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 Bioenergetics 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 Biophysics, Stockholm University, 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, Structural biology. 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