodies, Scientific Reports 7 (2017) 41519

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BK-DEC splice variant forms a functional BKCa channel in the inner mitochondrial membrane Shur K. Kucman1, Justyna Jędraszko1, Piotr Bednarczyk2, Adam Szewczyk1, Bogusz Kulawiak1 1Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology Polish Academy of Science, Warsaw, Poland 2Faculty of Wood Technology, Department of Physics, Warsaw University of Life Sciences, Warsaw, Poland

Ischemia of brain or heart tissue is the one of the most common causes of death worldwide. In the inner mitochondrial membrane several potassium channels have been identified whose activation lead to cytoprotection during ischemic event. It was found that activation of mitochondrial large conductance calcium activated potassium channel (mitoBKCa) preserves brain and heart muscle cells.

Recently, the molecular identity of the mitoBKCa channel was described. A BK-DEC splice variant of BKCa-type channels α subunit has been demonstrated to localize in mitochondria. However it is not known whether this isoform is able to form a functional channel in mitochondria. In our study we used HEK293T cells transfected with cDNA encoding BK-DEC splice variant. Electrophysiological recordings with use of mitoplast isolated from transfected cells revealed presence of the large conductance and voltage dependent ion channel. This type of channel was not present in mitoplasts isolated from untransfected cells. We found that recorded channel showed all basic 2+ pharmacological properties typical for the mitoBKCa channels described previously. The channel was Ca sensitive, its activity was stimulated by potassium channel opener NS1619 and inhibited by paxilline, well known mitoBKCa channel inhibitor. Additionally, kinetics and conductance of observed channel were very similar to the mitoBKCa channel. Based on collected data we conclude that BK-DEC splice variant forms a functional channel in the inner mitochondrial membrane of HEK293T cells.

This work was supported by the Polish National Science Centre grant No.2015/18/E/NZ1/00737 and the Nencki Institute of Experimental Biology.

P7 a/11 Calcineurin-homologous proteins bind with high affinity to the CBD-region of the human Na+/H+ exchanger NHE1 Shuo Liang1,2, Simon Fuchs1,2, Sierra C. Hansen1, Marie Markones3,4, Evgeny Mymrikov1,4, Heiko Heerklotz3,4,5, Carola Hunte1,4 1Institute for Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Freiburg, Germany 2Faculty of Biology, University of Freiburg, Freiburg, Germany 3Department of Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany 4BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany 5Leslie Dan Faculty of Pharmacy, University of Toronto, Canada

Homeostasis of the intracellular pH is critical for cell vitality. A key player in this process is the Na+/H+ exchanger NHE1, which exports excess protons in exchange to sodium ions. The interaction of NHE1 with calcineurin B homologous proteins (CHPs) is important for full functionality of this transporter. Inherited loss-of-function mutations that are linked to diseases are known for NHE1 as well as for a CHP isoform. Three human CHP isoforms exist. They bind to the cytoplasmic regulatory domain of NHE1. The exact binding site of CHP3 has been debated. In order to clarify this question and to gain insights in the so far unknown energetics of the interaction, we produced and purified human CHPs as well as defined regions of the regulatory domain of NHE1 and characterized their interaction with biochemical and biophysical methods. We found that CHP3 binds with high affinity and specifically to the N-terminal stretch of the regulatory domain of NHE1 - the CBD region - and not to its C-terminal part - the CTD region. We will also present the characterization of the interaction with CHP1 and CHP2. The data provide insights into the molecular mechanisms that underlie the regulatory interaction between CHPs and NHE1.

P7 a/12 A specific mechanism based on alternative 5’UTRs controls the VDAC1 translation in D. melanogaster Angela Messina1,3, L. Leggio1,2, F. Guarino2,3, A. Magrì1, S. Reina1,2, V. Specchia4, F. Damiano4, M.F. Tomasello5 1Department of Biological, Geological and Environmental Sciences, University of Catania, Catania, Italy 2Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy 3National Institute of Biostructures and Biosystems (INBB), Catania, Italy 4Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce, Italy 5IBB-CNR, Institute of Biostructure and Bioimaging, Section of Catania, Catania, Italy

The eukaryotic porin, also called Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane [1]. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L [2]. The main VDAC isoform, called VDAC1 (or Porin1), is expressed from the first gene of the cluster [3]. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5′ untranslated regions (UTRs) [2]. We studied the influence of the two 5′ UTRs, 1A-5′ UTR and 1B-5′ UTR, on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained expressing in D. melanogaster SL2 cells constructs containing the two 5′ UTRs upstream of the luciferase reporter gene. A short region of 15 nucleotides in the 1B-5′ UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. Overall, our results suggest that contacts between the 5′ UTR and specific 18S rRNA sequences could modulate the translation of the alternative Drosophila 1B-VDAC mRNA [4]. Interestingly, this mechanism is independent of the coding region cloned downstream of the 5′-UTR. Furthermore, our results show that the unproductive 1B-VDAC mRNA is able to respond to 1A-VDAC transcript levels, and thus it might work as a molecule signaling the need for activation of mitochondrial biogenesis [4].

References 1. V. Shoshan-Barmatz et al., Mol Aspects Med 31(3) (2010) 227-85 2. M. Oliva et al., Mol Gen Genomics 267 (2002) 746–756 3. A. Messina et al., FEBS Lett. 384(1) (1996) 9-13 4. L. Leggio et al. Sci Reports (2018) 8:5347

P7 a/13 Role of the essential protein LETM1 in mitochondrial cation regulation Karin Nowikosky, Shane Austin, Christina Pfeiffer Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Wien, Austria

Both high throughput screening and targeted research have demonstrated the essentiality of LETM1 for cell fitness and survival [1, 2]. The undisputed vital function of LETM1 is to maintain the mitochondrial osmotic balance. Whether this occurs by extrusion of excess mitochondrial K+ or Ca2+ against their electrophoretic uptake, was a matter of disputes that lasted over the last ten years, While mitochondrial calcium plays an enormous role in the regulation of metabolism and cell death, the role of mitochondrial potassium has not received much attention. The latest achievements in the field of mitochondrial calcium were the molecular identification of the mitochondrial uniporter and co-factors and of the sodium/calcium exchanger. The current consensus is that sodium/calcium exchanger is the most important mitochondrial Ca2+ release pathway. So the burning question is why a calcium/proton exchanger with a subsidiary role would still be essential to sustain viability. We present the most important findings on LETM1 and demonstrate that as a key regulator of mitochondrial K+ it also affects mitochondrial Ca2+ fluxes.

[1] V.A. Blomen, P. Majek, L.T. Jae, J.W. Bigenzahn, J. Nieuwenhuis, J. Staring, R. Sacco, F.R. van Diemen, N. Olk, A. Stukalov, C. Marceau, H. Janssen, J.E. Carette, K.L. Bennett, J. Colinge, G. Superti-Furga, T.R. Brummelkamp, Gene essentiality and synthetic lethality in haploid human cells, Science, 350 (2015) 1092-1096. [2] T. Wang, K. Birsoy, N.W. Hughes, K.M. Krupczak, Y. Post, J.J. Wei, E.S. Lander, D.M. Sabatini, Identification and characterization of essential genes in the human genome, Science, 350 (2015) 1096-1101.

P7 a/14 Novel psoralen-derivatives with increased solubility in cancer treatment Roberta Peruzzo1, Michele Azzolini2,3, Matteo Romio4, Katrin A Becker5, Andrea Mattarei4, Mario Zoratti2,3, Erich Gulbins5, Cristina Paradisi4, Luigi Leanza1, Ildikò Szabò1,2 1Department of Biology, University of Padua, Padua, Italy 2CNR Institute of Neurosciences, Padua, Italy 3Department of Biomedical Sciences, University of Padua, Padua, Italy 4Department of Chemical Sciences, University of Padua, Padua, Italy 5Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany Charles Perrault

Ion channels are emerging as new oncological targets. Indeed, several ion channels show a different expression pattern in normal and cancer cells. The potassium channel Kv1.3 shows multiple sub-cellular localization and it is functional in both the plasma membrane and the inner mitochondrial membrane. Pharmacological inhibition of the mitochondrial channel (mtKv1.3), but not of the plasma membrane channel by membrane permeant blockers, Psora-4, PAP-1 and clofazimine, triggered apoptosis in different cancer cells. Cell death occurred even in the absence of Bax and Bak, by inducing mitochondrial membrane depolarization, production of mitochondrial ROS and release of cytochrome c. Downregulation by siRNA of Kv1.3 prevented all these effects, indicating specificity. Since membrane permeant Kv1.3 inhibitors are characterized by poor water solubility, in order to increase their bioavailability as well as their solubility, we have recently synthesized a few more soluble PAP-1 derivatives. The new derivatives have been found to selectively kill cancer cells in vitro and even in vivo in a mouse melanoma preclinical model, without inducing any side effect. Surprisingly, these new compounds were also able to inhibit Complex I activity, suggesting a possible physical interaction between mtKv1.3 and Complex I.

[1] Leanza et al. Inhibitors of mitochondrial Kv1.3 channels induce Bax/Bak-independent death of cancer cells, EMBO Mol Med (2012) [2] Leanza et al., Mitochondrial ion channels as oncological targets, Oncogene (2014) [3] I. Szabò and M. Zoratti, Mitochondrial channels: ion fluxes and more, Phys. Rev (2014) [4] Pardo and Stühmer, The roles of K+ channels in cancer, Nat Rev Cancer (2014) [5] Leanza et al., Targeting a mitochondrial potassium channel to fight cancer, Cell Calcium (2015) [6] Peruzzo et al., Impact of intracellular ion channels on cancer development and progression, Eur. Biophys. J. (2016) [7] Leanza et al., Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo, Cancer Cell (2017)

P7 a/15 Following kinetic processes of membrane proteins in real time using fluorescence microscopy Thomas Schick1,2, Olivier Biner1,2, Christoph von Ballmoos1 1University of Bern, Department of Chemistry and Biochemistry, Bern, Switzerland 2Graduate School for Cellular and Biomedical Sciences, Bern, Switzerland

We have employed rapid fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins (MPs) into a common lipid bilayer, containing different proteins. With this technique, we functionally co-reconstituted different oxidases and ATP synthase from E. coli into unilamellar vesicles. Furthermore, we have used the same methodology to reconstitute MPs into giant unilamellar vesicles (GUVs) by fusion of small positively charged proteoliposomes with negatively charged GUVs. With the help of functional assays and fluorescence microscopy, we have confirmed the functional incorporation of three different MPs into the GUV membrane.[1] Our next goal was to measure transmembrane transport processes of MPs in GUVs directly under the fluorescence microscope. In comparison to small liposomes, GUVs do not suffer from disadvantages like high membrane curvature and small interior volume (concentrations change quickly in a 50 nm liposome). Furthermore, GUVs are large enough to be monitored by fluorescence microscopy. Here, we present our current efforts to in situ monitor transmembrane transport events. These are on one hand proton pumping events of MP complexes like the ATP synthase or cytochrome c oxidase employing soluble, lipid anchored pH sensitive probes. On the other hand we want to follow other transmembrane transports such as sodium/proton antiport using pH- or sodium sensitive dyes or the translocation of sugar molecules by converting sugar import into a fluorescent read out, easily detectable using optical microscopy.

[1] O. Biner, T. Schick, Y. Muller, C. von Ballmoos, Delivery of membrane proteins into small and giant unilamellar vesicles by charge- mediated fusion, FEBS letters, (2016).

P7 a/16 Loss of mitochondrial phosphate carrier SLC25A3 in skeletal muscle provides evidence for compensatory but lower capacity phosphate uptake into mitochondria Erin L. Seifert, Lauren Anderson-Pullinger, Valentina Debattisti, Yana Sharpadskaya, György Hajnόczky MitoCare Center for Mitochondrial Imaging Research and Diagnostics, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, USA

The mitochondrial Pi carrier (PiC) encoded by SLC25A3 has been assumed to be the main or only transport mechanism of Pi uptake into mitochondria for ATP synthesis and buffering of matrix Ca2+. This can now be directly tested with the recent availability of in vivo models of altered PiC expression. We depleted PiC in adult mouse skeletal muscle (skm) for the first time using a mouse with floxed SLC25A3 alleles coupled to an skm-specific Tamoxifen-inducible Cre. Despite >95% depletion of PiC in skm, mice with relatively acute PiC loss (3 wks) were indistinguishable from littermate controls in terms of body and skm weight, and home cage behavior, though serum lactate was mildly elevated (3 mM vs. 1.8 mM in control). When challenged to an aerobic treadmill running regime, PiC-depleted mice could run at increasing speed, though they fatigued sooner (50 mins of running vs. 75 mins in control). Maximal oxidative phosphorylation (oxphos), tested as O2 consumption with saturating [succinate] and [ADP] in skm mitochondria, was absent despite higher maximal uncoupled O2 consumption, observations that are fully consistent with a Pi limitation on oxphos. However, when pyruvate (+malate) or fatty acid (+malate) was supplied, oxphos was stimulated, reaching 35% and 50% of control, respectively. Ca2+ uptake by PiC-depleted skm mitochondria was unimpaired, though matrix free [Ca2+] was higher for the same level of Ca2+ uptake indicating lesser buffering of matrix Ca2+. Finally, 3-wk PiC loss was not accompanied by higher transcript levels of genes of one carbon metabolism or of most other genes that together characterize a stress response reported in mouse and cell models of mitochondrial DNA depletion. Altogether our observations suggest that acute PiC loss can be compensated by an alternative route of Pi transport into mitochondria in skm. Yet, lesser ATP synthesis and matrix Ca2+ buffering capacity occur and likely contribute to early fatigue. Funding: R01 GM123771 to ELS and GH.

P7 a/17 Purification of a seven-subunit Mrp-type sodium-proton antiporter Julia Steiner, Leonid Sazanov Institute of Science and Technology Austria

Multi resistant and pH homeostasis proteins (Mrp) are a type of monovalent cation-proton exchangers distributed across a wide phylogenetic range [1]. These antiporters are integral membrane proteins and belong to the group of secondary active transporters. Mrp antiporters are involved in pH homeostasis, cholate efflux and resistance and in the pathogenesis of Pseudomonas aeruginosa, a leading cause of nosocomial infections in hospitals. What sets the Mrp-type antiporter apart from other sodium-proton antiporters is that it is composed of seven subunits, MrpA-G in contrast to other cation-proton antiporters, which function as single membrane proteins. Moreover, Mrp displays no homology to other monovalent cation-proton antiporters but interestingly MrpA, -D and -C show primary sequence similarity to the subunits NuoL, NuoM/N and NuoK, respectively, of complex I, which is part of the electron transport chain in mitochondria [2]. This homology reinforces the hypothesis that the present day complex I is built from distinct smaller units with specialized functions. The overall goal of this project is the ascertainment of the protein structure of the hetero-oligomeric sodium-proton antiporter Mrp using cryo-electron microscopy. The structure will help to obtain deeper insights into the transport cycle of sodium-proton antiporters. Moreover, it will give valuable knowledge about the evolutionary relationship with complex I and thus about the functional mechanism of its antiporter-like subunits. The poster shows the expression and purification of Mrp as well as screening of different conditions to obtain sufficient, soluble and stable protein applicable for further structural characterization.

1. M. Ito, M. Morino T. A. Krulwich, Mrp antiporters have important roles in diverse bacteria and archaea, Frontiers in Microbiology 8 (2017) 2325 2. L. A. Sazanov, A giant molecular proton pump: structure and mechanism of respiratory complex I, Nature Reviews Molecular Cell Biology 16 (2015) 375-88

P7 a/18 Delivery of functional genetic code into human mitochondrion Natalya Verechshagina1, Daria Kurochkina1, Natalya Nikitchina1, Nikita Shebanov1, Masashi Tanaka2, Konstantin Orishchenko1,3, Ilia Mazunin1 1 Laboratory of Molecular Genetics Technologies, Immanuel Kant Baltic Federal University, Kaliningrad, Russia 2 Department of Clinical Laboratory, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan 3 Laboratory of Cell Technologies, Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia

Scientists have known a lot about protein but next to nothing about nucleic acid import systems into human mitochondrion. As a consequence, they can create and use protein-only systems such as mitochondria-targeted restriction endonucleases, TAL-effector nucleases and zinc-finger nucleases but not, for instance, RNA-guided endonucleases. Moreover to activate homology-directed repair or homology recombination systems the DNA molecule should be imported into mitochondrion as a template for that. We propose that dsDNA could be imported into human mitochondrion in link with DNA-binding protein using the TIM/TOM pathway. To test this speculation we have created recombinant protein which consists of a TAL effector fused with a mitochondrial target signal in its N-tail. The system called mitoTALE includes two parts: the modified DNA-binding protein and a binding site at the 5'-end of the imported dsDNA molecule. We also propose that the negative charge of dsDNA molecule will be enough to use the common cationic lipid nucleic acid transfection reagents. Another way to deliver the mitoTALE-dsDNA complex into the cells is fusing mitoTALE with an engineered super negatively charged GFP - if dsDNA is too short and charge is not sufficiently negative.

P7 a/19 Dynamics of Mitochondrial Calcium Uniporter expression at different gestational age Polina Vishnyakova1, Nadezhda Tarasova1,2, Maria Volodina1,3, Iulia Sukhanova1,4, Natalya Kan1, Mikhail Vysokikh1,4, Gennady Sukhikh1 1National Medical Research Center for Obstetrics, Gynecology and Perinatology, Moscow, Russian Federation 2Molecular Medicine Institute, I.M. Sechenov First Moscow State Medical University, Moscow, Russia Federation 3Centre for Cognition and Decision Making, National Research University Higher School of Economics, Moscow, Russian Federation 4Moscow State University, Moscow, Russian Federation

Mitochondria play a crucial role in buffering and regulation of cytoplasmic calcium (Ca2+) concentration. That helps to regulate a number of cellular events, including apoptosis, Ca2+-mediated signaling and activity of Krebs cycle enzymes. Mitochondrial calcium uniporter, located in the inner mitochondrial membrane, transports Ca2+ to the mitochondrial matrix. Mitochondrial calcium uniporter consists of pore-forming subunit (MCU) and a few regulatory subunits (MICU1, MICU2, MCUb, SMDT1). Calcium transport through the placenta during pregnancy plays an important role in the correct development of the fetus. Thus, in the present study we estimated the relative expression level of mitochondrial calcium uniporter subunits in two components of feto-maternal system: placenta and myometrium. We obtained placental and myometrial tissues from 50 women, divided into four groups depending on gestational age at the moment of delivery: 22-27, 28-32, 33-36 and 37-40 (control group) weeks. Samples were analyzed by Real-Time Quantitative PCR and Western blot. Our results revealed an intriguing pattern of MCU expression level and it`s regulatory subunits during the gestation. Our findings allow to assume an important role of placental mitochondria in Ca2+ buffering for more effective Ca2+ transport at the third trimester. In myometrium we found the opposite changes mRNA in MCU, MCUb and SMDT1 expression. This may indicate the role of the mitochondrial calcium stores in the organization of coordinated contractions of the myometrium during labor. Calcium metabolism contributes to the pathogenesis of one of the «great obstetrical syndromes» - preterm birth. Thus a better understanding of the Ca2+ transport mechanisms could contribute to the search for new targets and therapy.

This work was supported by the Russian Foundation for Basic Research with grant RFBR No. 16-29-07436 and RFBR No. 17-54- 570004. Work of P.V. was supported by the President's scholarship (SP-4132.2018.4).

P7 a/20

Metal ion-substituted protoporphyrins as a tool to study regulation of the activity of mitoBKCa channel by hydrogen sulfide Agnieszka Walewska, Piotr Koprowski, Adam Szewczyk Laboratory of Intracellular Ion Channels, Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

Mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is one of the main potassium channels localized in the inner membrane of mitochondria. MitoBKCa is formed by a DEC splice variant of KCNMA1 gene and it is a tetrameric protein composed of four α subunits. Each α subunit consists of a short N-terminus, seven transmembrane segments and a large C-terminus containing + two RCK (regulating conductance of K ) domains located in the mitochondrial matrix. Various modulators of the activity of mitoBKCa are known, including activator such as carbon monoxide and inhibitor heme or its oxidized form hemin (Fe(III)-protoporphyrin IX).

It is known that the activity of mitoBKCa, similarly to that of plasmalemmal BKCa channels is inhibited by hemin, which binds to –CXXCH– motif located between two RCK domains. It was also previously shown that a gasotransmitter hydrogen sulfide (H2S) increased plasmalemmal BKCa channel activity. H2S mechanism of action includes persulfide formation with -SH groups of cysteines, sulfide-metal interactions in heme proteins and redox reactions. In this study, we performed patch-clamp experiments on mitoplasts derived from mitochondria of astrocytoma U-87 MG cells to measure activity of single mitoBKCa channels.

First, we applied sodium hydrosulfide (NaHS) as a H2S donor, what had no impact on mitoBKCa channel activity. Next, we applied hemin and other metal ion-substituted protoporphyrins IX followed by NaHS. Here we found that NaHS activated the hemin-inhibited mitoBKCa channels but also for example channels inhibited by Zn(II)-protoporphyrin IX. On the other hand, Mg(II)-protoporphyrin IX did not influence the channel activity. In summary, we observed various and complex effects of applied protoporphyrins on the activity of mitoBKCa, which seemed to depended on the metal center ion.

This work was supported by Polish National Science Center, grant no. 2015/17/B/ NZ1/02496.

P7 a/21 The structure-function analysis of mitochondrial calcium uniporter (MCU) using a yeast expression system Takenori Yamamoto1,2, Mizune Ozono1,2, Akira Watanabe1,2, Kosuke Maeda1,2, Yusuke Ido1,2, Akiko Yamada3, Hiroshi Terada4, Yasuo Shinohara1,2 1Institute for Genome Research, Tokushima University, Tokushima, Japan 2Faculty of Pharmaceutical Sciences, Tokushima University, Tokushima, Japan 3School of Detistry, Tokushima University, Tokushima, Japan 4Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan

The mitochondrial calcium uniporter (MCU) complex is a highly selective calcium channel. This complex consists of a pore-forming subunit, MCU, and its regulatory subunits, e.g., EMRE and MICUs, etc. MCU has DXXE motif located on the outer surface of the inner membrane; the motif is critical for the calcium uptake function. However, the functional roles of the amino acid regions other than DXXE motif in MCU have not yet been well understood. We here showed that two coiled-coil regions in MCU, which are highly conserved among various species of MCUs, were critical for the mitochondrial calcium uptake function. Although yeast mitochondria lack calcium uptake function, the expression of mammalian MCU and EMRE enables the reconstitution of the calcium uptake function in yeast mitochondria [1]. Using the yeast reconstitution system of calcium uptake function, we found that the mutations of two coiled-coil regions of mouse MCU caused the significant decrease of the MCU- and EMRE-dependent calcium uptake into mitochondria. The decrease of calcium uptake function by the mutations of coiled-coil regions was independent of the functions of the regulatory subunits other than MCU. These findings suggested that the two coiled-coil regions of MCU play an important role in the pore-forming functions of MCU itself.

[1] T Yamamoto, R Yamagoshi, K Harada, M Kawano, N Minami, Y Ido, K Kuwahara, A Fujita, M Ozono, A Watanabe, A Yamada, H Terada, Y Shinohara. Analysis of the structure and function of EMRE in a yeast expression system. Biochim Biophys Acta. 1857 (2016) 831-839

P7 a/22

Role of dcu C4-dicarboxylate transporters in H2 production during fermentation of glucose or glycerol Lusine Karapetyan1, Satenik Mirzoyan1, Antonio Valle, Jorge Bolivar, Armen Trchounian1,2, Karen Trchounian1,2 1Department of Biochemistry, Microbiology and Biotechnology, Yerevan State University, Yerevan, Armenia 2Scientific-Research Institute of Biology, Yerevan State University, Yerevan, Armenia 3University of Cadiz, Cadiz, Spain

There are various protein complexes in the bacterial cell membrane, such as proton F0F1-ATPase and dcu family carriers. dcu family + + carriers are responsible for transporting C4-dicarboxylates (succinate, fumarate, malate, etc.), in parallel transferring H ions. H ions are also transported across the membrane by other systems, including the proton ATPase. To understand the relationship between the work of these complexes and their effects on H2 production E. coli wild type and mutants with defects in dcu system have been studied. Bacterial were grown at pH 6.5 and 5.5 during fermentation of glucose or glycerol. In the case of glucose utilization at pH 6.5, in dcuD mutant H2 production rate was increased by 26% compared to wild type and other mutants. Whereas, when glycerol is utilized, the wild type H2 production rate is ~ 50% more compared to other mutants. At pH 5.5 in the presence of glucose or glycerol H2 production rate was similar. The relationship of the proton ATPase and dcu transporter has been studied using 0.2 mM N,N’-dicycloheylcarbodiimide

(DCCD) specific inhibitor of proton ATPase under fermentative conditions. As a result, it has inhibited the production of H2 in both pHs with glucose and glycerol. An exception was the dcuACB mutant, which grown in the presence of glucose at pH 6.5, in which the production of H2 was inhibited ~ 90% in case of high DCCD concentration. It can be assumed that D subunit of dcu is related to proton

ATPase and the H2 producing hydrogenase 3, DcuD and proton ATPase are interacting together for regulating and maintaining the proton and h2 cycling and thus proton motive force generation.

P7 a/23 Karen Trchounian1,2, Bella Hakobyan1, Satenik Mirzoyan2, Armen Trchounian1,2 1Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology 2Scientific-Research Institute of Biology, Yerevan State University, Yerevan, Armenia

Understanding Escherichia coli formate channels working direction during fermentation of the mixture of glucose, glycerol and formate at pH 7.5

Escherichia coli generates molecular hydrogen (H2) via oxidation of formate with formate hydrogen lyase complex. It is known that E. coli transports formate through FocA and FocB formate channels. During anaerobic fermentation FocA has an important role in regulating intracellular formate level during anaerobic fermentation. Role of FocB is not clear yet. It is shown that E. coli focA, focB formate channels depending on the conditions (external pH, carbon sources) work either in exporting or importing directions. Particularly at pH 7.5 when cells were grown in the presence of the mixture of glucose and glycerol when in the assays glucose was added mainly in focB single and focA focB double mutants H2 production was increased compared to wild type suggesting that in all cases channels are working towards exporting direction. But in glycerol assays H2 production in focA was increased ~3-fold and in focB decreased ~1.7-fold. Interestingly in externally added formate assays H2 production was opposite in the same mutants compared to glycerol assays. Only in focA focB double mutant H2 production was increased ~ 2.5 fold. When cells were grown in the presence of mixture of glucose, glycerol and formate and in the assays glucose was added mainly in focA and focB single mutants H2 production increased ~3-fold and 2.5-fold, respectively, compared to wild type. In contrast to glucose and glycerol conditions, here in glycerol assays no any significant role of formate channels has been detected. But in formate assays in focA single and focA focB double mutants H2 production was increased ~1.3- and ~1.7-fold respectively, compared to wild type. Moreover, in focB mutant it was decreased ~2-fold. Taken together the data suggest that the presence of external formate changes the working direction of formate channels. This might be due to maintaining, H2 and proton cycling and thus cytoplasmatic pH.

P7 b/1 The effect of 2-ketobutyrate on mitochondrial substrate level phosphorylation David Bui, Christos Chinopoulos Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

The reaction catalyzed by succinate-CoA ligase in the mitochondrial matrix yields a high-energy phosphate when operating towards hydrolysis of the thioester bond of succinyl-CoA. This process is more widely known as mitochondrial substrate-level phosphorylation (mSLP). The catabolism of several amino acids converge to succinyl-CoA but through different biochemical pathways. Among them, threonine and methionine catabolize to succinyl-CoA through the common intermediate, 2-ketobutyrate (2-kb). During the course of this pathway, 2-kb passes obligatorily through an ATP-consuming step substantiated by propionyl-CoA carboxylase. In the present work we tested the hypothesis that catabolism of 2-kb negates mSLP due to this ATP-consuming propionyl-CoA carboxylase step. mSLP, by means of recording the directionality of the adenine nucleotide translocase while measuring membrane potential in the presence of rotenone, was evaluated in isolated mouse liver and brain mitochondria. 2-kb supported the development of only a moderate membrane potential, compared to NADH or FADH2-linked substrates. The 2-kb-induced membrane potential was inhibited by rotenone, atpenin and arsenite, implying the involvement of complex I, complex II and a dehydrogenase –most likely branched chain keto-acid dehydrogenase, respectively. Co-addition of 2-kb with NADH- or FADH2-linked substrates yielded no greater membrane potential than in the presence of substrates alone. However, in the presence of NADH-linked substrates, 2-kb prevented mSLP in a dose-dependent manner. Our results imply that despite that 2-kb eventually leads to succinyl-CoA formation, obligatory metabolism through propionyl-CoA carboxylase associated with the expenditure of ATP, abolishes mSLP. Thus, the provision of metabolites converging to 2-kb may be a useful means for manipulating mSLP without using pharmacological or genetic tools.

P7 b/2 TRAP1 regulation in cancer metabolism: identification of new interactors Giuseppe Cannino1, Ionica Masgras1, Marco Gaspari2, Paolo Bernardi1, Andrea Rasola1 1Dipartimento di Scienze Biomediche, Università di Padova, Italia 2Dipartimento di Medicina Sperimentale e Clinica, Università della Magna Grecia di Catanzaro, Italia

The metabolic rewiring of cancer cells is a multifaceted process, and how mitochondria contribute to it remains poorly defined. We demonstrated that the mitochondrial chaperone TRAP1 binds and inhibits succinate dehydrogenase. The consequent accumulation of succinate causes HIF1α stabilization even under normal oxygen tension, setting a pseudohypoxic phenotype instrumental in prompting tumor growth. In addition, the mitochondrial fraction of ERK phosphorylates TRAP1 enhancing its oncogenic effect. In analogy with its cognate chaperone Hsp90, it is possible that TRAP1 has multiple interacting partners endowed with relevant functions in the oncogenic process. In order to identify novel TRAP1 interactors, we used a mass spectrometry analysis on TRAP1 immunoprecipitated by a human glioblastoma cell model, using cells where TRAP1 was knocked-out with a CRISPR/Cas9 approach as a negative control. Among the potential TRAP1 partners fished out by MS, we found proteins involved in OXPHOS, tricarboxylic acid cycle and glutaminolysis. Interestingly, we found some subunits of the ATP synthase, the central enzymatic complex in the process of energy conservation. Our preliminary data indicate that the absence of TRAP1 markedly inhibits the activity of ATP synthase dimers.

P7 b/3 Mitochondria ROS blocker OP2-113 downregulates the insulin receptor substrate-2 (IRS-2) and inhibits lung tumor growth Nivea Dias Amoedo1,2, Laetitia Dard1,2, Mariana Figueiredo Rodrigues1,2, Benoît Rousseau1,3, Julien Izotte1,3, Nathalie Dugot-Senan1,4, Stéphane Claverol1,5, Marc Bonneu1,5,6, Didier Lacombe1,2,7, Rodrigue Rossignol1,2,8 1INSERM U1211, Bordeaux Bordeaux, France 2Bordeaux University, Bordeaux, France 3Transgenic Animal facility A2, University of Bordeaux 4TBMCORE INSERM US005-CNRS 3429 Bordeaux, France 5Functional Genomics Center (CGFB), Proteomics Facility, Bordeaux, France 6Bordeaux-INP Avenue des Facultés 33405 Talence Cedex, France 7CHU Bordeaux, Haut-Lévèque Hospital, Pathology department, Bordeaux 8CELLOMET, Functional Genomics Center (CGFB), Bordeaux, France

Lung cancer is the leading cause of cancer death worldwide and the mean 5-year survival rate after a non-small cell lung cancer (NSCLC) diagnosis is approximately 18%. Genetically defined precision medicine remains limited to small groups of NSCLC patients, fostering the need to identify novel targets and approaches to manage lung cancer. Among the different emerging strategies for cancer therapy, targeting the production of reactive oxygen species by the mitochondrion (mtROS) may be promising since mtROS contribute to cancer cell growth and metastasis. Here, we evaluated the impact of the OP2-113 mtROS blocker (OP2-Drug SAS) on lung cancer cells biology in vitro and in vivo using A549 human lung adenocarcinoma cells. First, OP2-113 altered cell proliferation rate and migration capacity in vitro, without any effect on cell respiration. The effect of OP2-113 was further investigated in vivo using an orthotopic mice model of human luciferase-expressing A549 lung adenocarcinoma cells. Mice were injected daily with 20mg/kg OP2- 113 and tumor growth was monitored in vivo using bioluminescence imaging. We observed a progressive inhibition of tumor growth in the group of mice treated with OP2-113. After three weeks, tumor size was reduced by a factor of three fold, with p<0.05. The underpinning mechanisms involved the reprogramming of A549 cells proteome by OP2-113. In particular, the OP2-113 treatment induced a strong downregulation of the candidate driver oncogene insulin receptor substrate-2 (IRS-2) and this effect was not observed with the mt-ROS scavenger MitoTEMPO. IRS2 was previously associated with cancer cells proliferation and survival but currently there is no anticancer agent that target IRS2. Our proof-of-concept preclinical study demonstrate that OP2-113 inhibits IRS2 expression and alters lung cancer cells growth in vitro and in vivo.

P7 b/4 Mitochondrial Src Kinase, A Potential Therapeutic Target for Breast Cancer Marie-Ange Djeungoue-Petga, Olivier Lurette, Stéphanie Jean, Marine Bou, Etienne Hebert-Chatelain Department of Biology, Université de Moncton, Moncton, Canada

Breast cancer is the second cause of cancer mortality for Canadian women. Several studies have shown that the tyrosine Src kinase is overexpressed in breast cancer tissue, where it is associated to increased invasiveness and metastatic potential of breast cancer cells, and with lower survival of patients. Several Src inhibitors are actually in clinical development for the treatment of cancer and tumors. However, their efficacy appears to be modest or different among the different subtypes of breast cancer. We demonstrated that Src kinase is localized in mitochondria (mtSrc), where it impacts on the organelle activity. The main goal of our study was to characterize the specific role of mtSrc in the metabolism and tumorigenic phenotype of breast cancer cells. To address this, we first evaluated the levels and activity of mtSrc in different breast cancer subtypes. Our results show that mtSrc is more active in breast cancer cells from the triple negative subtype. To explore the specific role of mtSrc, we expressed a Src mutant specifically targeted to mitochondria in triple negative breast cancer cells. We observed that mtSrc induces tyrosine-phosphorylation of several mitochondrial proteins, which is sufficient to impair oxygen consumption in breast cancer cells. Apoptosis and necrosis are also triggered in breast cancer cells upon expression of mtSrc Surprisingly, these effects are not mediated by the cytochrome c oxidase subunit II, which was previously shown to be targeted by mtSrc signaling breast cancer cells. Overall, our study demonstrate that mitochondrial Src kinase modulates metabolic and neoplasic phenotypes of breast cancer cells. Through the identification of new pathophysiological processes important for tumorigenesis, this work will benefit the development of potent and more specific therapies for breast cancer.

P7 b/5 Decisive role of mitochondrial substrate level phosphorylation on the survival of glutaminolytic cancer cells Judit Dóczi, Gergely Horváth, László Tretter, Vera Ádám-Vizi, Christos Chinopoulos Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary

Cancer cells require altered metabolism to efficiently utilize nutrients such as glucose and glutamine for intensive proliferation. Murine cancerous cell lines were categorized as glycolytic or glutaminolytic according to their specific characteristics of oxygen consumption rate and extracellular acidification rate after glucose or glutamine withdrawal. Survival of cell lines was analyzed after abolition of mitochondrial substrate level phosphorylation by silencing the expression of succinyl-coA ligase invariant G1 subunit (SUCLG1) by siRNA targeting. Survival of cancerous cell lines categorized as glutaminolytic was dramatically decreased 48 hours after silencing the expression of SUCLG1 subunit. Similar drop of survival rate in cell lines categorized as glycolytic was not observed 48 hours after silencing of SUCLG1 expression.

P7 b/6 Decisive role of mitochondrial substrate level phosphorylation on the survival of glutaminolytic cancer cells Pauline Esteves1,2, Laetitia Dard1,2, Aurélia Brillac1,2, Christophe Hubert1,2, Benoît Rousseau1,3, Elodie Dumon1,2, Julien Izotte1,3, Jean-William Dupuy1,4, Marc Bonneu1,4,5, Didier Lacombe1,2, Nivea Amoedo1,2 and Rodrigue Rossignol1,2,6 1Bordeaux University, Bordeaux, France 2INSERM U1211, Bordeaux, France 3Transgenic Animal Facility A2, University of Bordeaux 4Functional Genomics Center (CGFB), Proteomics Facility, Bordeaux, France 5Bordeaux-INP Avenue des Facultés 33405 Talence Cedex, France 6CELLOMET, Functional Genomics Center (CGFB), Bordeaux, France

The basic understanding of the biological effects of eukaryotic translation initiation factors (EIFs) remains incomplete. Here, we analyzed the function of EIF3F, which is commonly found to be overexpressed in human lung adenocarcinoma, and discovered that EIF3F promotes oxidative phosphorylation, cell migration and metastasis in vivo. The underpinning molecular mechanisms involved the upregulation of a cluster of 34 metastasis-promoting genes including Snail2 (SLUG), as revealed by proteomics combined with immuno- affinity purification of EIF3F and ChIP-seq/Q-PCR analyses. The interaction between EIF3F and Signal Transducer and Activator of Transcription 3 (STAT3) controlled the EIF3F-mediated increase in Snail2 expression and cellular invasion, which were specifically abrogated using the STAT3 inhibitor nifuroxazide. Our findings demonstrate the role of EIF3F in the molecular control of oxidative phosphorylation, cell migration, invasion and metastasis.

P7 b/7 Application of stable isotope metabolomics to discover new drug targets for breast cancer treatment Aleksandr Klepinin2, Song Zhang1, Ivan Vuckovic1, Tuuli Kaambre2, Slobodan Macura1, Petras Dzeja1 1Departments of Cardiovascular Medicine and Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 2National Institute of Chemical Physics and Biophysics, Tallinn, Estonia

Stable isotope metabolomics offers new perspectives to discover alterations in the dynamics of energetic and metabolic signaling circuits in cancer cells. Adenylate kinase (AK) and creatine kinase (CK) are ubiquitous enzymes which have critical role in cell energy metabolism and ATP distribution. In cells diminished CK pathway can be rescued by AK pathway which demonstrates the flexibility of phosphotransfer network. However, deletion of the bottleneck AK2 isoform in intermembrane space cannot be compensated and it is embryonically lethal. Stable isotope 18O-based dynamic metabolomics approach was applied to determinate changes in AK metabolic flux by measuring ATP β-phosphoryl turnover rates. Phosphocreatine (PCr), which reflects CK pathway, was detected by HPLC. For this purpose, 3 different cell lines were used: two breast cancer cell lines MCF7 and MDAMB231 and MCF10A was used as a control. Labeling experiments demonstrate that in MCF10A the rate of 18O incorporation into ATP β-phosphoryls, reflecting AK velocity, was close to cellular ATP turnover rate (γ-ATP labeling). Moreover, high PCr level was also determined in breast epithelial cells, indicating that the epithelia cells use both AK and CK pathway to maintain cell energy homeostasis. However, in breast cancer cells diminished PCr level was associated with downregulation of mitochondria CK (MtCK). Furthermore, labeling experiments showed that in breast cancer β-ATP[18O] labeling was 20-30% lower compared to γ-ATP[18O] reflecting diminished adenylate kinase flux which was associated with downregulation of AK1. Diminished CK pathway in breast cancer cells was compensated by overexpression of AK2 and AK6. Thus, during breast epithelia cell malignant transformation MtCK may be replaced by AK2. In conclusion, our study detected previously unforeseen metabolic transitions in cancer cells and revealed that AK network, especially AK2 and AK6 isoenzymes could be potential drug targets for breast cancer treatment.

P7 b/8 Progression series of murine fibrosarcoma separates proliferative and invasive transformation characteristics Michaela Kripnerová1, Martin Leba2, Jitka Kuncová3, Martin Pešta1, Jiří Hatina1 1Institute of Physiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic 2Department of Cybernetics, Faculty of Applied Sciences, University of West Bohemia, Univerzitní 8, Plzeň, Czech Republic 3Institute of Physiology, Faculty of Medicine in Plzeň, Charles University, Czech Republic

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour, ranging from rather indolent lesions with good prognosis to rapidly metastasizing fatal tumours. We established a unique single background progression series of murine sarcoma cell lines: JUN2, slowly proliferating nonmotile and noninvasive; JUN3, rapidly proliferating, motile and invasive and JUN2fos3, with slow proliferation, but pronounced motility and invasiveness. Interestingly, it is especially the migratory ability and not that much the other transformation traits that determines the prevailing metabolic phenotype of the cells. For the highly motile cell lines JUN-2fos-3 and JUN-3, their dependency on oxidative phosphorylation is increased and, importantly, their respiration determined by high-resolution respirometry (O2k Oroboros, Austria) is significantly more effective than the respiratory chain of JUN-1 and JUN-2 cells. On the contrary, physiological respiration of the noninvasive and poorly transformed JUN-2 cells is very similar to the theoretical maximum utilization of oxygen and shows a very small reserve for uncoupling respiration. Besides being highly transformed, the JUN-3 cell line exhibits the highest anchorage-independent growth and sarcosphere forming capacity, and could provide a convenient source of sarcoma stem cells. We believe that this progression series could be very valuable for deciphering crucial aspects of sarcoma progression. Supported by the Czech Grant Agency project 17-17636S and the Specific Student Research Project no. 260394/2017 (CHU).

P7 b/9 Bioenergetic changes in breast cancer cells by lithocholic acid Edit Mikó1,8, András Vida1,8, Tünde Kovács1, Gyula Ujlaki1, György Trencsényi2, Judit Márton1, Zsanett Sári1, Patrik Kovács1, Anita Boratkó1, Zoltán Hujber9, Tamás Csonka3, Péter Antal-Szalmás4, Mitsuhiro Watanabe10, Imre Gombos11, Balazs Csoka12, Borbála Kiss7, László Vígh11, Judit Szabó5, Gábor Méhes3, Anna Sebestyén9, James J. Goedert13, Péter Bai1,6,7, 1Departments of Medical Chemistry 2Departments of Medical Imaging 3Departments of Pathology 4Departments of Laboratory Medicine 5Departments of Microbiology 6Research Center for Molecular Medicine 7Dermatology, Faculty of Medicine, University of Debrecen, Hungary 8MTA-DE Lendület Laboratory of Cellular Metabolism, Debrecen, Hungary 91st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary 10Department of Internal Medicine, School of Medicine, Keio University Endo, Fujisawa-shi, Kanagawa, Japan 11Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 12Deptartment of Anesthesiology, Columbia University Medical Center, New York, USA 13National Cancer Institute, National Institutes of Health, Bethesda, USA

Breast cancer is characterized by reductions in the diversity of the gut flora. We recently provided evidence that bacteria through the bacterial metabolites can modulate the behavior of breast cancer cells. We assessed the effects of a bacterial metabolite, lithocholic acid (LCA), in concentrations corresponding to its physiological tissue concentration on cellular models of breast cancer. LCA can only be synthesized by gut bacteria, therefore, LCA-evoked changes implicate the involvement of bacteria. LCA rendered breast cancer cells hypermetabolic that was evidenced by Seahorse analysis and by steady-state and pulse-chase metabolomics. Bioenergetic changes were in correlation with the reduction of other hallmarks of cancer (e.g. EMT, antitumor immunity and proliferation). We also provided evidence that bacterial LCA production is reduced in early breast cancer suggesting the involvement of low LCA levels in the pathogenesis of breast cancer.

Our work is supported by: NKFIH K123975, PD124110, FK128387, GINOP-2.3.2-15-2016-00006, EFOP-3.6.3-VEKOP-16-2017-00009) and the Hungarian Academy of Sciences (Momentum fellowship and PROJEKT2017-44)

P7 b/10 Exploring the roles of mitochondrial dynamics protein Opa1 in melanocytes and melanoma Omori Akiko1, Migliorini Domenico1, Rambow Florian2, Scorrano Luca1 1Venetian Institute of Molecular Medicine, Padova, Italy 2VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium

Analyzing mitochondrial function and mouse models of hair graying processes serve as useful systems to uncover mechanisms involved in aging and the maintenance of tissue homeostasis [1]. The processes how selective differentiation occurred in melanocyte stem cells, moreover, how mitochondrial dynamics are involved in melanocyte genesis during hair cycles have not been defined yet. Here we showed that Optic atrophy 1(Opa1), a main regulator for inner mitochondrial membranous fusion and cristae structures [2]., is essential for providing differentiated melanocyte during the hair follicle cycle. Indeed, Opa1 ablation in mouse reduced differentiated melanocytes during hair cycles and resulted in graying hair. Conversely, the increase of Opa1 in melanoma appears to be efficient for colony formation in soft agar assay. Thus, Opa1 can be an essential regulator of melanocytes and regarded as potential therapeutic target against melanoma.

1.Harris ML, FufaTD, Palmer JW, Joshi SS, Larson DM, Incao A, Gildea DE, Trivedi NS, Lee AN, Day CP, Michael HT, Hornyak TJ3, Merlino G; NISC Comparative Sequencing Program, Pavan WJ. A direct link between MITF, innate immunity, and hair graying. Plos Biology, (2018)16(5) 2. Cogliati S, Frezza C, Soriano ME, Varanita T, Quintana-Cabrera R, Corrado M, Cipolat S, Costa V, Casarin A, Gomes LC, Perales- Clemente E, Salviati L, Fernandez-Silva P, Enriquez JA, Scorrano L. Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cell. Sep 26;155(1)(2013)160-71.

P7 b/11 Mechanisms of coupling of glycolysis and oxidative phosphorylation in colon cancer. Do we progress in understanding of the Warburg effect? Zulfiya Orynbayeva2, Oya Altinok1,2, Juan L. Poggio2, David E. Stein2, Nathaniel W. Snyder3 1School of Biomedical Engineering and Health Sciences, Drexel University, Philadelphia (USA) 2Department of Surgery, Drexel University College of Medicine, Philadelphia (USA) 3Drexel University Autism Institute, Philadelphia (USA)

Since 1920th it is known that upon malignant transformations cells acquire an elevated glycolysis, the so-called Warburg Effect. However, growing number of studies demonstrate that many cancer types have highly oxidative mitochondria. Our group demonstrated that among those are prostate and colon cancer, which mitochondria have elevated metabolic parameters. Then, what is the purpose of the Warburg Effect and how cells benefit high glycolytic lactate production? The lactate shuttling mechanism is emerging as a central in bridging between glycolysis and OxPhos. This has been shown in heart, skeletal muscles and neurons, i.e. highly energetic tissues. The potential mechanisms that cooperate in glucose oxidation are the mitochondria malate-aspartate shuttle (MAS), consisting of glutamate- aspartate (Aralar/Citrin) and malate-α-ketoglutarate transporters, and lactate dehydrogenase which is supposedly localized in mitochondria inter-membrane space. Cooperation of these shuttles is required in generating of respiratory substrate NADH in mitochondria matrix which otherwise is membrane impermeable. We demonstrated that cancer cells employ MAS and mitochondrial LDH to control over NADH/NAD+ homeostasis to enable aerobic conversion of glycolytic lactate back to pyruvate to support elevated mitochondria activity.

P7 b/12 The phosphotransfer network and metabolic plasticity of colon cancer cells Ljudmila Ounpuu, Aleksandr Klepinin, Elen Niemeister, Heiki Vija, Tuuli Kaambre National Institute of Chemical Physics and Biophysics, Tallinn, Estonia

The specific eradication of cancer stem cells (CSCs) holds significant promise for colon cancer treatment. However, targeting of CSC surface biomarkers or stemness maintaining signalling pathways might also impair normal stem cells. Therefore more specific targets are required. Our study provides evidence that adenylate kinase (AK) enzymes may serve as specific regulators of metabolic remodelling in CSC-like cells. Previously we showed that AK network is up-regulated in post-operative colon cancer tissues [1] as well as in carcinoma derived CSC (2102Ep) [2] suggesting an increased AK activity as a distinct feature of malignant transformation. In the present study, we were able to re-establish AK homeostasis by inducing differentiation of colon cancer cells (Caco-2) with sodium butyrate. Interestingly, the availability of glutamine and glucose in cell culture medium affected AK activity upon cell differentiation. Moreover, the correlation between AK and lactate dehydrogenase activities under different cellular growth conditions was revealed. Our study introduces AK as a potential target for anti-CSCs therapies and highlights the importance of further elucidation of AK role in the metabolic reprogramming of colon cancer.

[1] V. Chekulayev, K. Mado, I. Shevchuk, A. Koit, A. Kaldma, A. Klepinin, N. Timohhina, K. Tepp, M. Kandashvili, L. Ounpuu, K. Heck, L. Truu, A. Planken, V. Valvere, T. Kaambre, Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes, Biochemistry and biophysics reports, 4 (2015) 111-125. [2] L. Ounpuu, A. Klepinin, M. Pook, I. Teino, N. Peet, K. Paju, K. Tepp, V. Chekulayev, I. Shevchuk, S. Koks, T. Maimets, T. Kaambre, 2102Ep embryonal carcinoma cells have compromised respiration and shifted bioenergetic profile distinct from H9 human embryonic stem cells, Biochim Biophys Acta, 1861 (2017) 2146-2154.

P7 b/13 Substrate oxidation differences in human glioma cells and their potential clinical significance Anna Sebestyén1, Zoltán Hujber1, Gábor Petővári1, Ildikó Krencz1, Titanilla Dankó1, Gergő Horváth2, Katalin Mészáros3, Hajnalka Rajnai1, László Tretter2, András Jeney1 11st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary 2Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, Hungary 3Hungarian Academy of Sciences - Momentum Hereditary Endocrine Tumours Research Research Group; Semmelweis University - National Bionics Program Budapest, Hungary

Astrocytes and neurons are in metabolic symbiosis. Different tumours use multiple substrates as salvage pathways. The reinterpretation of Warburg effect, especially in gliomas suggesting that other substrates than glucose can also be oxidised. Gliomas are still incurable aggressive heterogeneous tumours. IDH mutations could be driver alterations in gliomas, however, IDH-mutant gliomas have better prognosis than wild-type. IDH mutations not only cause 2-hydroxyglutarate (2HG) production but also initiate metabolic changes. The alterations of bioenergetic characteristics and different substrate oxidation capacities in glioma cells were analysed using in vitro treatments, metabolic analyses (Seahorse, protein expression studies – in human samples, as well – and liquid chromatography – mass spectrometry). Potential use of gamma-aminobutyric acid (GABA) shunt and its - beside glutamine, malate and lower rate glucose - oxidation were detected in succinic acid semialdehyde dehydrogenase (SSADH) expressing glioma cells. Moreover, SSADH overexpression was found in almost all studied human cases with no significant association between clinico-pathological parameters (e.g. IDH mutation). Based on these and the in vitro proliferation studies, we suggest that the characteristic SSADH overexpression and the potential GABA oxidation may have special importance in metabolic adaptation of glioma cells. However, this GABA shunt related effect could be influenced by available substrates and functions of wild-type or mutant IDH1 enzymes – IDH1 mutation and 2HG oncometabolite treatment were associated with reduced GABA and glutamine oxidations in vitro. Our results demonstrate that SSADH expression – an IDH mutation independent in vivo characteristic of human glioma cells – provides a possibility for GABA oxidation. This may have special importance in survival, proliferation and metabolic adaptation of glioma cells.

Supported by Bolyai Fellowship, STIA 2017 and ÚNKP-17-2/3.

P7 b/14 Mitochondrial 2HG production as a function of IDH2 and HOT in breast cancer cells Katarína Smolková1, Jitka Špačková1, Aleš Dvořák2, Libor Vítek2 and Petr Ježek1 1Institute of Physiology, Department of Mitochondrial Physiology, Czech Academy of Sciences, Czech Republic 2Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Czech Republic

Cancer metabolic alterations result from complex genetic and epigenetic adjustments, and include also mitochondrial pathways glutaminolysis, reductive carboxylation (RC), 2-hydroxyglutarate (2HG) production, and NADPH synthesis. We studied complex mechanisms that promote mitochondrial enzymes isocitrate dehydrogenase 2 (IDH2) and hydroxyacid-oxoacid transhydrogenase (HOT) towards 2HG production in breast cancer cell lines. We demonstrate that IDH2 enzyme produces oncometabolite 2HG in vitro, as assumed from analysis of enzymatic products of isolated recombinant wild-type IDH2. Our analysis of metabolic flux shows that mitochondrial production of 2HG by wild-type IDH2 is largely dependent on mitochondrial NADPH balance, because induction of mitochondrial NADPH by dm-L-malate or overexpression of NADPH-producing enzymes induce IDH2-dependent 2HG synthesis. In addition, we demonstrate that active interplay and competition between IDH2 and HOT for substrate (2OG) exist; overexpression of superactive mutant of glutaminese 1, which induces 2OG production, favours HOT if NADPH levels are low. Moreover, we demonstrate that IDH2 is a direct substrate of mitochondrial deacetylase SIRT3, and that distinct regulations by SIRT3 towards oxidative vs. reductive IDH2 activity exist. The analysis of metabolic flux shows that mitochondrial production of 2HG by wild-type IDH2 depends on SIRT3 presence and activity, as supposed by overexpression of wild-type SIRT3 and SIRT3-inactive mutant. The observed 2HG- producing activity is regulated by SIRT3, as an acetylation surrogate IDH2 mutant K413Q tends to decrease levels of 2HG in vitro. Taken together, our findings impact the understanding of breast cancer etiology, since breast cancer express broad range of IDH2, HOT and SIRT3 levels, and exhibit distinct metabolic phenotypes, including 2HG levels. Supported by Grant Agency of the Czech Republic 16-04788S to P.J.

P7 b/15 Different regulation of mitochondrial respiration in human colorectal and breast cancer clinical samples Laura Truu, E. Rebane-Klemm, I. Shevchuk, L. Ounpuu, A. Koit, V. Chekulayev and T. Kaambre Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia

Energy metabolism is characterized by many contradictions, concluding that malignant tumors prefer glycolysis to a more energy efficient oxidative phosphorylation. Majority of these conclusions are the result of in vitro studies on cell culture models, without taking into account factors arising from the tumor microenvironment. We compare the bioenergetics of Caco-2 cell line, human colorectal cancer (HCC) postoperational healthy and cancer tissue samples and also colon polyps. Caco-2 cells as well as HCC tissues exhibit stimulated mitochondrial biogenesis. For the interpretation of our data about the regulation of respiration, we used the model of protein supercomplex Mitochondrial Interactosome (MI). Kinetic method can be useful for quantifying the role of components of MI, maximal respiration rate and regulation of mitochondrial outer membrane permeability. The mitochondria of Caco-2 cells and HCC tissues displayed greater functional activity of respiratory complex (C)II compared to CI, whereas in normal colon tissue an inverse pattern in the ratio of CI to CII activity was observed. In HCC tissue, energy efficiency of flux control is performed at the level of respiratory chain complexes I-IV, whereas in Caco-2 cells at the level of CIV and ATP synthasome. The tumor formation does not diminish mitochondrial respiration capacity. Taking into account the correlation between increased O2 consumption and tumor progression, we suppose that the suppression of mitochondrial biogenesis may be a promising target for colorectal cancer therapy. The differences between cancer clinical material and respective cell cultures could represent an adaptive response to distinct growth conditions; signify the importance of proper validation of results obtained from in vitro models before their extrapolation to the more complex in vivo systems.

P8 a/1 The study of proton transfer in photosystem II Krzysztof L. Buzar, Ana-Nicoleta Bondar Freie Universität Berlin, Department of Physics, Theoretical Molecular Biophysics, Berlin, Germany

The proton transfer in Photosystem II is the crucial element of the proteins main function, which is the light driven water splitting reaction. Recent crystallographic studies of photosystem II [1-2] reveal interactions between the dimers of the protein suggesting dimer- dimer interactions having impact on the release of protons from the reaction centre. [3] To properly investigate the intrinsic influence of dimers on this process, simulations of dimer-dimer system is required. The system proposed for this study would consist of over 2.5 million atoms. To this aim we perform force-field parametrization of the manganese metal cluster of the oxygen evolution complex via quantum mechanical calculations and classical mechanical calculations of the dimer of dimers of photosystem II protein.

1. Y.Umena, K.Kawakami J.R.Shen, N.Kamiya, Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature. 473 (2011) 55-60 2. J.Hellmich, M.Bommer A.Burkhardt, M.Ibrahim, J.Kern, A.Meents, F.Müh, H.Dobbek, A.Zouni, Native-like photosystem II superstructure at 2.44 Å resolution through detergent extraction from the protein crystal. Structure (2014) 1607-1615 3. B.Daum, D.Nicastro, J.Austin, J.R.McIntosh, W.Kühlbrandt, Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea. The Plant Cell (2010) 1299-1312

P8 a/2 Evaluating the importance of cyclic electron flow around photosystem I in microalgae Suzanne Ferté1, Laure Guillou2, Francis-André Wollman1, Benjamin Bailleul1 1IBPC, UMR7141, CNRS, Paris, France 2Station Biologique de Roscoff, Roscoff, France

It is commonly assumed that in plants and green algae, cyclic electron flow (CEF) around photosystem I (PSI) plays a crucial role in optimizing photosynthesis. It allows the PSI and the cytochrome b6f to contribute to the electrochemical proton gradient, with no net product. It is therefore believed to have two main roles, (i) regulating the photosynthetic control and non-photochemical quenching in photosystem II (PSII), and (ii) providing the extra ATP required for carbon fixation. However, several decades of research did not provide a clear-cut answer about the mechanism, extent, regulation and conservation of CEF among different photosynthetic clades. This is mostly due to the absence of a consensus method to estimate this flow in physiological conditions. The most accepted approach compares the quantum yield of PSII and the one of PSI, accessible through absorption changes associated to the redox state of P700. But we could show that the latter method tends to underestimate the quantum yield of PSI, leading to aberrant conclusions regarding CEF. We propose an alternative method based on the electrochromic shift of photosynthetic pigments, which allowed us to estimate the extent and modes of regulation of CEF in various phytoplankton clades, in the laboratory and in the field.

P8 a/3 TO b OR NOT TO b: DIRECT REDUCTION OF CYTOCHROME b-563 BY FERREDOXIN IN HIGHER PLANT PHOTOSYNTHETIC CYCLIC ELECTRON FLOW IN VITRO? Nick Fisher1, Vanessa Quevedo1 and David M. Kramer1,2 1MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan, USA 2Dept. of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA

The linear electron flow reactions of plant and algal photosynthesis result in the production of ATP and NADPH at a fixed ratio of ~1.3:1. This falls short of the 1.5:1 requirement for the carbon-fixing reactions of the Calvin-Benson-Bassham (CBB) cycle. Cyclic electron flow (CEF) around Photosystem I (PSI), which generates ATP without NADPH production, is one way in which the organism may attempt to address this shortfall. During CEF, electrons from photoexcited PSI are transferred back into the thylakoid plastoquinone (PQ) pool through the action of ferredoxin:plastoquinone reductase (FQR), generating reduced PQ and ultimately returned to the oxidizing site of PSI through (protonmotive) cytochrome (cyt) bf complex activity. The identity of the FQR is a topic of much debate. Three pathways have been proposed to fulfill this role in higher plants, namely, i) the protonmotive Fd/NADPH:plastoquinone dehydrogenase (Ndh) complex I; ii) the antimycin A (AA)-sensitive pathway which may involve the participation of the PGRL1- and PGR5 proteins; and iii) the PQ reduction (Qi) site of the cyt bf complex. In this latter pathway, which bypasses the regular Q-cycle operation of the bf complex and may involve the unusual high-spin haem ci at Qi, electrons from reduced

Fd are transferred to heme bH at Qi and then onto PQ, with associated proton uptake from the stroma.

Here we demonstrate that, in vitro, reduction of heme bH by NADPH-reduced Fd is not observed in osmotically shocked spinach chloroplasts under aerobic conditions in DCMU- and tridecyl stigmatellin-inhibited preparations, whilst demonstrating CEF competency through a pmf-dependent acridine dye fluorescence quenching assay. Under anaerobic conditions approximately 40% of the cytochrome bL+bH content is observed to go reduced on a minutes timescale (despite rapid reduction of the PQ pool), and remain at this level for the duration of the experiment. As such, the inherent FQR activity of the bf complex in vitro under aerobic conditions is assumed to be neglible. The implications of this observation for CEF processes is discussed. This work was supported by Grant DE-FG02-11ER16220 from the Photosynthetic Systems program from Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy (to DMK.)

P8 a/4 DEM - the dynamic exchange membrane model. Polymorphism of lipid phases in plant thylakoid membranes Győző Garab1,2, Bettina Ughy1, Petar H. Lambrev1, László Vígh1 1Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2Faculty of Science, University of Ostrava, Ostrava 1, Czech Republic

The functional state of all energy-converting biological membranes is a bilayer, which warrants the build-up of the electrochemical potential gradient for protons and its utilization for ATP synthesis. In the light of this, it is not easy to understand that non-bilayer lipids constitute about half of their lipid contents. It has been proposed that non-bilayer lipids, for their segregation capability, regulate the protein-to-lipid ratio in these, densely packed membranes; it has also been hypothesized that the non-bilayer propensity of their lipids - via allowing a dynamic exchange between the bilayer and non-bilayer lipid phases - contributes to the structural and functional flexibility of membranes [1]. We have addressed this problem by investigating the phase behavior of plant thylakoid membranes. The co- existence of a non-bilayer, isotropic phase and the bilayer in fully functional isolated thylakoid membranes has been first demonstrated by using 31P-NMR measurements [2]. Our recent 31P-NMR experiments, in addition to the bilayer phase, have revealed the presence of two isotropic phases and an inverted hexagonal (HII) phase; the heterogeneity of lipid phases has also been confirmed by time-resolved fluorescence spectroscopy of the lipophilic dye merocyanine 540 [3,4]. Substantial variations in the lipid-phase behavior of the thylakoid membranes exposed to different treatments (e.g. low pH, ionic strength, osmolarity, temperature, catalytic hydrogenation) and reversible changes, in particular, are in good agreement with the DEM – in which lipocalins and fusion channels are also proposed to play key roles.

1. G. Garab, K. Lohner, P. Laggner, T. Farkas (2000) Trends Plant Sci 5: 489–494 2. S.B. Krumova, C. Dijkema, P. de Waard, H. Van As, G. Garab, H. van Amerongen (2008) BBA 1778: 997–1003 3. G. Garab, B. Ughy, P. de Waard, P. Akhtar, U. Javornik, et al. (2017) Sci Rep 7: 13343 4. C. Kotakis, C. Akhtar, O. Zsiros, G. Garab, P. Lambrev (2018) Photosynthetica 56: 254–264

P8 a/5 Effect of high-light acclimation on photosynthetic apparatus of Norway spruce Václav Karlický1,2, Michal Štroch1,2, Irena Kurasová1,2, Zuzana Materová1, Kristýna Večeřová2, Bettina Ughy3, Otmar Urban2, Győző Garab1,3, Vladimír Špunda1,2 1Faculty of Science, University of Ostrava, Ostrava, Czech Republic 2Global Change Research Institute CAS, Brno, Czech Republic 3Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

The photosynthetic apparatus of Norway spruce displays several specific features differing from typical land plants. Absence of Lhcb3 and Lhcb6 in light-harvesting complexes (LHC) of photosystem II (PSII) was found in the gymnosperm genera Picea and Pinus (family Pinaceae), resulting in different PSII supercomplex structure and macro-organization. Further, the significantly different lipid composition of spruce thylakoid membrane might contribute to higher PSII thermal stability and more pronounced acceleration of light-induced violaxanthin deepoxidation at elevated temperatures in spruce needles, as compared to Arabidiposis or barley. In this work, we have primarily focused our attention on high-light (HL) acclimation of the photosynthetic apparatus of spruce. Circular dichroism spectra of thylakoid membranes isolated from HL-acclimated seedlings revealed strongly diminished psi-type circular dichroism bands, suggesting a reduced degree of the long-range order of PSII-LHCII supercomplexes compared to the control. Also, in vitro 77K fluorescence spectroscopy of chlorophyll a revealed that, in contrast to control membranes, in HL thylakoid membranes the emission ratio of PSI/PSII and the absorption cross-sections of PSII and PSI were very similar under stacking and unstacking conditions, indicating a suppressed lateral segregation of the two photosystems. The induction of non-photochemical quenching of chlorophyll fluorescence (NPQ) was found to be fast in both HL-acclimated and control seedlings, although the magnitude of NPQ was significantly reduced, suggesting sustained energy dissipation in HL-acclimated leaves. These results are discussed by also taking into account the effects of HL acclimation on the composition of photosynthetic pigment-system (a surprisingly pronounced increase of lutein) and of the thylakoid lipids (a considerable reduction of MGDG content but increased relative content of SQDG) - unusual features in typical angiosperms.

P8 a/6 Light-induced conformational changes in Photosystem II core complexes revealed by rapid-scan Fourier Transfrom Infrared spectroscopy Melinda Magyar1, Alberto Mezzetti2,3, Gábor Sipka1, Qingjun Zhu4, Guangye Han4, Petar H. Lambrev1, Winfried Leibl2, Jian-Ren Shen4,5, Győző Garab1,6 1Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2Institute for Integrative Biology of the Cell, UMR 9198, CEA Saclay, France 3Laboratoire de Réactivité de Surface UMR 7197, Sorbonne University, Paris, France 4Photosynthesis Research Center, Chinese Academy of Sciences, Beijing, China 5Photosynthesis Research Center, Okayama University, Okayama, Japan 6 Faculty of Science, University of Ostrava, Ostrava 1, Czech Republic

In our earlier work [1] we investigated chlorophyll-a fluorescence transients elicited by trains of single-turnover saturating flashes (STSFs) in dark-adapted isolated plant thylakoid membranes and cyanobacterial PSII core complexes (PSII-CCs) in the presence of

DCMU, allowing only one stable charge separation. We have shown that the fluorescence rises Fo-to-F1 and F1-to-Fmax, induced by the first STSF and by the consecutive multiple STSF excitations, respectively, originate from the reduction of QA, and conformational changes - hypothesized to be coupled to the dielectric relaxation in the reaction center matrix, as in purple bacterial reaction centers [2]. In order to provide direct experimental evidence on the occurrence of STSF-induced conformational transitions we performed time- resolved, rapid-scan FTIR measurements on DCMU-treated cyanobacterial PSII-CCs. Our experiments revealed distinct transient - spectra associated with the Fo-F1 and F1-Fmax states, i.e. between the conformational transitions coupled with the QA-to-QA and the charge-separated to the light-adapted state, respectively. The latter transients, in the amide I region also, were almost fully reversible on a timescale commensurate with the Fmax-to-F1 decay but were inhibited below -10 °C, showing their origin in reorganizations in the protein moiety; these transitions were also different from the STSF-induced conformational changes associated with the reduction of QB.

References: (1) M. Magyar, G. Sipka, L. Kovács, B. Ughy, Q. Zhu, G. Han, V. Špunda, P.H. Lambrev, J-R. Shen, G. Garab, Rate-limiting steps in the dark-to light transition of Photosystem II - revealed by chlorophyll-a fuorescence induction, Scientific Reports 8 (2018) 2755 (2) M. Malferrari, A. Mezzetti, F. Francia, G. Venturoli, Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy, Biochimica et Biophysica Acta 1827 (2013) 328- 339

P8 a/7 Mechanistic insights into Calredoxin function in the presence and absence of calcium Giulia Maria Marchetti1, Ratana Charoenwattanasatien2, Karen Zinzius1, Henning Mootz2, Genji Kurisu3 and Michael Hippler1 1Institute of Plant Biology and Biotechnology, University of Muenster, Germany 2Institute of Biochemistry, University of Muenster, Germany 3Institute for Protein Research, Osaka University, Japan

Calredoxin (CRX) is a thioredoxin protein that displays a calcium-dependent redox activity in the chloroplast stroma of Chlamydomonas reinhardtii. It is involved in ROS detoxification in the chloroplast [1] and in the regulation of the central carbon metabolism of C. reinhardtii. It consists of a calcium-binding domain connected to a thioredoxin domain via a flexible linker. The structure of CRX with bound calcium has already been obtained via protein crystallography [1]. To understand the calcium-dependent thioredoxin activity, we aim to solve the CRX structure in absence of calcium. Conformational changes in presence or absences of calcium were revealed via NMR analysis after methionine labelling. Further low-resolution information was obtained using SAXS and via an engineered biosensor where YFP and CFP are linked to the thioredoxin and calcium-binding domain of CRX, respectively. In order to obtain clearly defined NMR peaks, the intein technology is being used to enable segmental labelling of the two domains and allow a better recognition of their signals in the NMR spectrum [2]. In particular, the two domains of CRX were fused to two split inteins and separately expressed in E. coli grown in differently isotopically labelled media. After purification, a splice reaction is performed with formation of a splice product similar to the native structure of CRX. After improvement of the purification method it would be possible to perform NMR analysis on the splice product and obtain different signals for the differently labelled domains, enabling the identification of the high resolution structure of CRX in presence and absence of calcium.

1. Hochmal, Ana Karina, et al. "Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast." Nature communications 7 (2016): 11847. 2. Liu, Dongsheng, et al. "Segmental isotopic labeling of proteins for nuclear magnetic resonance." Methods in enzymology 462 (2009): 151-175.

P8 a/8 Direct measurement of the excited states of carotenoids in LHCII trimers using fs-stimulated Raman Spectroscopy Francesco Saccon3, Juan M. Artes Vivancos1,2, Yusaku Hontani1, Miroslav Kloz1,4, Alexander Ruban3, Ivo van Stokkum1, Rienk van Grondelle1 and John T.M. Kennis1 1Department of Biophysics, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands 2Chemistry Department. UMass Lowell 3Queen Mary University of London. School of Biological and Chemical Sciences, London, United Kingdom 4ELI Beamlines, Prague

Light harvesting comprises the primary processes during photosynthesis when the excitation energy from photons is seized by pigments in light harvesting complexes (LHCII) and efficiently delivered to photosystems [1]. The ultrafast early events in light harvesting have been studied with time-resolved spectroscopy techniques, but the identity and role of some dark excited states remain still to be clarified. Here, we applied a state-of-the-art technique called femtosecond stimulated Raman spectroscopy [2] to isolated LHCII from higher plants. By exciting in the carotenoid region and applying target analysis, we were able to separate the contributions of the different pigments in LHCII and determine the pathways of energy transfer within the single proteins. Our work shows the potential of this novel technique for the study of the energy equilibration dynamics in photosynthetic complexes, in the light harvesting state as well as during dissipative regulatory processes.

1. G.D. Scholes, G.R. Fleming, A. Olaya-Castro, R. van Grondelle, Lessons from nature about solar light harvesting., Nat. Chem. 3 (2011) 763–774 2. P. Kukura, D.W. McCamant, R. A. Mathies, Femtosecond stimulated Raman spectroscopy., Annu. Rev. Phys. Chem. 78 (2006) 5953–5959

P8 a/9 Comparison of plastid ultrastructure under isoosmotic polyethylene glycol and salt stress Katalin Solymosi2, Beata Mysliwa-Kurdziel1, Annamária Kósa2 1Department of Plant Physiology and Biochemistry, Jagiellonian University, Krakow, Poland 2Department of Plant Anatomy, ELTE - Eötvös Loránd University, Budapest, Hungary

Ionic imbalance of plastids often results in ultrastructural alterations, among which the swelling of the thylakoid lumen is quite often reported under essential metal deficiency or excess (e.g. [1]) as well as under drought and salt stress [2] and is often related to decreased photosynthetic activity of the stressed plants. However, it is still not very well understood which kind of molecular mechanisms can lead to the swelling in case of drought stress which represents water deficiency, osmotic stress induced by polyethylene glycol (PEG) treatment which induces water loss from the tissues, or salt stress during which excess ions are accumulating inside the tissues. In this work we critically and quantitatively compared the ultrastructural alterations induced by isoosmotic PEG and salt treatment of excised leaves of dark-grown wheat plants along with the alterations of the native pigment-protein complexes. This way specific ionic and osmotic components of the stress could be distinguished. In addition, we also present results of an alternative fixation method of salt stressed samples that may circumvent the osmotic shock caused by the addition of standard transmission electron microscopic fixatives with low osmolarity and thus better preserves the native structures under stress conditions.

References: 1. K. Solymosi, M. Bertrand, Soil metals, chloroplasts, and secure crop production: a review, Agronomy for Sustainable Development 32 (2012) 245-272. 2. Abdelkader AF, Aronsson H, Solymosi K, Böddi B, Sundqvist C, High salt stress induces swollen prothylakoids in dark-grown wheat and alters both prolamellar body transformation and reformation after irradiation. Journal of Experimental Botany 58 (2007) 2553-2564.

This work was supported by the Bolyai János Research Scholarship (to K.S.) and by OTKA FK 124748.

P8 a/10 Ferredoxin:NADP oxidoreductase; connected to the first and the last steps of photosynthetic reactions Bettina Ughy1, Pierre Setif2, Ghada Ajlani2 1Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2Institute for Integrative Biology of the Cell, CNRS, CEA, Gif-sur-Yvette, France

FNR (Ferredoxin:NADP oxidoreductase) is the enzyme that catalyzes the last step of linear photosynthetic-electron transfer; providing NADPH for anabolism. In many cyanobacteria, FNR is attached to the light-harvesting complex, the phycobilisome (PBS). The N- terminal domain of this FNR is similar to PBS linkers and is responsible for FNR attachment to PBS [1]; we named this isoform FNRL.

Here we show that PBS from loss- and gain-of-function mutants, of Synechocystis sp. PCC6803, contained less and more FNRL, respectively. Moreover, the amount of FNRL and that of a PBS-rod linker inversely correlate suggesting that these proteins share a PBS- binding site, in line with biochemical results from Synechococcus sp. PCC7002 [2]. Furthermore, we compare light-induced NADPH evolution in WT and the mutants and provide viewpoints for FNRL function. In addition to an expected higher and lower light-induced NADPH accumulation, we show that although electrons are efficiently used for NADPH production, competing processes alternatively use them.

1. WM Schluchter, DA Bryant, Molecular characterization of ferredoxin-NADP+ oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002, Biochemistry 31 (1992) 3092-102. 2. C Gomez-Lojero, B Perez-Gomez, G Shen, WM Schluchter, DA Bryant, Interaction of ferredoxin:NADP+ oxidoreductase with phycobilisomes and phycobilisome substructures of the cyanobacterium Synechococcus sp. strain PCC 7002, Biochemistry 42 (2003) 13800–11.

P8 a/11 Impact of donor- and acceptor-side inhibition upon the light tolerance of photosystem II Sam Wilson, Alexander V. Ruban School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom

Photoinhibition is the light-induced decrease in the photosynthetic yield, the primary target of which is the reaction centre of photosystem II (PSII) [1]. Currently, there is no clear consensus on the exact mechanism of photodamage induced by visible light. However, it is clear that photodamage can occur through limitations on both the donor- and acceptor-sides of PSII [2]. The former mechanism relies on disruption to the oxygen-evolving complex. Consequently, the lifetime of P680+ is extended, which inevitably oxidises the nearby D1 protein [3]. The latter mechanism is caused by electron transport limitations at the PSII acceptor side. This leads to the formation of 3P680, forming triplet oxygen, which in turn bleaches P680 and leads to further degradation of D1 [3]. Using a novel chlorophyll fluorescence methodology, reaction centre closure can be measured and quantified alongside photoprotection. This is achieved through estimation of the redox state of QA, using the parameter of photochemical quenching in the dark (qPd) [4]. Through the use of electron acceptors, donors, and inhibitors, a functional, in vivo system has been built, in which the different pathways of photoinhibition can be induced and controlled. By elucidating the signature of each photoinhibitory pathway, their effect on light tolerance and their interplay with photoprotective mechanisms has been quantified, modelled, and distinguished.

1. S.B. Powles, Photoinhibition of photosynthesis induced by visible light, Annu. Rev. Plant Physiol., 35 (1984) 15–44. 2. I. Vass, Molecular mechanisms of photodamage in the Photosystem II complex, Biochim. Biophys. Acta - Bioenerg., 1817 (2012) 209–217. 3. P. Pospíšil, Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II, Biochim. Biophys. Acta - Bioenerg., 1817 (2012) 218–231. 4. A. V. Ruban, Quantifying the efficiency of photoprotection, Philos. Trans. R. Soc. Lond. B. Biol. Sci., 372 (2017) 20160393.

P8 a/12 Colocalization of Autofluorescent and YFP Tagged Photosynthetic Membrane Proteins Imaged by CLSM Gábor Steinbach1, Adéla Strašková2, Eva Kotabová2, Félix Schubert3, Josef Komenda2, Martin Tichý2, Radek Kaňa2 1Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2Institute of Microbiology, Czech Academy of Sciences, Třeboň, Czech Republic 3Department of Mineralogy, Geochemsitry and Petrology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary

Cyanobacteria are the simplest model organisms to understand the photosynthetic machinery and thylakoid membranes of higher plants. Thylakoids are the place of the light-photosynthetic reactions that involve light absorption, charge separation and electron/proton transfer. The efficient exciton/electron transfer is enabled by protein megacomplexes. The pigment-proteins complexes represented by photosystem I (PSI), photosystem II (PSII) and phycobilisomes (PBS) form heterogeneous mosaic of microdomains. The partial separation of PSI and PSII in cyanobacteria thylakoids and microdomain’s stability may be required for efficient excitation/electron transfer, regulated according to cellular demands and environmental conditions. Separation of PSI, PSII-PBS and PSI-PSII-PBS supercomplex areas were confirmed on wild type Anabaena sp. 7120 cyanobacteria cells via cryo-imaging [1] and on YFP-tagged (PsaF subdomain of PSI) Synechocystis sp. PCC 6803 cells. Additional mobility measurements (fluorescence recovery after photobleaching – FRAP) proved that the low mobility of both photosystems when almost 50% of photosystems being immobile insures a limited diffusion of thylakoid membranes. [2] Based on our results we assume that the cyanobacterial microdomains are an evolutionary precursor for the organization of photosystems into granal/stromal thylakoids due to thylakoid membrane stacking in higher plant chloroplasts.

1. G. Steinbach, F. Schubert, R. Kaňa, Cryo-imaging of photosystems and phycobilisomes in Anabaena sp. PCC 7120 cells, Journal of Photochemistry and Photobiology B: Biology 152 (2015) 395–399 2. A. Strašková, G. Steinbach, E. Kotabová, J. Komenda, M. Tichý, R. Kaňa, Stable photosynthetic microdomains in thylakoid membrane of cyanobacteria, Plant Cell, submitted

P9 /1 Large-scale analyses of PTP dynamics on individual mitochondria Camille Colin1, Philippe Diolez2, Stéphane Arbault1 1Univ. BORDEAUX, ISM, NSysA Group, ENSCBP, Pessac, France 2Univ. BORDEAUX, IHU, Liryc, CRCTB, Inserm, Pessac, France

Studies of mitochondria bioenergetics are usually based on large population and mean analyses but these approaches do not allow to discriminate between sub-population dynamics and quantitation. The goal of the present work is to target single mitochondria analyses and study the activity of the mitochondrial Permeability Transition Pore (mPTP) [1], which is hypothesized to control mitochondrial dynamics. Experiments are based on fluorescence microscopy of individual mitochondria on a large-scale. We developed an automated method based on Trackmate-ImageJ [2] to localize and monitor fluorescence changes of single mitochondria. They were extracted from mouse hearts, loaded with a membrane potential-sensitive dye (TMRM) and sedimented on a cover glass for monitoring. Following additions of energetic substrates and calcium, transient variations of single mitochondria membrane potential (loss and recovery) were reproducibly observed. We found two major shapes of fluorescence transients related to the mPTP opening: 1) A spike-shape response resulting in a rapid membrane depolarization followed by a longer recovery; 2) A longer duration spike initiated by a fast opening, followed by a stationary phase and recovery. These two behaviors were not correlated with fluorescence variations in terms of loss or increase. Frequency and kinetics of mPTP openings were modulated by addition of calcium and H2O2. Overall, we developed a semi-automatic approach to track by fluorescence fast membrane potential changes of a large number of single mitochondria. We applied this method to characterize mPTP openings and we observed two major transient responses: a fast spike shape and a long-lasting spike that will be characterized in near future.

1. Agarwal A et al (2016), “Transient Opening of the Mitochondrial PTP Induces Microdomain Calcium Transients in Astrocyte Processes.” Neuron 2. Tinevez, JY. et al. (2016), "TrackMate: An open and extensible platform for single-particle tracking". Methods

P9 /2 Role of F-ATP synthase f subunit in dimer formation and PTP modulation Chiara Galber1, Giovanni Minervini1, Giuseppe Cannino1, Andrea Carrer1, Silvio Tosatto1, Giovanna Lippe2, Valentina Giorgio1 and Paolo Bernardi1 1Department of Biomedical Sciences and CNR Neuroscience Institute, University of Padova, Italy 2Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Italy

The mitochondrial F-ATP synthase is a large multisubunit complex of 600 kDa organized into a catalytic part (F1) and a membranous moiety (Fo) linked by central and peripheral stalks. Recently, it has been demonstrated that purified dimers of F-ATP synthase added to a lipid bilayer form channels matching features of the permeability transition pore (PTP) [1]. The PTP is a mitochondrial mega-channel which induces cell death through the sudden membrane permeabilization to solutes and collapse of membrane potential. Although many candidates have been proposed as components of the PTP, there is now the evidence that the F-ATP synthase is its major constituent [2], even if the subunits directly involved in PTP formation and regulation remain undefined. We have proposed that channel formation takes place in F-ATP synthase dimers at the interface between two monomers. Structural data of the enzyme suggest an important role of f subunit in dimer stabilization in Yarrowia lipolytica and Saccharomyces cerevisiae. Based on bioinformatics analysis we have focused on f subunit to characterize its role as a possible pore-forming site. Electrophysiological studies are under investigation. We have used the CRISRP-Cas9 and the shRNA interference technologies to generate human cells lacking or with a decreased level of subunit f, respectively. We will report our progress at characterizing cell viability, mitochondrial morphology, F-ATP synthase catalysis and PTP features in these models.

1. V. Giorgio, S. von Stockum, M. Antoniel, A. Fabbro, F. Fogolari, M. Forte, G.D. Glick, V. Petronilli, M. Zoratti, I. Szabó, G. Lippe, P. Bernardi, Dimers of mitochondrial ATP synthase form the permeability transition pore, Proc Natl Acad Sci USA,110 (2013) 5887-92 2. P. Bernardi, A. Rasola, M. Forte, G. Lippe, The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology, Physiol. Rev. 95 (2015) 1111-1155.

P9 /3 The inhibitor protein IF1 modulates the permeability transition pore in a human tumorigenic cell model Valentina Giorgio1, Victoria Burchell1, Chiara Galber1, Giulia Valente1, Valeria Petronilli1, Giovanna Lippe2, Paolo Bernardi1 1Department of Biomedical Sciences and CNR Neuroscience Institute, University of Padova, Italy 2Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Italy

The mitochondrial protein IF1 is the natural inhibitor of F-ATP synthase. IF1 is encoded in ATPF1 gene which is located in chromosome 1 and 4 of human and mouse genomes. Alternative splicing of the human IF1 mRNA transcripts can generate three isoforms that differ in length and sequence. The longest mature protein consists of 84 amino acids, a N-terminus necessary for its inhibitory property and a C-terminus required for formation of its active dimeric form. It is well established that binding of the mature IF1 to the catalytic F1 domain of F-ATP synthase inhibits ATP hydrolysis and is optimal at low pH. It has also been suggested that IF1 binding may stabilize dimers of the F-ATP synthase. Our previous work has demonstrated that purified F-ATP synthase dimers added to a lipid bilayer form channels matching the electrophysiological features of the permeability transition pore (PTP), a mitochondrial mega-channel which is activated by matrix Ca2+ and ROS. Long-lasting openings of the channel induce cell death through the release of pro-apoptotic factors. We have found that silencing of IF1 in a human tumorigenic cell line decreases F-ATP synthase dimer/oligomer stability, promotes PTP opening and prevents tumorigenic capacity in vitro.

P9 /4 Regulation of the Mitochondrial Permeability Transition Pore by Arginine Residues of F-ATP Synthase Lishu Guo1, Michela Carraro1, Geppo Sartori1, Andrea Urbani1, Valeria Petronilli1, Christoph Gerle2, Ildikò Szabò3, Paolo Bernardi1 1Department of Biomedical Sciences, University of Padova, Italy 2Institute for Protein Research, Osaka University, Japan 3Department of Biology, University of Padova, Italy

Arginine-glyoxal adducts can lead to either suppression or induction of permeability transition (PT) of isolated rat liver mitochondria (1). We find that purified bovine F-ATP synthase reconstituted into planar lipid bilayers displays channel activity after the addition of p- hydroxyphenyl glyoxal (OH-PGO) in presence of Ca2+, even in the absence of BZ423 and of oxidants, previously shown to be required for Ca2+-induced activation of the channel. OH-PGO is thus a strong inducer of PTP, and these results indicate that the reactive arginine(s) are located in the F-ATP synthase. Phenylglyoxal (PGO) is the most extensively used reagent for the site-specific chemical modification of arginine in proteins. We show that phenylglyoxal (PGO) affects the PT in a species-specific manner: inhibitory effects in mouse and yeast, inducing effects in human and Drosophila. Subunits e and g are essential for the dimerization of F-ATP synthase and mitochondrial cristae formation (2). Remarkably, we demonstrate that mitochondria from a yeast mutant strain lacking subunit g (ΔATP20) is resistant to the PT inhibitory effects of PGO, and that the effect is phenocopied in a subunit g R107A mutant. Thus, the effect of PGO on the PT is specifically mediated by R107, the only subunit g arginine that is conserved across species. These findings identify the target of PGO and strongly indicate that the PT is mediated by F-ATP synthase.

Reference 1. M. Johans, et al., Modification of permeability transition pore arginine(s) by phenylglyoxal derivatives in isolated mitochondria and mammalian cells: Structure-function relationship of arginine ligands, J. Biol. Chem. 280 (2005) 12130–12136

2. H. Guo, S. A. Bueler, J. L. Rubinstein, Atomic model for the dimeric FO region of mitochondrial ATP synthase, Science 358 (2017) 936-940

P9 /5 Deletion of subunits of human ATP synthase and impact on the mitochondrial permeability transition Jiuya He, Joe Carroll, Shujing Ding, Ian M. Fearnley and John E. Walker Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, United Kingdom

Mitochondrial permeability transition refers to the opening of a non-specific pore, the permeability transition pore (PTP), in the inner membrane. PTP opening is triggered by elevation of mitochondrial matrix calcium levels, and can be inhibited by cyclosporin A.

Mitochondrial ATP synthase has been proposed to form the PTP [1]. The enzyme has a hydrophilic catalytic F1 portion in the matrix, bound to the Fo membrane domain. The two domains are linked by a central stalk, and a peripheral stalk that provides an essential part of the enzyme’s stator. ATP formation is driven by rotation of a ring of c-subunits and the attached central stalk, powered by a trans- membrane proton motive force. In mitochondria, rows of ATP synthase dimers curve the inner membrane into the cristae architecture. It has been suggested that the ATP synthase provides the PTP, either via a pore at the dimer interface or when dimers dissociate [1,2], or by exposure of a pore in the central cavity of the c-ring [3]. Using CRISPR-Cas9 directed gene disruption in human HAP1 cells, the PTP was found to be maintained when the c-subunit was removed completely [4]. Similarly, the individual removal of either the peripheral stalk b-subunit or of the oligomycin sensitivity conferral protein abolished dimer formation, but did not prevent the PTP from opening [5]. 0 The PTP also persisted in a 143B ρ cell line which lacks mitochondrial DNA. Therefore, the Fo domain subunits ATP6 and ATP8, which are encoded in the mitochondria genome, are not involved in forming the pore [4]. The five remaining membrane subunits in the Fo domain have been removed individually in HAP1 cells, the vestigial complexes have been characterized [6], and the ability of these cells to form a PTP has been investigated. Also, in a single cell line, the c-ring and the central stalk component, the δ-subunit, have been removed together, and the impact on the assembly of ATP synthase and PTP opening have been examined.

References [1] V. Giorgio, S. von Stockum, M. Antoniel, A. Fabbro, F. Fogolari, M. Forte, G.D. Glick, V. Petronilli, M. Zoratti, I. Szabó, G. Lippe, P. Bernardi, Dimers of mitochondrial ATP synthase form the permeability transition pore, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 5887- 5892. [2] M. Bonora, C. Morganti, G. Morciano, G. Pedriali, M. Lebiedzinska-Arciszewska, G. Aquila, C. Giorgi, P. Rizzo, G. Campo, R.

Ferrari, G. Kroemer, M.R. Wieckowski, L. Galluzzi, P. Pinton, Mitochondrial permeability transition involves dissociation of F1Fo ATP synthase dimers and C-ring conformation, EMBO Rep. 18 (2017) 1077-1089. [3] K.N. Alavian, G. Beutner, E. Lazrove, S. Sacchetti, H.A. Park, P. Licznerski, H. Li, P. Nabili, K. Hockensmith, M. Graham, G.A.

Porter, E.A. Jonas, An uncoupling channel within the c-subunit ring of the F1Fo ATP synthase is the mitochondrial permeability transition pore, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 10580-10585. [4] J. He, H.C. Ford, J. Carroll, S. Ding, I.M. Fearnley, J.E. Walker, Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase, Proc. Natl. Acad. Sci. U.S.A. 114 (2017) 3409–3414. [5] J. He, J. Carroll, S. Ding, I.M. Fearnley, J.E. Walker, Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase, Proc. Natl. Acad. Sci. U.S.A. 114 (2017) 9086–9091. [6] J. He, H.C. Ford, J. Carroll, C. Douglas, E. Gonzales, S. Ding, I.M. Fearnley, J.E. Walker, Assembly of the membrane domain of ATP synthase in human mitochondria, Proc. Natl. Acad. Sci. U.S.A. 115 (2018) 2988-2993.

P9 /6 Effect of anions on Cyclophilin D binding to F-ATP synthase: Implications for the permeability transition pore Giovanna Lippe1, Gabriele Coluccino1, Valentina Giorgio2, Federico Fogolari3, Valeria Petronilli2, Paolo Bernardi2 1Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Italy 2Department of Biomedical Sciences and CNR Neuroscience Institute, University of Padova, Italy 3Department of Mathematics, Computer Sciences and Physics, University of Udine, Italy

The mitochondrial F-ATP synthase is an amazing H+-driven molecular motor, which provides most cellular ATP under aerobic conditions and is regulated by a variety of matrix proteins, including Cyclophilin D (CyPD). CyPD binds to the OSCP subunit of F-ATP synthase, causing a decrease of the specific enzymatic activity that is reversed by cyclosporin A (CsA). In mammalian mitochondria CyPD is the best-characterized protein modulating the permeability transition pore (PTP) and the receptor for the PTP inhibitor CsA. The finding that it interacts with the F-ATP synthase set the foundation for the hypothesis that F-ATP synthase can turn into a Ca2+-activated energy- dissipating channel that favors cell death. The debate is still open as to whether and how F-ATP synthase can form the PTP. In the present study we have analysed the effects of several anions on CyPD binding to F-ATP synthase. Indeed, inorganic phosphate (Pi) desensitizes the PTP to opening, except in mammalian mitochondria, where it acts as a PTP inducer in the absence of CsA. Intriguingly, arsenate (As) and vanadate (Vi) can replace Pi as PTP inducer but not as PTP inhibitors [1]. All these anions favoured CyPD binding to F-ATP synthase, strongly suggesting that the PTP-inducing effect of anions observed in mammalian mitochondria depends on CyPD binding to this enzyme complex.

References: 1. E. Basso, V. Petronilli, M.A. Forte, P. Bernardi, Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation, J. Biol. Chem. 283 (2008) 26307-11

P9 /7 The role of ATP synthase megachannel in mitochondrial permeability transition Nelli Mnatsakanyan, Han-A Park, Jing Wu, Paige Miranda, Elizabeth A. Jonas Department of Internal Medicine, Yale University, New Haven, USA

Mitochondrial permeability transition (mPT) is one of the main causes of necrotic and apoptotic cell death during neurodegenerative diseases and stroke. The opening of the mitochondrial permeability transition pore (mPTP) leads to mitochondrial inner membrane permeabilization and dissipation of membrane potential, followed rapidly by cell death. Despite the vital importance of mPTP in controlling cell life and death pathways, the molecular structure and identity of mPTP is not yet fully understood. We have growing evidence that F1Fo ATP synthase c-subunit ring forms a large conductance ion channel the gating of which is performed by the F1 hydrophilic portion of ATP synthase. We observed significant decrease in ATP synthase F1 subunit levels under glutamate-induced excitotoxic conditions, which is prevented by the mPTP inhibitor cyclosporine A. This suggests that structural disassembly of ATP synthase subdomains unmasks the c-subunit channel, placing mitochondria at increased risk for permeability transition. We have now generated a mutant c-subunit channel with a markedly reduced conductance that we find protects from excitotoxic death of hippocampal neurons. In addition, in our recent studies we have successfully overexpressed and purified human ATP synthase c-subunit from E. coli, free of any potential contamination by other mitochondrial proteins. When human c-subunit purified from E. coli is reconstituted into artificial lipid bilayers, recordings reveal a large multi-conductance channel with the biophysical characteristics of mPTP. We are currently studying the role of c-subunit leak channel in mPT by using ATP synthase c-subunit CRISPR knockdown and knockout mouse embryonic stem cells. We find that ATP synthase c-subunit CRISPR knockdown cells have significantly smaller conductance channel activity compared with the wild type cells. These findings will provide us with an increased understanding of the molecular composition and structure of mPTP.

P9 /8 A mitochondrial therapy for Duchenne muscular dystrophy Anna Stocco1, Marco Schiavone1, Alessandra Zulian1, Valeria Petronilli1, Justina Sileikyte2, Michael Forte2, Francesco Argenton3, Luciano Merlini4, Patrizia Sabatelli5, Paolo Bernardi1 1Department of Biomedical Sciences and CNR Neuroscience Institute, University of Padova, Padova, Italy 2Vollum Institute, Oregon Health and Science University, Portland, OR, USA 3Department of Biology, University of Padova, Padova, Italy 4Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 5CNR Institute of Molecular Genetics and Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli-IRCCS, Bologna, Italy

Duchenne muscular dystrophy (DMD) is a life-threatening X-linked muscle disease caused by mutations in the dystrophin gene. It is characterized by progressive degeneration of muscle fibers and an effective, or generally applicable therapy is lacking. Studies in animal models (hamster, chicken and mouse) demonstrated the presence of mitochondrial dysfunction during the disease pathogenesis. Opening of the mitochondrial Permeability Transition Pore (PTP, a mitochondrial high-conductance channel), due to an increase of calcium concentration in both sarcoplasm and mitochondria, was observed to be one of the main mechanisms involved in DMD pathogenesis [1]. Since the mdx-/- mouse (the best-characterized mouse model of the disease) displays a rather mild dystrophic phenotype, we took advantage of the severe sapje zebrafish mutant, which lacks dystrophin and shows ultrastructural muscle defects close to those of DMD patients [2]. Our aim is to explore in vivo a possible mitochondrial therapy targeting the PTP with non- immunosuppressive derivatives of cyclosporine A and new PTP inhibitors developed by our research group. As previously observed in muscle biopsies from DMD patients, we show that sapje zebrafish (i) display a dramatic disruption of muscle structure, (ii) a strong decrease of respiratory reserve capacity and (iii) are prone to mitochondrial dysfunction due to opening of the PTP, whose features in zebrafish are the same as those of mammals. Treatment with the FDA-approved cyclophilin inhibitor Alisporivir - a cyclosporin A derivative that desensitizes the PTP but does not inhibit calcineurin - and the new PTP inhibitor MF1 led to a striking recovery of muscle structure, motor impairments and respiratory function [3]. Improvement of sapje zebrafish survival was observed as well. As Alisporivir has an excellent safety profile, it could be used in combination with MF1 for treatment of DMD.

1. J. Reutenauer, O.M. Dorchies, O. Patthey-Vuadens, G. Vuagniaux, U.T. Ruegg, Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy, British Journal of Pharmacology 155 (2008) 574-584 2. D. Bassett, P.D. Currie, Identification of a zebrafish model of muscular dystrophy, Clinical Experimental Pharmacology and Physiology 31(2004) 537-540 3. M. Schiavone, A. Zulian, S. Menazza, V. Petronilli, F. Argenton, L. Merlini, P. Sabatelli, P. Bernardi, Alisporivir rescues defective mitochondrial respiration in Duchenne Muscular Dystrophy, Pharmacological Research 125 (2017) 122-131

P10 /1 Towards unidirectional reconstitution of membrane proteins into liposomes Andrea M. Amati1,2, Christoph von Ballmoos1 1Department for Chemistry & Biochemistry, University of Bern, Switzerland 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland

For functional studies, membrane proteins (MPs) are often purified and integrated (reconstituted) into systems which mimic their natural environment in a membrane as e.g. liposomes, allowing to investigate its function and structural aspects without any disturbing background from their native environment. The most important problem during MP-reconstitution is the often random orientation of the MP in the liposomal membrane after reconstitution. For functional studies of the MP of interest and quantitative analysis of its properties, unidirectional orientation in the liposomal membrane is required. Previous work of other groups did not include a final and universal approach [1, 2] and procedures have to be individually optimized for an enrichment of enzyme orientation. In most cases, however, orientation cannot be influenced and is thought to depend on the 3D structure of the protein. We are currently developing and establishing a universal method to force unidirectional reconstitution of MPs by the aid of a molecular unit that can be attached to every protein. Once our method is fully established, the interplay of two or more membrane proteins can be investigated more quantitatively.

[1] J.L. Rigaud, D. Levy, Reconstitution of membrane proteins into liposomes, Methods in enzymology, 372 (2003) 65-86. [2] R. Tunuguntla, M. Bangar, K. Kim, P. Stroeve, C.M. Ajo-Franklin, A. Noy, Lipid bilayer composition can influence the orientation of proteorhodopsin in artificial membranes, Biophysical journal, 105 (2013) 1388-1396.

P10 /2 The mitochondria-targeted antioxidant SkQ1 can carry adenosine 3′,5′-cyclic monophosphate, but not guanosine 3′,5′-cyclic monophosphate, through artificial and natural membranes Yuri N. Antonenko1, Alexander M. Firsov1, Irina G. Rybalkina2, Elena A. Kotova1, Tatyana I. Rokitskaya1, Sergei D. Rybalkin2 1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 2Department of Pharmacology, University of Washington, Seattle, USA

In a recent publication [1], we have demonstrated for the first time that a conjugate of decyltriphenylphosphonium with plastoquinone (SkQ1) can serve as a carrier of adenosine 3,5-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms. The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3,5-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium lacking a quinone moiety, was effective in carrying cAMP. The SkQ1-induced transfer of cAMP, but not cGMP, across the plasma membrane of human platelets, from outside to inside the cells, was detected by phosphorylation of the vasodilator stimulated phosphoprotein. Possible impact of SkQ1 on cAMP signaling pathways in living cells will be discussed.

1. A.M. Firsov, I.G. Rybalkina, E.A. Kotova, T.I. Rokitskaya, V.N. Tashlitsky, G.A. Korshunova, S.D. Rybalkin, Y.N. Antonenko, A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes, Biochim. Biophys. Acta - Biomembranes 1860 (2018) 329-334

P10 /3 Generation of a novel, personalised in vitro model to assess the impact of mitochondrial DNA variation upon bioenergetic function and susceptibility to hepatotoxicity Amy Ball1, Ana Alfirevic2, Jon Lyon3, Amy Chadwick1 1MRC Centre for Drug Safety Science, University of Liverpool, UK 2The Wolfson Centre for Personalised Medicine, University of Liverpool, UK 3GlaxoSmithKline, R&D Ware, UK

Background: Mitochondrial dysfunction is associated with idiosyncratic drug-induced liver injury (iDILI), a major cause of drug withdrawal. Variation in the mitochondrial genome (mtDNA) has been found to induce changes in mitochondrial function. We hypothesise that inter-individual variation in mtDNA underlies cases of iDILI via changes in mitochondrial function which can alter susceptibility to injury. We therefore aimed to develop an in vitro model to test the effect of mtDNA variation pre-clinically. Methods: We performed the next-generation mtDNA sequencing of 384 healthy volunteer samples and identified mitochondrial sub- haplogroups. A sample of volunteers then donated fresh blood from which platelets (containing mtDNA) were isolated and fused with HepG2 rho-zero cells (liver carcinoma cell line devoid of mtDNA) to generate HepG2 transmitochondrial cybrids. Cybrids provide a controlled nuclear DNA background upon which to study the effects of mtDNA variation. Cybrids were then analysed for differences in mitochondrial function using high throughput imaging and extracellular flux analysis in permeabilised (to test individual respiratory complexes) and whole cells. Analysis was performed at both basal state and following treatment with compounds associated with iDILI. Results/Conclusion: We have identified 291 sub-haplogroups in our cohort and have generated transmitochondrial cybrids from multiple mitochondrial haplogroups. Preliminary data has shown a significant difference in nefazodone (associated with iDILI)-induced ATP depletion (IC50 9.98 μM vs 19.42 μM) and differential complex IV expression between haplogroups B and H respectively. To the best of our knowledge, this is the first time HepG2 cybrids have been generated. Importantly, this provides a level of personalisation in one of the most commonly used cell-lines in hepatotoxicity testing, enabling mtDNA variants which increase susceptibility to iDILI to be identified pre-clinically.

P10 /4 The electron-bifurcating hydrogenase Hnd from Desulfovibrio fructosovorans Arlette Kpebe, Chloé Guendon, Martino Benvenuti, Amani Rebai, Emilien Etienne, Victoria Fernandez, Bruno Guigliarelli, Carole Baffert, Myriam Brugna Laboratoire de Bioénergétique et Ingénierie des Protéines, Aix-Marseille Université, CNRS, Marseille, France

Hydrogenases are enzymes able to catalyze in a reversible way both the oxidation of H2 and the reduction of protons at a bimetallic FeFe or NiFe active site. The diversity of these hydrogenases is naturally present in our model organism, the sulfate reducing bacterium Desulfovibrio fructosovorans which has a complex hydrogenase system composed of six different enzymes. This study focuses on the cytoplasmic FeFe hydrogenase Hnd. The four subunits of this enzyme share strong sequence similarity with subunits of multimeric FeFe hydrogenases that can perform electron bifurcation [1,2]. The newly discovered process called flavin-based electron bifurcation can be regarded as a third mode of energy conservation [3,4]. In order to characterize Hnd at the molecular level, the hnd operon encoding the four subunits of the complex was cloned. A Strep- tagged recombinant form of the hydrogenase was produced and purified. We present here results of the biochemical characterization of the enzyme. We show that Hnd is an electron bifurcating enzyme that couples the reduction of both NAD+ and a ferredoxin to the oxidation of dihydrogen. A recombinant form of the hydrogenase catalytic subunit was also produced, purified and characterized.

1. G.J. Schut, M.W. Adams, The iron-hydrogenase from Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production, J. Bacteriol. 191 (2009) 4451-4457 2. K. Schuchmann, V. Müller, A bacterial electron-bifurcating hydrogenase, J. Biol. Chem. 287 (2012) 31165-31171 3. W. Buckel, R.K. Thauer, Energy conservation via electron bifurcating ferredoxin reduction and proton/Na(+) translocating ferredoxin oxidation, Biochim. Biophys. Acta 1827 (2013) 94-113 4. W. Buckel, R.K. Thauer, Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review, Front. Microbiol. 9 (2018) 401

P10/5 Conformational states of coenzymes FAD and NADH monitored by ultrafast spectroscopy Géza I. Groma1, Ferenc Sarlós1, Zoltán Násztor1, Áron Sipos1, János Horváth1, Rita Nagypál1, András Dér1, Zsuzsanna Heiner1, Thomas Rowland2, Jérémie Leonard2, Stefan Haacke2, 1Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2University of Strasbourg, CNRS, Institut de Physique et Chimie des Matériaux de Strasbourg

In living cell coenzymes FAD and NADH exist in both free and protein-bound forms. Early studies indicated that in solutions both coenzymes populate folded as well as unfolded conformations, corresponding to the arrangements of their two rings. The excited-state lifetime of these small molecules is very sensitive to the interring interactions, hence can exactly monitor the populations of the different conformations. Here we show that in aqueous solution of FAD the fluorescence kinetics of the flavine group is an excellent monitor of the Hofmeister effect. This phenomenon is based on the ability of the different anions to control the aggregation/conformation state of the molecules: the group of the so called chaotropes promotes closed conformations while the kosmotrops favor open ones. Applying a special method for the analysis of fluorescence kinetics we identified an open conformation with τ1 = 2.5 ns and three closed ones, corresponding to τ2 =

80 ps, τ3 = 10 ps and τ4 = 2 ps. The presence of kosmotropic and chaotropic anions did not change the fluorescence lifetimes but extensively controlled their relative amplitudes, exactly according to the Hofmeister effect as supported by our molecular dynamic calculations. In high contrast to these lifetimes, in flavocytochrome C sulfide dehydrogenase the covalently bound FAD showed a single fluorescence decay component of <200 fs, indicating an extreme strong interaction with the enzyme. In the folded conformation of NADH an effective excited-state energy transfer (EET) occurs from the adenine to the nicotinamide group. Our transient absorption measurements revealed that the EET is as fast as ~70 fs. It was found that even this extremely high rate, over a sub-nm inter-ring distance, can be well described by a Förster-type mechanism. The EET is followed by a 1.7 ps vibrational and a 650 ps electronic relaxation. This work was supported by the GINOP-2.3.2-15-2016-00001 grant of the NRDI Office, Hungary.

P10 /6 Microbial oxidative sulfur metabolism: the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus Marianne Guiral1, Souhela Boughanemi1, Pascale Infossi1, Marielle Bauzan2, Agnès Hirschler-Réa3, Artemis Kosta4, Marie-Thérèse Giudici-Orticoni1 1Institut de Microbiologie de la Méditerranée, Laboratoire de Bioénergétique et Ingénierie des Protéines UMR 7281, Marseille, France 2Institut de Microbiologie de la Méditerranée, Plateforme de fermentation, CNRS, Marseille France 3Institut Méditerranéen d’Océanologie, IRD-AMU, Marseille, France 4Institut de Microbiologie de la Méditerranée, Plateforme de Microscopie, Marseille France

A heterodisulfide reductase (Hdr)-like enzyme is postulated to mediate oxidation of sulfur compounds in a number of sulfur-oxidizing archaea and bacteria. Nevertheless, no biochemical data were available so far about this enzyme in these micro-organisms. In methanogens, the HdrABC enzyme couples the endergonic reduction of ferredoxin to the exergonic reduction of the heterodisulfide CoM-S-S-CoB, using the electron bifurcation mechanism. The Hdr-like complex in sulfur-oxidizers is predicted to have a completely different function, although potentially it might also bifurcate electrons. Genes for an Hdr-like enzyme were identified in Aquifex aeolicus, a chemolithoautotrophic bacterium that uses inorganic sulfur compounds or hydrogen as energy source. We provide biochemical evidence that this enzyme is present in this sulfur-oxidizing prokaryote (cultivated with thiosulfate or S0) and demonstrate that Hdr-like is associated, presumably monotopically, with the membrane fraction. The Hdr proteins form a stable complex composed of five subunits, HdrA, HdrB1, HdrB2, HdrC1 and HdrC2, present at about 240 kDa on native gel. This Hdr-like complex is the first to be purified from a sulfur-oxidizing micro-organism [1]. 0 In addition, we established the growth of A. aeolicus under various conditions of H2, S2O3 or S (either sulfur compounds or H2 used as energy source) and fixed CO2 /O2 concentration and we investigated the levels and activities of different respiratory enzymes, including Hdr in these growth conditions. We propose a revised model for dissimilatory sulfur oxidation pathways in A. aeolicus, with Hdr predicted to generate sulfite [1].

1. S. Boughanemi, J. Lyonnet, P. Infossi, M. Bauzan, A. Kosta, S. Lignon, M.-T. Giudici-Orticoni, M. Guiral, Microbial oxidative sulfur metabolism: biochemical evidence of the membrane-bound heterodisulfide reductase-like complex of the bacterium Aquifex aeolicus, FEMS Microbiol Lett (2016) 363(15),pii: fnw156

P10 /7 Exploring the Energetics of Ammonia Oxidizing Archaea Logan H. Hodgskiss, Christa Schleper Archaea Biology and Ecogenomics Division, Univ. of Vienna, Austria

In the past decade, ammonia oxidizing archaea (AOA) have proven to be ubiquitous in soil environments. AOA are members of the Thaumarchaeota phylum and rely on ammonia oxidation as an energy source while growing autotrophically. Because of this, the core physiology of AOA contributes to both the global nitrogen and carbon cycles. However, little is known about the function and regulation of their primary metabolic processes. The recent isolation and genome annotation of an AOA from soil, Nitrososphaera viennensis, has set the stage to explore these questions. In pure culture, N. viennensis grows optimally at 42 ºC and is dependent on organic α-keto acids, such as pyruvate, for the detoxification of reactive oxygen species. Various growth experiments, based on a thermodynamic analysis, have been performed while varying the amount of nitrogen and inorganic carbon available for energy production and growth respectively. Additionally, the limitation of inorganic carbon has been shown to restrict the growth of N. viennensis in the absence of pyruvate. The pyruvate that is present likely creates a source of inorganic carbon via abiotic reactions with reactive oxygen species rather than acting as a source for mixotrophic growth. The dependence of N. viennensis on pyruvate has also been explored through the analysis of a mutant strain that is no longer dependent on pyruvate for growth and has a single point mutation in the nuoL subunit of Complex I, a known source of reactive oxygen species in microorganisms. The results from the mutant strain, along with the growth responses under varying substrate conditions, should assist in elucidating the ecological role that AOA play in the environment.

P10 /8 Non-heme Fe cofactor insertion into respiratory nitric oxide reductase from Paracoccus denitrificans Maximilian Kahle1, Josy ter Beek1, Jonathan P. Hosler2, Pia Ädelroth1 1Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden; 2Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, United States

Nitric oxide reductase (cNOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide to nitrous oxide, a key step in bacterial denitrification. The enzyme is expressed from an operon norCBQDEF and is purified as a NorCB heterodimer. In this study we investigate the function of the nor accessory genes norQDEF and their role in cNOR assembly. We found that cNOR can be expressed also in absence of the nor accessory genes yielding a stable NorCB dimer that contains all heme cofactors. However, the produced protein is inactive and lacks the non-heme iron cofactor FeB. Mutational analysis of the nor accessory genes showed that only NorQ and NorD are essential for active cNOR and that both proteins are most likely involved in Fe insertion into cNOR. This is remarkable since Fe cofactor binding in other metalloproteins often occurs spontaneously. NorQ belongs to the large bacterial family of MoxR AAA+ ATPases, proteins that were suggested to work as molecular chaperons primarily mediating metal insertion events. Our current work focuses on the structural and functional characterization of the NorQ and NorD accessory proteins and their involvement in cNOR maturation.

P10 /9 Graphene-functionalized interface: ballistic electron transport Elena Lacatus Polytechnic University of Bucharest, Romania

Designing biocompatible layers of biosensing devices means to functionally integrate them with the biological support, beyond the adhesion and decohesion functionality [1,2], through a biomimetic modeling of the interface signaling. Graphene –functionalized biosensing layers are using either porous-elastomer matrices, or are 3D printed directly on the biological support (skin) [3]. Modeling and simulating the carrier transport mechanism was considered for the both sides of the sensing interface and within the functionalized biomimetic interface layer, through the ballistic transport of electrons. A model of the entanglement of electrons from biosensor interface and GO-based layer [4], having a ballistic transport of electrons and quantum sensitivity describes the biosensing phenomena, is completing the electrochemical model with a real-time quantum entanglement.

References 1. E. Lacatus, Self-Assembled Biofunctionalized Graphene Oxide Models for Nanomedicine, Materials Today: Proceedings, Volume 4, Issue 11, Part 2, 2017, ISSN: 2214-7853, p. 11554-11563, DOI: 10.1016/j.matpr.2017.09.066, (2017) 2. E. Lacatus, Charge carrier transfer in functionalized biomimetic sensing nanostructures, DOI: 10.1016/j.bbabio.2016.04.265, Biochimica et Biophysica Acta (BBA) - Bioenergetics Volume 1857 (2016) 3. E. Lacatus, Modeling a multilayered graphene biosensing structure, 6th International Conference on Advanced Nanomaterials (ANM), Aveiro, Portugal, July 20-22, 2015, Materials Today-Proceedings, Volume: 3 Issue: 8 Pages: 2635-2645 (2016) 4. E. Lacatus, Modeling Bio-Sensing Functionalized Graphene Building Blocks under Environmental Stimuli, 10.13140/RG.2.2.15582.54088, COMSOL Technical Papers and Publications, COMSOL Conference 2016, Munich, ISBN: 978-0- 9910001-3-5; ISSN: 2372-2215 (2016)

P10 /10 Towards Fast Simulation Methods Based on Quantum Mechanics Pedro E. M. Lopes Fastcompchem.com

Computational chemistry was initially based on quantum mechanical methodologies, but it proved impossible to study large systems, such as large biologic macromolecules. This prompted the development of molecular mechanics where systems are described at the atomic level using empirical force fields. Nonetheless, methods based on force fields have severe problems that are well known. For example, these methods have problems describing the rich coordination chemistry of transition metals or physical phenomena such as charge transfer. The requirement of specific parametrization also limits their applicability. There is clearly a need to develop new computational methods based on quantum mechanics to study large and heterogeneous systems. Quantum based methods are typically limited by the calculation of Electron Repulsion Integrals (ERIs) and diagonalization of large matrices. Initial work focused on the development of fast techniques for the calculation of ERIs. The first generation of our methodology was published recently [1]. Subsequently, the second and third generations of the algorithms were developed and are several orders of magnitude faster. After the third generation of the ERI, work has begun on the implementation and testing of algorithms for diagonalization of large sparse matrices. The goal is to use the new algorithms for diagonalization and ERIs computation in a new generation of semi- empirical methods for biologic simulations, materials science and drug-design. The new computational method is based on a highly parameterized tight-binding Hamiltonian that incorporates terms for electron-electron repulsions, electron-nucleus attractions and dispersion. Results from the diagonalization of large sparse matrices will be presented. The test systems will be small solvated proteins (ex. Crambin). The timings of the diagonalization algorithms will be presented and discussed. Special emphasis will be placed on the calculation of important properties that cannot be captured by classical force fields, thus highlighting the need for fast computational methods based on quantum mechanics. The parameterization strategies of the semi-empirical method will be discussed.

1. P.E.M. Lopes, Fast calculation of two-electron-repulsion integrals: a numerical approach, Theor Chem Acc 136 (2017) 112

P10 /11 Remodeled cardiolipin modify the biophysical properties of the mitochondrial inner membrane in response to the protonmotive force Luis Alberto Luévano-Martínez Department of Parasitology, Instituto de Ciências Biomédicas. Universidade de São Paulo, Brazil

Cardiolipin (CL), a key lipid component of the inner mitochondrial membrane, is synthesized in situ as a saturated lipid and after its synthetized it is remodeled to more unsaturated species by a cardiolipin-specific phospholipase A2 (cld1 in yeast) and the transacylase, tafazzin. Previous studies show that yeast lacking this phospholipase cld1 accumulates saturated cardiolipin without any effect on the bioenergetics properties of those mitochondria [1, 2]. This absence of bioenergetic phenotypes leads important cues about why this Eukaryotic-specific lipid modifying pathway appeared and was retained by positive selection in Metazoans. In this work, I compared the biophysical properties of the mitochondria inner membrane from wild type yeast cells (with mostly unsaturated CL species) with those of a cld1Δ (with saturated CL species). Liposomes made with isolated wild type lipids were found to be more fusogenic and prone to the formation of hexagonal inverted phases than liposome made from the knockout strain. Interestingly, I observed that modulating the protonmotive force, each component of this force seems to exert different effects on phospholipid hydration and packing of mitochondrial inner membrane phospholipids. Moreover, I found that although bioenergetic parameters are not affected by the lack of CL remodeling, mitochondrial lipids from wild type cells are more responsive to the presence of a protonmotive force, thus modifying their biophysical characteristics

References: [1] M.G Baile, M Sathappa, Y.W. Lu, E. Pryce, K. Whited, J.M. McCaffery, X. Han, N.N. Alder, S.M. Claypool, Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast. J. Biol. Chem. 289 (2014) 1768–1778. [2] C. Ye, W. Lou, Y. Li, I.A. Chatzispyrou, M. Hüttemann, I. Lee, R.H. Houtkooper, F.M. Vaz, S. Chen, M.L. Greenberg. Deletion of the cardiolipin-specific phospholipase Cld1 rescues growth and life span defects in the tafazzin mutant: implications for Barth syndrome. J Biol Chem. 289(6) (2014) 3114-3125.

P10 /12

+ Role of F0F1-ATPase in H flux by Escherichia coli during lactose fermentation at different pHs Satenik Mirzoyan2, Armen Trchounian1,2, Karen Trchounian1,2 1Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology, Yerevan State University, Yerevan, Armenia 2Scientific-Research Institute of Biology Faculty of Biology, Yerevan State University, Yerevan, Armenia

E. coli is able to ferment lactose, which further inside the cell breaks down to galactose and glucose.The lactose entry into the bacterial cell is accompanied by 3 protons during respiratory conditions but fermentative conditions is not studied well. E. coli BW25113 wild type cells were grown with 5 g/l lactose concentration at different pH values (5.5-7.5). To understand the role of F0F1-ATPase N,N´- dycyclohexylcarbodiimide (DCCD) specific inhibitor has been applied, which becomes specific under anaerobic conditions. H+ export was determined during lactose fermentation at pH 7.5 and the flux was 1.1 mM/min. DCCD inhibited the H+ export and it was + + shown the H is imported into the cells with the 0.3 mM/min flux. The data clearly show that F0F1-ATPase is responsible for all H export at this pH. Moreover, at pH 6.5 no any import of H+ was detected. Especially, the H+ flux was 1 mM/min and DCCD inhibited the H+ flux + by 65%. At this pH H are only exported from the cell and it is suggested that, besides F0F1-ATPase, there are other systems exporting H+. Interestingly at low pH (pH 5.5) the H+ flux was very low 0.1 mM/min which might be due to that lactose utilization at pH 5.5 is higher compared to other pHs and the external environment is acidic so the cells are not pumping protons out to maintain the cytoplasmatic pH + + stable. Moreover, DCCD inhibited H flux was the same as without inhibition which shows that no any role of F0F1-ATPase in H flux is considered. Taken together, it might be concluded that during lactose fermentation depending on external pH the H+ flux value is changed. + Moreover, role of F0F1-ATPase in H export is shown at pH 7.5 and partially at pH 6.5 but not at pH 5.5. In addition, it is suggested that at slightly acidic and acidic pHs there are other H+ exporting systems and one of them might be E. coli [Ni-Fe] hydrogenases which are + can export H itself or convert it to H2.

P10 /13 Surface-enhanced Raman spectroscopy as a new tool to study peculiarities of mitochondria bioenergetics Evelina I. Nikelshparg1, Nadezda A. Brazhe1, Adil A. Baizhumanov1, Leonid I. Deev1, Anna A. Semenova2, Asya S. Sarycheva2, Eugene A. Goodilin2,3, Olga Sosnovtseva4, Georgy V. Maksimov1 1Faculty of Biology, Moscow State University, Moscow, Russia 2Faculty of Materials Sciences, Moscow State University 3Faculty of Chemistry, Moscow State University 4Faculty of Medical and Health Sciences, Copenhagen University

Raman spectroscopy has been applied to study heme conformation in cytochromes for a long time. However, the intensity of Raman signal from oxidized hemes is too low to perform Raman study of intact non-treated mitochondria. We created plasmonic nanostructured surfaces (NSS) to investigate cytochromes inside living mitochondria via surface-enhanced Raman spectroscopy (SERS). SERS provides great enhancement of Raman signal from biomolecules in a short distance to plasmonic nanostructures. Placing isolated mitochondria onto NSS does not cause neither membrane disruption nor uncoupling. SERS spectra from mitochondria on NSS represents spectra of cytochrome C heme. We demonstrated that application of substrate–uncoupler–inhibitor titration protocol resulted in significant changes of cytochrome C heme properties and redox state. All results were in a good agreement with oximetry and ATP production measurements. That makes SERS a new additional tool to investigate mitochondria bioenergetics. Furthermore, we showed that the SERS signal is sensitive to morphological properties of mitochondria such as distant between inner and outer membrane, which can be dependent on traits of an organ used for mitochondria isolation or modulated by osmolality and ionophore application. EIN acknowledges financial support from RFBR grant mol_a № 18-34-00503.

P10 /14 How mitochondrial (ultra)structure affects mitochondrial function Elianne P Bulthuis, Cindy EJ Dieteren, Jori AL Wagenaars, Job Berkhout, Laura FB Hesp, Peter HGM Willems, Merel JW Adjobo-Hermans, Werner JH Koopman Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud Center for Mitochondrial Medicine, Radboudumc, Nijmegen, The Netherlands

Mitochondria are cellular compartments with a highly dynamic structure. Evidence was provided that the shape and volume of the mitochondrial matrix can affect the kinetics of mitochondrial reaction diffusion systems [1]. It is important to quantitatively understand such reaction systems because human pathologies are generally associated with changes in mitochondrial (ultra)structure. To this end, we previously combined live-cell FRAP (fluorescence recovery after photobleaching) analysis with 3D random-walk diffusion modeling to determine the solvent-dependent diffusion constant of fluorescent proteins in HEK293 cells. This demonstrated that mitochondrial cristae substantially hinder protein diffusion [2]. Here we address the hypothesis that cristae allow size-dependent protein diffusion and thereby function as a molecular sieve. To this end, we generated HeLa cell lines that stably expressed GFP-concatemers of different length in the mitochondrial matrix (GFP1, GFP2, GFP3 and GFP4). Whereas GFP1 and GPF2 were fully mobile, GFP3 and GFP4 became progressively immobilized. The latter was enhanced in cells treated with chloramphenicol (CAP), an inhibitor of mitochondrial DNA translation that also modulated mitochondrial matrix architecture. We conclude that larger proteins cannot freely diffuse within the mitochondrial matrix, suggesting that mitochondrial internal structure affects protein diffusion in a size-dependent manner.

References: 1. L. Lizana, B. Bauer, O. Orwar, Controlling the rates of biochemical reactions and signaling networks by shape and volume changes, Proc. Natl. Acad. Sci. U.S.A., 105 (2008) 4099-4101 2. C.E.J. Dieteren, S.C.A.M. Gielen, L.G.J. Nijtmans, J.A.M. Smeitink, H.G. Swarts, R. Brock, P.H.G.M. Willems, W.J.H. Koopman, Solute diffusion is hindered in the mitochondrial matrix, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 8657-8662

P10 /15 Protons at the membrane water interface Ewald Weichselbaum, Peter Pohl Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria

The efficiency of cellular energetics crucially depends on proton diffusion along biological membranes. However, the origin of proton affinity to the membrane surface so far remained enigmatic. We were able to show that proton binding by surface anchored moieties is not involved [1]. On the contrary, the peculiar structure of surface water adjacent to a hydrophobic interface ensures that interfacial proton migration is very much favored as compared to the diffusion in the perpendicular direction [2]. We now quantified the substantial ‡ Gibbs activation energy barrier ΔG r that opposes proton surface to bulk release by photo-releasing protons from a membrane patch at different temperatures and monitoring their arrival at a distant patch. The results disproved that quasi-equilibrium exists between protons ‡ in the near-membrane layers and in the aqueous bulk. Instead, non-equilibrium kinetics is consistent with this experiment. ΔG r only contains a minor enthalpic contribution that roughly corresponds to the breakage of a single hydrogen bond [3]. To reconcile the delayed proton surface-to-bulk release with proton’s weak bonding to surface water molecules, we also accounted for the contribution of membrane surface charge.

[1] A. Springer, V. Hagen, D.A. Cherepanov, Y.N. Antonenko, P. Pohl, Protons migrate along interfacial water without significant contributions from jumps between ionizable groups on the membrane surface, Proc. Natl. Acad. Sci. U.S.A, 108 (2011) 14461-14466. [2] C. Zhang, D.G. Knyazev, Y.A. Vereshaga, E. Ippoliti, T.H. Nguyen, P. Carloni, P. Pohl, Water at hydrophobic interfaces delays proton surface-to-bulk transfer and provides a pathway for lateral proton diffusion, Proc. Natl. Acad. Sci. U.S.A, 109 (2012) 9744-9749. [3] E. Weichselbaum, M. Österbauer, D.G. Knyazev, O.V. Batishchev, S.A. Akimov, T.H. Nguyen, C. Zhang, G. Knör, N. Agmon, P. Carloni, P. Pohl, Origin of proton affinity to membrane/water interfaces, Scientific Reports, 7 (2017) 4553.

P10 /16 The Energy Metabolism of Leishmania as a Drug Target Eduardo Rial, Alejando Lastra-Romero, Paula Martínez-de-Iturrate, Montserrat Nácher-Vázquez, Luis Rivas Centro de Investigaciones Biológicas, CSIC, Madrid, Spain

Protozoan parasites belonging to the genus Leishmania cause important human and animal infections, termed leishmaniasis, a neglected tropical disease affecting an estimated 12 million people around the world. Its life cycle includes two major stages. The flagellated promastigote dwells inside the gut of its vector, the sandfly, that transmits the parasite by biting its vertebrate host. The promastigotes are incorporated into the phagolysosome of the macrophage where they differentiate into the non-motile aflagellated intracellular amastigotes. Inside the phagolysosome the amastigotes proliferate to eventually cause the lysis of the macrophage and infect new cells. During its life cycle, Leishmania has to adapt to different environments both in the insect vector and in the vertebrate host and this involves profound changes in its energy metabolism [1]. Treatment of leishmaniasis is currently limited to chemotherapy and the few available drugs are threatened by rising resistance and severe side effects. Since the development of new drugs is urgently needed [2], the metabolic adaptations are an appealing target due to their essential role in parasite survival. The aim of our work is (1) to develop strategies to investigate the energy metabolism of Leishmania and (2) to apply these tools in the quest for new drugs that impair the metabolic adaptations used by the parasite to thrive inside the vertebrate host. Characterization of the energy metabolism of the two parasite forms and its application to the investigation of drug action will be presented.

1. T. Naderer, M.J McConville, The Leishmania-macrophage interaction: a metabolic perspective, Cell Microbiol. 10 (2008) 301-308 2. B. Zulfiqar, T.B. Shelper, V.M. Avery, Leishmaniasis drug discovery: recent progress and challenges in assay development, Drug Discov. Today 22 (2017) 1516-1531

P10 /17 A novel hydroxylamine oxidoreductase from a thermoacidophilic volcanic methanotroph for survival of high ammonia stress Wouter Versantvoort1, Arjan Pol1, Huub JM Op den Camp1, Mike SM Jetten1, Boran Kartal2 and Joachim Reimann1 1Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen, AJ, The Netherlands 2Microbial Physiology Group, MPI for Marine Microbiology, Bremen, Germany

Aerobic methanotrophs make a living by respiring methane to CO2. The first step in this process is the O2-dependent conversion of methane to methanol by either a soluble or a membrane-bound methane monooxygenase (s- or pMMO respectively). pMMO can also oxidize ammonia to hydroxylamine. However, the produced hydroxylamine is a potent inhibitor of methanol dehydrogenase, necessitating its rapid turnover to prevent the inhibition of the energy-conserving steps in respiration. Interestingly, many methanotrophic bacteria encode a hydroxylamine oxidoreductase (HAO) in their genomes. HAOs play a crucial and central role in the metabolism of aerobic and anaerobic ammonia-oxidizing bacteria by oxidizing hydroxylamine to NO [1][2]. Here, were purified a HAO from the methanotrophic bacterium Methylacidiphilum fumariolicum SolV that lives in volcanic regions at pH 2 and 65 °C. The SolV HAO possesses the characteristic P460 active site chromophore and catalyzes the rapid oxidation of hydroxylamine to NO. This NO is further converted to nitrite via an unknown enzyme, as nitrite is the end product of ammonia oxidation in SolV [3]. Although this methanotroph HAO is functionally identical to the previously characterized HAOs, its physiological role in the metabolism of (volcanic) methanotrophs is most likely different. We propose that HAO in methanotrophs allows them to thrive under high environmental ammonia concentrations, such as those observed in volcanic mudpots, by preventing the accumulation of inhibitory hydroxylamine levels.

[1] J.D. Caranto & K.M. Lancaster, Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase, PNAS 114 (2017) [2] W.J. Maalcke et al., Structural basis of biological NO generation by octaheme oxidoreductases, JBC 289 (2014) [3] S.S. Mohammadi et al., Ammonia oxidation and nitrite reduction in the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, Front. Microbiol. 8 (2017)

P10 /18 Measuring real-time bioenergetic behaviour of electrically stimulated muscle cells Anthony Wynne, Charles Affourtit School of Biomedical Sciences, University of Plymouth, United Kingdom

Bioenergetic failure links to altered mitochondrial function and underpins a wide range of medical disorders. It is becoming increasingly clear that decreased ‘bioenergetic health’ is an early warning sign of cell dysfunction and consequent disease. Cellular bioenergetics can be measured in intact cells by extracellular flux analysis. Such measurement will allow disease diagnosis at earlier stages than currently possible. Moreover, it will allow mechanistic understanding of disease pathology thus enabling identification of novel drug targets that should improve treatment. A general challenge of cell-based bioenergetics assays is to perform them in cells that reflect the physiological environment of the systems they model. Driven by our research on skeletal muscle [1, 2], we have adapted existing extracellular flux technology [3]. This innovative adaptation now allows real-time measurements of mitochondrial function in skeletal and cardiac muscle models facing physiological contractile workloads that can be controlled by electrical stimulation. Although initially conceived for use on an extracellular flux platform, our invention could, in principle, be applied to any plate reader and may thus offer invaluable alternatives for current drug discovery and safety screens. Moreover, application of the novel technology to human skeletal and cardiac muscle cells would lower the dependency of such screens on animal models.

1. Affourtit, C. (2016) Mitochondrial involvement in skeletal muscle insulin resistance – a case of imbalanced bioenergetics. BBA – Bioenergetics 1857, 1678-1693 2. Affourtit, C., Bailey, S. J., Jones, A. M., Smallwood, M. J. and Winyard, P. G. (2015) On the mechanism by which dietary nitrate improves human skeletal muscle function. Front. Physiol. 6:211 3. Wynne A. G., Fry, N. and Affourtit C. Measuring the bioenergetic behaviour of electrically stimulated skeletal muscle cells. Patent filed to the UK Intellectual Property Office (Application Number 1714476.7) on 8th September 2017.

P10 /19 Mitochondrial uncouplers induce CpG methylation of the ICAM1 gene promoter in endothelial cells Roman A. Zinovkin2, Anastasia I. Kalashnikova1, Anastasia S. Prikhodko2, Ludmila A. Zinovkina1 1Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia 2Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

Epigenetics is one of the mechanisms to regulate gene expression. DNA methylation at CpG sites of gene promoters provides long-term transcriptional repression. Mitochondrial uncoupling is known to decrease expression of some cell adhesion molecules (e.g. ICAM1, Intercellular Adhesion Molecule 1) in endothelial cells. We have tested the theory that this decrease is mediated via epigenetic changes in promoter region of ICAM1 gene. Genomic DNA was purified from endothelial cells incubated with various mitochondrial uncouplers of oxidative phosphorylation. The bisulfite-treated DNA was used for subsequent PCR, molecular cloning and sequencing. We have found significant increase in CpG methylation level in the DNA of the cells treated with mitochondrial uncouplers.

This work was supported by Russian Foundation for Basic Research, project №18-04-01110.

P10 /20 Mitochondria: An old organelle with new functions Zakaria A. Almsherqi Department of Physiology, National University of Singapore, Singapore

The most prominent role of the mitochondria is to produce the energy currency of the cell. Moreover, the mitochondria play a central role in many other metabolic tasks, such as cell signalling, regulation of the membrane potential, apoptosis, calcium signalling, regulation of cellular metabolism and steroid synthesis. However, recent data show that mitochondria could have unique functions in response to cellular stress/demand. Recently developed new techniques show that mitochondrial membranes are highly pleomorphic and their organization can vary tremendously. The classical lamelliform may transform into vesicular, tubular or cubic membrane (CM) morphology as a consequence to metabolic demands and/or pathological stress. CM represents highly curved, three-dimensional nano-periodic structures that correspond to mathematically well-defined triply periodic minimal surfaces. An example of highly organized cubic membrane morphologies has been observed in the inner mitochondrial membranes of tree shrew’s retinal tissue known as megamitochondria (MG). These mitochondria are unique in their size and ultrastructural inner membrane arrangement. Using transmission electron microscopy (TEM) images, analytical modelling and computer-generated TEM micrographs, we investigated the optical properties of MG observed in the retina of Tupaia species. Our 3D simulation of MG membrane arrangements shows that MG might act as (1) ball-lens focuses the light onto the photoreceptor as well as (2) selectively filtering out high energy photons allowing the rest of light spectrum to transmit. In this context, MG might act as a ‘biophotonic’ filter offering a protective mechanism against exposure of short wavelengths of UV for the retina. Interestingly, a closely similar MG observed in Amoeba Chaos upon starvation would have a different function to play. These mitochondria may offer an optimal intracellular membrane organization for intracellular macromolecules protection against oxidative damage.

P10 /21 Resolving the mechanism of proton pumping coupled to hydride transfer in E. coli Transhydrogenase Simone Graf1,2, Sangjin Hong3, Robert Gennis3, Christoph von Ballmoos1 1University of Bern, Department of Chemistry and Biochemistry, Bern, Switzerland 2Graduate School for Cellular and Biomedical Sciences, Bern, Switzerland 3Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, USA

The proton translocating transhydrogenase (TH) is an important enzyme involved in maintaining the redox states of the NAD and NADP pools that is found in the inner membranes of mitochondria and cytoplasmic membrane of bacteria. In the forward direction, the transhydrogenase uses the proton motive force (pmf), established by the respiratory chain, to catalyze hydride transfer from NADH to NADP+, generating NADPH which is required in many biosynthetic reactions or the removal of reactive oxygen species (ROS) which is vital in living organisms. In the non-natural reverse reaction the enzyme acts as a proton pump that is capable of establishing a pmf.[1] Structurally, the TH consists of three domains (dI-III), two of which (dI, dIII) are hydrophilic and responsible for nucleotide binding, while dII is embedded in the membrane and responsible for proton transport. In the membrane, the protein is organized as a dimer, with dIII being in asymmetric conformation as evidence by the recently solved structure of the Thermus thermophilus dII domain and of the holoenzyme. It is proposed that swiveling of dIII between two conformations contributes to the binding change mechanism that is thought to couple hydride transfer to proton translocation. The exact mechanism, however is unknown, and more functional and structural work is required.[2] Here we present our present progress in investigating the functional properties of the Escherichia coli TH in proteoliposomes. Reconstituting the protein into lipid vesicles, we study the proton translocation in both directions in dependence of membrane potential and proton gradient generated by other enzymes such as the ATP-synthase. In order to elucidate how the pmf can support the binding change mechanism, we have also co-reconstituted TH with the ATP synthase into proteoliposomes. This experimental setup allows us to mimic the physiological situation, in which a pmf is established and maintained across the membrane. We show that the pmf generating capacity of TH in the reverse direction is capable to support ATP synthesis. Currently, mutant variants are assessed with these assays to get further insights into the molecular mechanism of this enzyme.

[1] J.B. Jackson, J.H. Leung, C.D. Stout, L.A. Schurig-Briccio, R.B. Gennis, Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel, FEBS Lett. 589 (2015) 2027–2033. doi:10.1016/j.febslet.2015.06.027. [2] J.H. Leung, L. a Schurig-Briccio, M. Yamaguchi, A. Moeller, J. a Speir, R.B. Gennis, C.D. Stout, Structural biology. Division of labor in transhydrogenase by alternating proton translocation and hydride transfer., Science. 347 (2015) 178–81. doi:10.1126/science.1260451.