Protein and Lipid Interactions Within the Respiratory Chain

Tobias Nilsson Protein and lipid interactions within the respiratory chain

Protein interactions and lipid within respiratorythe chain Studies using membrane-mimetic systems

Tobias Nilsson

Tobias Nilsson Recieved his M.Sc. in Biochemistry from the department of Biochemistry and Biophysics at Stockholm University in 2014. Stopped drinking coffee in 2009. This thesis was produced on tremendous amounts of tea.

ISBN 978-91-7797-845-9

Department of Biochemistry and Biophysics

Doctoral Thesis in Biochemistry at Stockholm University, Sweden 2019 Protein and lipid interactions within the respiratory chain Studies using membrane-mimetic systems Tobias Nilsson Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Friday 22 November 2019 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B.

Abstract Energy conversion from nutrients to ATP is a vital process in cells. The process, called oxidative phosphorylation (OXPHOS) is performed by a combination of membrane-bound proteins. These proteins have been studied in great detail in the past, however much is still unknown about how they interact with each other. Studying the OXPHOS proteins in their native environment can be difficult due to the complexity of living cells. By isolating parts of the OXPHOS system and inserting them into membrane-mimetic systems it is possible to investigate their functions in a controlled environment. In the work presented here, we co-reconstituted several of these proteins into liposomes made from synthetic lipids. We demonstrated production of ATP at steady-state conditions with the ATP synthase, driven by proton pumping by cytochrome bo3. Introduction of anionic lipids decreased the coupled activity and we could correlate this effect to weaker interactions between ATP synthase and cytochrome bo 3 in the membrane. We also reconstituted cytochrome c oxidase (CytcO) from Saccharomyces cerevisiae with Respiratory supercomplex factor 1 (Rcf1) into liposomes and submitochondrial particles (SMPs). Loss of Rcf1 has previously been found to result in a lower CytcO activity. We found that activity could be restored upon co-reconstitution of CytcO with Rcf1, but only after unfolding and re-folding of the latter, which shows that Rcf1 can adopt two configurations in the membrane.

Keywords: Cytochrome c oxidase, ATP synthase, Bioenergetics, membrane-mimetics, Rcf1, liposomes, oxidative phosphorylation, lipids, protein-protein interactions.

Stockholm 2019 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-173617

ISBN 978-91-7797-845-9 ISBN 978-91-7797-846-6

Department of Biochemistry and Biophysics

Stockholm University, 106 91 Stockholm

PROTEIN AND LIPID INTERACTIONS WITHIN THE RESPIRATORY CHAIN

Tobias Nilsson

Protein and lipid interactions within the respiratory chain

Studies using membrane-mimetic systems

Tobias Nilsson ©Tobias Nilsson, Stockholm University 2019

ISBN print 978-91-7797-845-9 ISBN PDF 978-91-7797-846-6

Printed in Sweden by Universitetsservice US-AB, Stockholm 2019 “It is well known that a vital ingredient of success is not knowing that what you're attempting can't be done.”

- Terry Pratchett

List of publications & manuscripts

I. Von Ballmoos, C., Biner, O*., Nilsson, T.*, & Brzezinski, P. ( 2016 ). Mimicking respiratory phosphorylation using purified enzymes. Biochimica et Biophysica Acta - Bioenergetics, 1857(4), 321–331.

II. Nilsson, T., Lundin, C. R., Nordlund, G., Ädelroth, P., Von Ball- moos, C., & Brzezinski, P. ( 2016 ). Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis. Scien- tific Reports, 6(March), 1–11.

III. Sjöholm, J., Bergstrand, J., Nilsson, T., Šachl, R., Von Ballmoos, C., Widengren, J., & Brzezinski, P. ( 2017 ). The lateral distance between a proton pump and ATP synthase determines the ATP- synthesis rate. Scientific Reports, 7(1), 1–12.

IV. Nilsson, T., Schäfer, J., Zhou, S., Ädelroth P., & Brzezinski, P. Ac- tivation of Cytochrome c Oxidase from Saccharomyces cerevisiae by Addition of Respiratory Supercomplex Factor 1. (Manuscript) *Equal contributions

Additional papers

• Knopp, M., Gudmundsdottir, J., Nilsson, T., König, F., Warsi, O., Rajer, F., Ädelroth, P. & Andersson, D. (2019) De novo emergence of peptides conferring antibiotic resistance. mBio, 10 (3) e00837- 19; DOI: 10.1128/mBio.00837-19 Contents

Introduction ...... 15

The respiratory chain ...... 16

Terminal oxidases ...... 19 Cytochrome c oxidase ...... 20 Ubiquinol oxidase ...... 23

ATP synthesis ...... 25 Oxidative phosphorylation ...... 25

F1FO ATP synthase ...... 26

Biological membranes ...... 29 Fluid mosaic model ...... 29 Membrane phase transitions ...... 30 Lateral proton translocation ...... 31 Lipid properties and membrane compositions ...... 31 Membrane-mimetic systems ...... 33 Arrangement of respiratory enzymes ...... 35

Respiratory supercomplexes ...... 37 Respiratory supercomplex factors ...... 39

Energy coupling ...... 41

Methods ...... 43 Protein reconstitution in liposomes ...... 43 Bioluminescence assay to measure ATP synthase rates ...... 43 Oxygen reduction rates ...... 44 Tunable resistive pulse sensing ...... 45 Detection of pH gradients ...... 46 Fluorescence correlation spectroscopy ...... 47

Main findings ...... 49

Populärvetenskaplig sammanfattning ...... 53

Acknowledgements ...... 55

References ...... 57

Abbreviations

CL – Cardiolipin Cryo-EM – Cryogenic electron microscopy cyt. c – Cytochrome c Cyt cO – Cytochrome c oxidase IMM – Inner mitochondrial membrane IMS – Intermembrane space PA – Phosphatidic acid PC – Phosphatidylcholine PE – Phosphatidylethanolamine PG – Phosphatidyl-glycerol pmf – Proton motive force PS – Phosphatidylserine Rcf – Respiratory supercomplex factor TMH – Transmembrane helix

14 Introduction

Just like all our home devices need electricity to run, cellular processes need to obtain energy from the molecule ATP. But just like electricity from our power outlets at home isn’t the actual source of energy, ATP is simply the converted form of energy derived from various food sources. The final steps of the process of converting the free energy stored in food to ATP takes place at biological membranes and are performed by a number of enzymes working in tandem. These were discovered and characterized during the last decades, however many aspects of their function are still unknown, especially in regards to how they interact with each other and how they are regulated. In the work presented in this thesis we have attempted to expand the knowledge of how this interplay and regulation may work by studying mainly other processes than the enzymes themselves.

In Paper I we developed a method for mimicking parts of the energy- conversion process in vitro by co-reconstituting terminal oxidases and the F1FO ATP synthase into a lipid bilayer, and showed that we could use this system to gain functional insights of the coupled system. We further utilized this approach in Paper II where we investigated the effect of the lipid composition on the coupled activity of these two enzymes, in an attempt to understand how protons move between them. Our results indicate that the lipid composition may be used by bacterial cells as means to regulate ATP synthesis. In Paper III we showed that the effect on ATP synthesis, seen at different lipid compositions, could be explained mainly in terms of changes in lipid-dependent protein-protein distance. In Paper IV we investigated the interplay between the terminal oxidase, cytochrome c oxidase (Cyt cO) and a small regulatory peptide, respiratory supercomplex factor 1 (Rcf1) from Saccharomyces cerevisiae . We found that when co-reconstituting the peptide into mitochondrial membranes with Cyt cO from a rcf1Δ strain the enzymatic activity of the Cyt cO increased. Our results suggest that Rcf1 could both enhance ligand binding of cytochrome c (cyt. c) and has a direct impact on the catalytic activity of Cyt cO.

Figure 1 Schematic representation of the aerobic respiratory chain. Electrons are delivered to Complexes I and II from NADH and succinate, respectively. The enzymes use the electrons to reduce coenzyme Q, which is in turn oxidized by Com- plex III, which delivers the electron to Complex IV via the soluble protein cyt. c. Blue arrows represent the electron flow between complexes, grey arrows show translocation of protons, however not the stoichiometry. Complex IV also takes up protons from the n-side (and electrons from the p-side) for the catalytic reaction. Complex III releases protons to the p-side from the oxidization of coenzyme Q. During the redox-cycle, two protons are also taken up from the n-side. The free energy derived from transport of protons from the n-side to the p-side is used to drive ATP synthesis, as protons flow back through the ATP synthase, shown by a grey arrow.

16 The respiratory chain

The respiratory chain, shown schematically in Figure 1, is a coupled enzymatic system found in aerobic organisms. In mammals it is found in the inner mitochondrial membrane (IMM) and is composed of four transmembrane redox-enzyme complexes. Three of these complexes contribute to the build-up of a transmembrane proton electrochemical gradient, often referred to as the proton motive force ( pmf ), which is in turn utilized by the F 1FO ATP synthases to drive catalysis of ADP to ATP. Although not part of the respiratory chain, the ATP synthase is sometimes referred to as Complex V.

Complex I of the respiratory chain, the NADH:Ubiquinone oxidoreductase, accepts electrons from NADH from the citric-acid cycle and donates them to the pool of ubiquinone, also known as co-enzyme Q, found inside the IMM. The enzyme also pumps four protons per NADH across the membrane, into the mitochondrial inner membrane space (IMS) 1.

Ubiquinone is also reduced by complex II, succinate dehydrogenase, which is a part of both the respiratory chain and the citric acid cycle. It receives electrons by oxididation of succinate to fumarate and it is the only respiratory-chain enzyme that does not translocate protons. However, as it contributes to maintaining the reduced “Q pool”, it is still considered to be a part of the respiratory chain. It also removes protons from the matrix, where the Q-binding site and proton uptake site is located 2,3 .

The reduced Q pool maintained by Complexes I and II is re-oxidized by Complex III, the cytochrome bc 1 complex, which delivers electrons from ubiquinone to cyt. c, a soluble globular 12-13 kDa heme protein found inside the IMS. During this step, four protons are released into the IMS from oxidation of coenzyme Q 4,5 . Cyt. c donates electrons to complex IV, cytochrome c oxidase, which reduces molecular oxygen to water. Cyt cO contributes to the pmf by taking up electrons and protons from opposite sides of the membrane and linking this redox reaction to proton pumping 6,7 .

18 Terminal oxidases

Terminal oxidases is a collective term for the last of the redox-enzymes in the respiratory chain. They have the vital role of donating electrons from the respiratory chain to O2 thereby maintaining an electron flux. The reaction catalyzed by these enzymes is electrogenic, i.e. they contribute to maintaining the pmf by pumping protons across the membrane. Terminal oxidases use molecular oxygen as an electron acceptor, as is the case for e.g. mitochondria and aerobic bacteria, but there are species of archea and bacteria with different metabolic strategies. Instead of terminal oxidases, there are enzymes using other substrates as final electron acceptors, such as sulfur, nitrogen compounds, halogenated organics or transition metals 8.

Figure 2 structures of terminal oxidases. The structures of the cytochrome bo3 11 (ubiquinol oxidase) from E. coli (Left, PMID: 1FFT) and aa 3 Cyt cO from S. cerevisiae 12 (Right, PMID: 6HU9). Subunits I, II and III are shown in yellow, teal and purple respectively. Supernumerary subunits are shown in green, with the exception of Cox12 and Cox13 in the yeast structure, shown in orange and pink, respectively. Arrows point at the Q-site and Cu A-site, where electrons are received. Subunit IV in the cytochrome bo 3 is also shown in green.

19 The superfamily of heme/copper terminal oxidases can be divided into 3 classes, the A-, B- and C-type oxidases 9,10 . A-type oxidases include many of the most studied oxidases, i.e. the cytochrome aa 3 oxidases found in mitochondria and some bacterial species like Rhodobacter sphaeroides , as well as the ubiquinol oxidases found, e.g. in Escherichia coli (E. coli ).

Cytochrome c oxidase

Cytochrome c oxidase is found in the IMM of eukaryotes such as Humans, Saccharomyces cerevisiae and in many aerobic bacterial species, such as Rhodobacter sphaeroides and Paracoccus denitrificans . It catalyzes reduction of O 2 to water, while also translocating protons from the negative n-side to the positive p-side of the membrane.

- + + O2 + 4 e (cyt. c) + 8 H (n-side) → 2 H 2O + 4 H (p-side)

While the oxidases differ in their subunit composition between organisms, especially between bacteria and eukaryotes, there are three conserved core subunits which contain the active site as well as most residues involved in the proton uptake pathways required for reducing O 2 to H 2O and pumping protons across the membrane 13,14 . The active site is composed of a heme- and a copper ion (Figure 3A). In eukaryotes the three core subunits are encoded in the mitochondrial DNA, while the remaining “supernumerary” subunits are encoded in the nucleus 15 .

The aa 3 oxidase in bovine heart mitochondria is composed of a total of 13 16 subunits , whereas the S. cerevisiae yeast aa 3 oxidase is composed of 11 17 subunits . In subunit II of e.g. the yeast aa 3 oxidase there is a binuclear cupper cluster, Cu A, located close to the cyt. c binding site. Electrons are transferred from cyt. c to Cu A. Subunit I contains the other three redox centers, heme a, heme a3 and the mononuclear Cu B. Subunit III does not participate in the catalytic reaction, but is an important structural subunit. Four electrons are required for the catalysis of O 2 to water.

Figure 3 Redox components of complex IV. ( A) Cupper-Heme catalytic site of aa 3 oxidase from S. cerevisiae . Hemes a and a3 shown in red, with the iron shown as a sphere and Cu A and Cu B sites shown as blue spheres. (B) Oxidised (Ubiquinone) and reduced (Ubiquinol) forms of Coenzyme Q. The brackets display the polymeric region which differs in length between organisms, between 6-10 repeats.

The electrons tunnel between the redox centers unidirectionally as the redox potential increases for each step 18–20 . Protons for pumping and catalysis are taken up by the D- and K-pathways (Figure 4), named after conserved residues Asp124 and Lys354, respectively (numbering based on P. denitrificans structure)21,22 . Mammalian Cyt cOs have been suggested to have a third proton pathway, the H-pathway 16,23 , however this pathway is not present in R. sphaeroides or P. denitrificans 24,25 Cyt cOs. The role of the H pathway, if any, is presently unclear 25 .

Figure 4 CytcO proton and electron pathways. Residues involved in the D- and K- pathways are shown in orange and green, respectively. The arrows show the direc- tionality of the proton uptake. Cupper atoms are shown in dark blue and hemes in red. Structure is 6HU9 12 .

22 Ubiquinol oxidase

E. coli lacks Complex III and cyt. c, so the terminal cytochrome bo 3 in the respiratory chain is instead a ubiquinol oxidase. Cytochrome bo 3 accepts electrons from ubiquinol Q 8 and in the process releases 4 protons per reduced O2 to the p-side of the membrane, from the oxidation of ubiquinols 26–28 . Subunits I, II and III share homology with the mitochondrial enzyme, however subunit II lacks both the Cu A cluster and a cyt. c binding site. Instead there is a ubiquinol binding site located in Subunit I, involving residues R71, D75 and H98, close to the membrane surface on the p-side of the membrane, suggested based on an analysis of a crystal structure, site directed mutagenesis and the use of spectroscopic techniques 11,29 . A second, “low-affinity” ubiquinol binding site (relative to the previously mentioned) has been suggested, based on co-purifications with between 0-2 Q 8 molecules, depending on detergent, as well as inhibition studies and mutagenesis studies. However, the location of this site (if any) has not yet been identified 28,30 . A hydrogen-bond stabilized ubisemiquinone reaction intermediate has been observed in the “high affinity” binding site 30 . It was suggested that the substrate in the high affinity site acts as a co-factor in place of the Cu A cluster and that the low affinity binding site is where the ubiquinol from the Q-pool binds to deliver electrons.

24 ATP synthesis

Oxidative phosphorylation

Oxidative phosphorylation refers to the process of producing ATP, coupled to enzymatic oxidation of nutrients, such as via the respiratory chain in aerobic organisms. Hydrolysis of ATP is utilized in a multitude of energy- demanding cellular processes. The ATP/ADP ratio in mouse heart is ~100 with the ATP concentration around 5 mM 31,32 . In E.coli the ATP/ADP ratio is around 3.2 and the ATP concentration is roughly 2.6 mM 33 .

Production of cytotoxic reactive oxygen species (ROS), mainly in the form - of superoxide (O 2 ), as a result of electrons leaking into side-reactions with molecular oxygen has been linked to the respiratory chain 34 . ROS formation in mitochondria is especially prominent at high proton gradients, during a state called reverse electron transfer, when electrons are delivered back to Complex I from Complex II via the Q-pool 35,36 . ATP synthase activity have been found to counter this effect, likely due to its role in regulating the pmf , as it uncouples ΔpH37 .

25 F1FO ATP synthase

The enzyme responsible for ATP production is called the F 1FO ATP synthase. In the ATP synthase the protons are transferred from the p- to to the n-side and the Gibb’s free energy released induces conformational changes that drive catalysis 38,39 . The enzyme complex is composed of two multimeric parts, the membrane-spanning F O and the soluble F 1. In E. coli , the F O part is composed of subunits a, b and c, and contains the proton pathway 40,41 . Protons are taken up between the a-subunit and the c-ring. The c-ring is composed of 8-15 c-subunits depending on organism, and during the process of proton uptake / release the c-ring rotates in a clockwise direction during ATP synthesis, as shown in Figure 5. Each c-subunit can hold one proton, meaning the ATP synthesis efficiency (H+ / ATP) differs between species, as each full rotation of the c-ring corresponds to three ATP produced. As the c-ring rotates, so does the central stalk, the γ-subunit in the soluble F 1 domain. The peripheral stalk composed of two b-subunits is part of the F 1 domain, as well as the F O domain, and acts together with the δ- subunit as an immobilized stalk that prevents the α- and β-subunits from rotating by associating to the N-terminal region of one of the α-subunits. Instead, the rotation of the γ-subunit induces conformational changes in the β-subunits, which hold the nucleotide binding sites. Each β subunit adopts a distinct, different conformation, which sequentially changes during rotation of the γ-subunit. In some organisms, such as fermenting bacteria, the ATP synthase can work in reverse in vivo , pumping protons to build up a membrane potential by hydrolyzing ATP. This reversed reaction can also occur in mitochondria, during drops in membrane potential under anaerobic conditions 42 .

43 Figure 5 Structure of F 1FO ATP synthase from E. coli (PMID: 5T4O ). Subunits are shown in different colours and are labelled in the figure. Their stoichiometry is also indicated as subscripts. Proton uptake and release sites and rotational direction of the c-ring are labeled with grey arrows. Note that the n- and p-sides are flipped compared to previous images because convention is to show the F 1 domain above the membrane

In mitochondria the ATP synthase is located in the bends at the tip of the cristae in highly ordered, localized structures discussed in more detail in the chapter on respiratory supercomplexes. The cristae is the name of the folded sheets of the IMM. In E. coli , the enzyme is distributed across the entire inner membrane where it diffuses in a Brownian fashion 44,45 .

28 Biological membranes

Biological membranes are mainly composed of amphipathic lipids, commonly phospholipids, forming a bilayer with a thickness of roughly 40- 50 Å. The hydrophilic regions of the bilayer form the surfaces and the middle hydrophobic region accounts for around 25-40 Å 46 . In Mitchell’s chemiosmotic theory the membrane was considered as an insulator 38,39 , but biological membranes have many other important roles. The separation of charges is important for many vital biological functions, such as oxidative phosphorylation, current transduction in neurons and signaling in muscle cells. The lipids themselves can also participate in the chemical processes of the cell, either by being involved in the chemistry directly, or by serving as regulatory or structurally stabilizing co-factors. Much of this is discussed in more detail in the following chapters.

Fluid mosaic model

The concept of biological membranes existing as two-dimensional liquid with proteins embedded in them was described by Singer & Nicolson in 1972 and is known as the “Fluid mosaic model” 47 . It was based on the concept of a lipid bilayer proposed in 1925 by Gorter & Grendel 48 , and served as an alternative to the membrane tri-layer (protein-lipid-protein) model, which did not include the concept of membrane-embedded proteins. Over the years, many findings have added to the fluid mosaic model, but the basic concepts still hold true. Besides free lipids and proteins, biological membranes can also contain aggregates such as protein/glycoprotein complexes and membrane-associated structures such as peripheral proteins or cytoskeletal components 49 . Lipids diffuse laterally in two dimensions within the leaflets of either bilayer, but are also able to diffuse from one leaflet of the bilayer to the other, a process termed “flip-flop”. While slow (~10 -15 s-1), the process can be accelerated, or reversed, by proteins known as “flipases” and “flopases”, which serve to maintain and regulate the asymmetry in the lipid bilayers 50–52 . In eukaryotes, membranes from different organelles exchange lipids with one another via lipid transport proteins at membrane contact sites, proximal zones where the two membranes come together at distances of <30 nm, held together by

29 proteins 53 . Many of these connections are directly associated with nanotube extensions of the endoplasmic reticulum (ER), the organelle where much of the lipid synthesis takes place 54 .

Membrane phase transitions

Biological membranes can exist in two distinct phases; a solid (gel) phase and a fluid (liquid) phase. These phases are temperature dependent and transition between them occurs when the temperature reaches the melting point ( Tm), which can be modulated by changing the fluidity of the membrane, e.g. via introducing sterols, changing the average saturation state of the lipids or the average length of lipid acyl chains. This strategy is utilized by many organisms to maintain a certain fluidity as the temperature of the environment changes. Changes in membrane fluidity can have severe consequences on membrane-transport proteins and ATPase activity 55–57 . It has been observed that in the same membrane one lipid species can exist in the gel phase while another simultaneously is found in the liquid phase 58 . Most biological membranes don’t exist in the gel phase, as it prevents lateral diffusion of both lipids and proteins. Diffusion in the membrane is important for several biochemical processes, for example for substrates and interaction partners finding each other, as is the case for diffusion of ubiquinol between respiratory complexes. Diffusion is also important in membrane self-repair by diffusing lipids to fill gaps.

30 Lateral proton translocation

The proton motive force is defined as

= (∆Ψ)−2.3 (∆pH)

Where (∆Ψ) is the electrical component and (∆pH) is the concentration component as a result of protons being transferred across the membrane, or removed from the n-side 38 . As mentioned above, the model proposed by Mitchell only considered the protons to be moved into the bulk solution on the p-side and the membrane serving as a permeability barrier. Williams suggested later that protons are instead released to the surface of the membrane on the p-side and transferred laterally along the membrane surface between the enzymes in oxidative phosphorylation 59 . The concept of lateral proton transfer has been studied extensively. Pioneering studies of purple membranes from Halobacterium salinarium showed that protons could migrate long distances across the membrane surface at rates approximately 10 times faster than that of bulk diffusion, with almost no loss of protons to the bulk solution 60,61 . Studies on Cyt cO have shown that the presence of a membrane accelerates proton uptake and proton transfer to the catalytic site of the enzyme, indicating a direct protonation mechanism between the membrane and the enzyme 62,63 . A mechanism has been suggested for the lateral proton transfer where the protons move by a protonation-deprotonation Grotthuss-type mechanism along an ordered “shell” of interfacial water molecules arranged at the membrane surface 64 .

Lipid properties and membrane compositions

Some lipids are found as co-factors or structural components between subunits in many membrane proteins. These are referred to as non-annular lipids. Annular lipids are the lipids that surround the hydrophobic region of the protein, forming its solvation layer. Both of these types of lipids have been found to be important for structural integrity and maintaining the activity of membrane proteins 65,66 . The remaining lipids in the membrane are called bulk lipids and are in constant exchange with the annular lipids. As mentioned above, lipid acyl chains can affect the fluidity of the membrane and lipid head groups can affect the surface properties of the membrane.

The hydrophilic head size, and to some extent the acyl chain structure, give the different lipids certain shapes that affect the membrane. Cylindrical lipids have a head size that allows the acyl chains to point out from the head

31 in a parallel fashion and are referred to as “bilayer-prone” lipids, as they promote bilayer formation. When the head groups are smaller or larger, the acyl chains either point away from or towards each other, respectively, giving the lipids a conical shape that induce curvature in the membrane. These “non-bilayer prone” lipids can still be present in membranes when mixed with bilayer prone lipids. For example, in E. coli up to 70 % of the membrane lipids are the non-bilayer prone phosphatidyl-ethanolamine (PE) 67,68 . The effect of destabilizing the bilayer structure is also counteracted by the high membrane protein density, as integral membrane proteins have been found to promote bilayer formation of non-bilayer prone lipids 65 . Integral membrane proteins and lipids tend to align themselves so that the polar head groups interface with hydrophilic regions of the protein and the acyl chains align themselves with the hydrophobic transmembrane domain. This property can either lead to stretching or compressing of the lipids in the membrane, or tilting of transmembrane helices in the proteins 65 . It is likely that these effects are related to the ability of membrane proteins to promote bilayer formation for non-bilayer prone lipids. In vitro it is difficult to form lipid bilayers with high contents of non-bilayer prone lipids as they tend to form aggregates rather than membranes.

Some phospholipids commonly found in biological membranes are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl- glycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA) and cardiolipin (CL) (Figure 6). Under physiological conditions, PC and PE are zwitterionic and PG, PS, PA and CL are anionic. The relative composition of these lipids differs greatly in different membranes. As mentioned above, E. coli membranes are mainly composed of PE, while the main component of most eukaryotic membranes is PC 58 (typically >50 %). Lipids can also be organelle specific, such as cardiolipin, mainly found in the IMM and synthesized from PG, which in turn uses PA as a precursor in the CL synthesis mechanism 69 . As already mentioned above, most lipids in eukaryotes are synthesized in the endoplasmic reticulum (ER) 70 , however, cardiolipin is synthesized in the mitochondria and depends on lipid-transport proteins like the intermembrane space PA-specific transporter Ups1 71–73 . Interestingly, CL is more prominent in the outer than the inner leaflet of the IMM 74 , which suggests it may stimulate the cristae formation, although the opposite may also be true as recent studies have shown that CL preferentially sorts into already curved membranes 75 . It has also been found to be important for the structure and function of Cyt cO76,77 , further showing how lipids serve more roles than just forming a barrier for ions.

Figure 6 Polar head groups of common lipids. The phosphate head groups for the zwitterionic lipids PC and PE as well as the anionic lipids PG, PA and CL are shown in black to emphasize the differences. In red are the glycerol backbones for the polar heads as well as the ester bond for the acyl fatty acids. Acyl chains are denoted with R for all cases, although the R-groups could be different both within the same lipid and between different lipid species, depending on organism, cell type or type of membrane. Variants of these lipids were used in the studies presented in this thesis.

Membrane-mimetic systems

Studying membrane proteins in vivo or in native membrane fractions can be quite challenging due to the sheer complexity of the system. Native membranes are densely packed with a multitude of proteins, lipids and other functional components that perform many regulated and often coupled processes. Purification of the proteins and solubilization in detergent can sometimes result in structural changes and disassociation of subunits.

By reconstitution of purified membrane proteins into a membrane, or membrane-like environment, some of the original functions can be restored and studied in a more minimalistic system. These membrane-mimetic systems include small membrane “nano discs” with a diameter in the range 10-17 nm, depending on surrounding scaffold 78,79 , flat membranes supported on solid surfaces 80–84 , membranes supported on spherical surfaces like mesoporous silica particles 85,86 or whole enclosed membrane compartments such as unilamellar liposomes. The properties required of the mimetic system can depend both on the protein that is studied and the measuring method. For instance, larger membrane-mimetic systems may scatter light,

33 which could limit the use of optical measuring approaches. For nanodiscs the light scattering properties more closely resemble that of detergent solubilized protein than of liposomes 79 .

While the mimetic systems lack many of the components present in vivo , this approach has the benefit of being simpler and containing only components that are involved in the reactions of interest. In other words, it’s possible to study effects that would be much more difficult to detect in a natural environment. However, it’s also important to consider if the results are representative of reality, or just an artifact from the use of a specific methodology.

One type of membrane mimetic systems, mentioned above, is unilamellar liposomes. Liposomes are spherical lipid bilayers with a diameter ranging between small (20-100 nm), large (100-1000 nm) or giant (>1 µM). There are different approaches to form liposomes, but in general, small liposomes are formed by sonicating lipid mixtures, large by extruding membranes through filter membranes with defined pore sizes and the giant liposomes are formed by repeatedly chock freezing and thawing large liposomes, forcing them to fuse. It is also possible to form giant vesicles by applying an AC current between two indium-tin oxide coated glass plates, forcing smaller liposomes in the reaction chamber to fuse 87,88 . This method is called electroformation. Liposomes can be made from (i) purified lipid extracts from natural sources (commonly soy beans or E. coli ) (ii ) mixing synthetic lipids, allowing control over the lipid composition. It’s also possible to introduce other components during liposome formation, such as labeled lipids (e.g. labeled with fluorescent probes) or fluorescent dyes that can then be removed externally, for instance to introduce a pH sensor on the inside of the liposomes, not present on the outside. One difference between membrane-mimetic systems and native membranes is the lower protein densities in the former. Membrane-mimetic systems also usually have a specific membrane curvature (or lack thereof) which may not represent the native environment. Furthermore, it is difficult to control the bilayer asymmetry in membrane-mimetic systems. Perhaps the most important problem associated with the use of the artificial systems is that it is difficult to control the protein orientation in the membrane. In native membranes, proteins always insert into their respective membranes in a defined orientation, controlled by the membrane insertion machineries 89–91 . However, in membrane-mimetic systems orientation can differ between different systems, or different reconstitution methods. For instance, when bovine heart Cyt cO was reconstituted into liposomes via dialysis, most Cyt cOs oriented with the cyt. c-binding site facing the outside. However, when reconstituted via sonication there was a mixed orientation 92 .

34 Successful incorporation of functional respiratory-chain and oxidative- phosphorylation enzymes have been reported, such as reconstitution of Complex I into liposomes for determination of proton-pumping stoichiometry 93,94 . Other examples of reconstitution experiments include; ATP synthase or Cyt cO in nano discs 79,95 , silica supported membranes containing Cyt cO85 , or bacteriorhodopsin and Cyt cO in liposomes 96 , to mention a few. Experiments on ATP synthase co-reconstituted with bacteriorhodopsin in the mid 90’s showed the strengths of the approach to co-reconstitute and quantitatively investigate the energy requirements for ATP synthesis and the coupling between a proton pump and a proton sink 97 . However, until recent years, few studies investigated co-reconstitution of isolated respiratory enzymes together with ATP synthase.

Arrangement of respiratory enzymes

Over the years it has been debated how the respiratory enzymes arrange themselves in the membrane. For bacteria, the models have ranged from free diffusion of all enzymes (fluid state model) to tightly associated superstructures (solid state model), eventually settling at a mix between the two (plasticity model). According to this model respiratory complexes are capable of alternating between the two states depending on environmental changes (for review, see 98 ).

36 Respiratory supercomplexes

In mitochondria as well as some bacterial species, such as the Gram-positive Mycobacterium smegmatis , the respiratory enzymes have been found to form large multimeric structures composed of combinations of the different complexes 99,100 . The function of these respiratory ”supercomplexes” have been speculated to be related to regulation of respiration, stabilization of respiratory complexes and prevention of ROS formation by reducing 101 electron leakage . For the bovine I1III 2IV 1 supercomplex it was suggested early on that the structure would enhance steady state activity as it could allow for quinone channeling between complex I and III for more efficient electron transfer 102,103 . However, this conclusion is not supported by more recent findings, which showed that when introducing an alternative oxidase (AOX) to respiratory membranes containing supercomplexes, ubiquinol reduced by complex I was re-oxidized more rapidly by AOX than by complex III associated in a supercomplex with complex I, supporting the presence of a Q-pool rather than substrate channeled quinols 104 .

The fraction of supercomplexes and free respiratory enzymes differs between different organisms 100,105 . The assembly and stabilization of the supercomplexes is influenced by a multitude of factors. For example, CL has been found to bind to supercomplexes composed of the bc 1 complex and 106–109 Cyt cO and the I 1III 2IV 1 supercomplex was found to be enriched with PC, PE and CL 103 . CL is important for structural stability of respiratory supercomplexes and PE stabilizes the free form of Cyt cO. Adjusting the PE/CL ratio could be a means for the mitochondria to regulate the ratio between free enzymes and supercomplexes 110 . It has also been shown that prolonged exercise in humans can shift the ratio towards more supercomplexes 105 . Supercomplex composition differs between organisms. In yeast, complex III and IV organize themselves in a tetrameric complex composed of III 2IV 2 (Figure 7A) as well as a trimeric complex composed of 111 III 2IV . In the mammalian mitochondria the most abundant supercomplex arrangement of these enzymes are together with Complex I, in a I 1III 2IV 1 “respirasome” 103,112,113 .

The ATP synthase in mitochondria is often organized in dimers, as seen in fungi, mammalian and plant cryogenic electron microscopy (Cryo-EM)

37 structures 114 . A recent Cryo-EM structure of porcine ATP synthase also show a tetrameric oligomerization and it was suggested that the dimerization is less prominent in mammals, as the subunit causing the rigid dimerization bridge in e.g. yeast is shorter in mammalian ATP-synthases 115 . The dimers are localized in rows in the cristae bends, as the curvature of the membrane and the angle between the dimers (Figure 7B) match. It has been suggested that the ATP dimers themselves induce this membrane curvature, because knocking out the dimerization subunit results in deformed phenotypes with little to no cristae formation 114,116 . However, it is also possible that the ATP synthase localization and membrane curvature are synergistic, as it has been shown that inducing curvature into GUVs containing the curved membrane protein amphiphysin-1, result in localization of the proteins in the induced curved domain 117 .

Investigation of the organization of oxidative-phosphorylation enzymes in E. coli found that the proteins are distributed in clusters, unevenly across the membrane. 118,119 However, the different enzymes were found to co-localize with multiple copies of themselves, but not with each other, indicating no presence of respiratory heteromeric supercomplexes like the I 1III 2IV 1 respirasome found in mitochondria.

Figure 7 Supercomplexes. (A) Respiratory supercomplex III 2IV 2 from S. cerevisiae composed of a dimer of complex III (green shades) and one associated complex IV on each side (blue shades). Adapted from Cryo-EM structure PMID: 6HU9 12 . (B) ATP synthase dimer, based on Cryo-EM structure (black outlines) with atomic average fitted model (green, PMID: 4B2Q) modelled from the x-ray structure of the bovine ATP synthase 116 .

38 Respiratory supercomplex factors

The respiratory supercomplex factors (Rcf) are part of the hypoxia-induced gene 1 (Hig1) family. Members of this family have been found to have elevated expression levels during stress conditions, like low oxygen or low glucose 120 . In S. cerevisiae there are three Rcf variants. Rcf1 is conserved in a number of species and the N-terminal region shares homology with e.g. the human protein Higd1a. Rcf2 is fungi specific 121 and Rcf3 share sequence homology with the N-terminal region of Rcf2 122 . The Rcf proteins interact 121,122 with both the bc 1 complex and Cyt cO , with Rcf1 showing the strongest interactions, and they are important for the stability of the III 2IV 2 supercomplex 121 . Rcf1 has been found to interact strongly with CytcO subunit Cox3 (c.f. subunit III described earlier) and a double knockout of both Rcf1 and Rcf2 resulted in a lower enzymatic Cyt cO activity, as well as impaired assembly of supernumerary subunits Cox12 and Cox13 (see Figure 2). The effect of single knock-outs was more pronounced for Rcf1. Studies have also shown that Rcf1 enhances binding of cyt. c to the supercomplex 123 and it was suggested that Rcf1 functions as a membrane anchor for cyt. c, similarly to the integral membrane protein variants of cyt. c found in R. sphaeroides and R. capsulatus 124,125 .

Figure 8 Rcf1 structure and TMH scheme. (A) NMR structure of Rcf1 dimer (PMID: 5NF8 126 ). The two monomers are colored in green and teal, anionic and cationic residues in red and dark blue, respectively. The dashed line emphasizes the interface. (B) Scheme showing how the Rcf1 protein would be oriented in the membrane. To the left is a monomer of the 5-TMH structure solved by NMR, showing the QRRQ motif in white circles and the ERK motif (purple circles) and K59, both suggested to interact with cyt. c. To the right is the monomeric 2-TMH structure predicted based on protease cleavage studies and homology comparison with human Higd1a.

39 Early predictions based on the assumption of structural homology to the human Higd1a suggested that Rcf1 adopts a two transmembrane helix (TMH) structure, with the N- and C-terminus both on the p-side of the membrane 121,127 . To support this prediction, protease cleavage profiles were shown using V8 protease, which cleaves after negatively charged residues present at the C- and N-terminus, but not in the suggested transmembrane loop. A recent NMR structure of purified Rcf1 in DPC micelles shows a 5- TMH dimeric structure (Figure 8) with the C- and N-terminus facing opposite directions 126 . The interaction sites between the two peptides in the dimer are found in the interface between the three extra TMHs, which contain a number of complementary charged residues.

Both these structures contained a Hig1 conserved QRRQ motif at the membrane interface and an ERK motif in the soluble region (Figure 8). The QRRQ motif has been shown to be important for association of Rcf1 to Cyt cO subunit Cox3, and possibly also to Cox2 128 . An ERK motif found in the soluble helix between THM 4-5 was shown to be a likely interaction site for cyt. c, together with K59 in the loop between TMH 2-3126 . TMH 2-3 in the NMR structure are homologous to the two TMHs in the Higd1a structure.

40 Energy coupling

In brown adipose tissue, prominent in hibernating mammals, human infants and to a lesser degree also in human adults 129,130 , heat is generated to regulate body temperature 131 by uncoupling protein 1 (Ucp1), as an alternative pathway for the proton motive force. It has also been suggested that weaker uncoupling mechanisms of the proton gradient (referred to as “mild uncoupling”) can be beneficial in mitochondria when the ATP demand is low, but the levels of reducing equivalents (e.g. NADH) are high 132 . Without this uncoupling, the reducing agents and oxygen could react directly to form reactive oxygen species and the membrane would also hyperpolarize, I.e. Δψ would be greatly elevated. Mitochondrial hyperpolarization and accumula- - tion of H 2O2 (product of O 2 by superoxide dismutase) have both been found to induce apoptosis 133–135 . Recent findings about homologues to Ucp1 present in more diverse tissue, namely Ucp2 and Ucp3, suggest that they are part of a superoxide-activated mechanism, where they leak protons as a re- - sult of elevated O 2 levels, reducing the membrane potential and ATP synthesis 136 .

Uncoupling agents at concentrations in the lower nanomolar range can be used to simulate respiration via proton leaks without abolishing ATP synthesis. Examples of such uncouplers are carbonyl cyanide p- trifluoromethoxyphenyl hydrazone (FCCP), carbonyl cyanide m- chlorophenyl hydrazine (CCCP) and SF6847. These uncouplers, especially in combination with other classes of uncouplers such as the K + uncoupler valinomycin, can also be used to gain kinetic and thermodynamic data on proton pumps and proton sinks. Dissipation of the electric component or the pH component of the proton-motive force can be done independently. In doing so, it is possible to quantify each component and its impact on the studied system. For example, it is possible to discern if an ATP synthase is sensitive only to the proton gradient, or if it also can produce ATP as a result of an electrical gradient.

42 Methods

Protein reconstitution in liposomes

There are many procedures available for reconstitution of membrane proteins into lipid membranes. Proteins can be included during liposome formation, with mixed lipid-detergent micelles or added after liposome formation by destabilization of the liposomes with low detergent concentrations 137,138 . In most cases the detergent must be removed, either via gel-filtration, biobeads (which adsorb lipids) or dialysis. The choice of method depends on the protein, the detergent or the experimental system. Reconstitution of mitochondrial Cyt cO and chloroplast ATP synthase into liposomes was reported already in the 1970s 139–141 . In the studies presented in this thesis we mainly used detergent destabilization of preformed liposomes, after addition of the membrane protein, followed in time by removal of the detergent via gel filtration or dialysis.

Bioluminescence assay to measure ATP synthase rates

A complication when measuring the activity of the ATP synthase is that neither the enzyme itself nor the substrates involved are spectroscopically active. Hence, a coupled assay to indirectly measure ATP production needs to be applied. One such method relies on the enzyme luciferase, which consumes ATP in a reaction that emits light with an emission maximum at 562 nm. The experimental conditions can be optimized to emit light at an intensity that is linearly proportional to the ATP concentration. The setup can be calibrated by adding an internal standard of small amounts of ATP to the sample before activating the enzyme.

Figure 9 ATP synthesis bioluminescence assay. (A) Scheme of the coupled enzymatic system. ATP synthase and cytochrome bo 3 were co-reconstituted into small (100 nm) liposomes. DTT added externally reduces the oxidase via ubiquinol Q 1. The oxidase pumps protons into the liposomes and the resulting pmf drives ATP synthesis. ATP is detected using luciferase, which emits light at 562 nm in a reaction using ATP, oxygen and luciferin, the latter also added externally. (B) A typical data trace. Rates were calculated from the average slopes of the three measurements after Q 1 addition. After recording a baseline the system was calibrated by addition of a well- defined amount of ATP. ATP production was initiated by addition of Q 1 and inhibited by addition of the oxidase inhibitor KCN.

Oxygen reduction rates

The steady-state activity of Cyt cO can be followed using a Clark-type oxygen electrode, which measures the O 2 concentration over time in a solution. As every oxygen molecule reduced requires four electrons and the oxidase concentration can be determined from a reduced-oxidized absorption spectrum of the enzyme, the activity (in e - / s / enzyme molecule) can be calculated. In reconstituted or native systems uncouplers are used to dissipate the ΔΨ and/or the ΔpH in order to obtain the maximum rates. Maximum turnover rates and K M-values can be determined by titrating e.g. cyt. c. The ratio of the uncoupled and coupled rate is referred to as the respiratory control ratio (RCR), which is used as a measure of how “tight” the membranes are (c.f. proton leaks). For normalization of the Cyt cO activity, relative orientation of the protein in the membrane needs to be considered, as reconstitution yields a mixture between proteins with the cyt. c-binding site facing inwards, and those with it facing outwards. How to measure activity of different orientation populations is discussed extensively in Paper I , and mentioned briefly in the “Main findings” chapter.

Figure 10 Oxygen reduction and proton pumping assays. (A) A typical data trace from a Clark type electrode. The oxidase is activated by addition of a mediator (e.g. cyt. c or ubiquinone) after recording a baseline in the presence of uncouplers, reducing agents and reducing equivalents. After recording O2 reduction over time, the activity is stopped by addition of the oxidase inhibitor KCN. (B) ACMA fluorescence quenching trace for measuring proton pumping by e.g. ATP synthase. Addi- tion of ATP induces pumping of protons to the inside of vesicles, resulting in quenching of the ACMA signal. Addition of valinomycin in the presence of K + results in an increase in pumping activity as the charge gradient is being equilibrat- ed. Addition of FCCP dissipates the ΔpH and the ACMA fluorescence signal returns to the baseline.

Tunable resistive pulse sensing

To measure the particle size distributions and concentrations for characterization of liposomes we used the method Tunable Resistive Pulse Sensing (TRPS). Two electrodes are separated by a membrane with a conical pore and an electrolytic buffer is used to form a current between the poles. As particles flow through the pore they block the flow of electrolytes, which is seen as a drop in the current. The amplitude is proportional to the size of the particle moving through the pore. A calibration using a silicate standard allows for calculation of the exact size of each particle. The flow rate of particles through the pore is proportional to the particle concentration. The membrane, and thus the pore, is stretchable and the pore opening can be tuned to only allow one particle through the pore at a time. This method has the advantage over dynamic light scattering in that it detects each particle separately, rather than approximates the average of all the particles in the solution.

Figure 11 Schematic of Tunable Resistive Pulse Sensing. When liposomes pass through the pore the current across the pore is blocked. The liposomes (orange) pass through the pore unidirectionally as indicated by the black arrow due to a pressure difference. The amplitude of the signal corresponds to the size of the particle and the frequency of the events corresponds to the concentration of the sample. By calibrat- ing the system with a particle standard, the diameter of each liposome and the liposome concentration can be calculated.

Detection of pH gradients

Fluorescent probes sensitive to changes in pH can be used to monitor proton pumping, or proton leaks, across biological membranes in real time. There are different probes suitable for different experimental setups, such as membrane non-permeable dyes like fluorescein, or the ratiometric 8- hydroxypyrene-1,3,6-trisulfonic acid (Pyranine), which can be used to reliably determine an absolute pH value in an enclosed hydrophilic environment, e.g. the inside of a liposome. Two non-permeable dyes with distinctly different excitation/emission profiles can be used to simultaneously measure pH on both the inside and outside of a membrane. There are also membrane-permeable probes, such as 9-Amino-6-Chloro-2- Methoxyacridine (ACMA), which can be used to follow in time the build-up and dissipation of a ΔpH up to 2 units 142 . The probe equilibrates between a free form and a membrane-bound form, where the membrane-bound form self-quenches. In its neutral state the probe is assumed to freely diffuse across the membrane. When a ΔpH is built up (inside of the membrane is acidified), quenching increases due to accumulation of the positively charged state in the aqueous inner compartment. ACMA is a sensitive dye and has previously been used to monitor transmembrane ΔpH in the range 0.1-1.5 units as a result of proton pumping by the ATP synthase in bacterial membranes from Rhodobacter capsulatus 143,144 .

46 Fluorescence correlation spectroscopy

Confocal microscopy is a type of fluorescence microscopy where a microscope is fitted with a pinhole between the sample and the detector to filter out emitted light not corresponding to a certain location centralized in the Z-plane. Filtering out some of the emitted light with the pinhole results in detection of a specific focal volume that is the only part of the sample observed, even though the whole sample is excited. The diameter of the pinhole determines the width of the focal volume. When using fluorescence correlation spectroscopy (FCS), fluorophores diffuse in and out of the focal volume and induce fluctuations in the fluorescence intensity.

The size and dwell time of the intensity fluctuations can give information about how many labeled particles are present on average in the focal volume and how fast they diffuse. Information about the fluorophore diffusion time can be determined from the auto-correlation curve, which is calculated based on the deviation of intensity from the mean for a time series at a time shift (τ). The auto-correlation function, when normalized, is defined as

〈() ∙ ( + )〉 () = 1 + 〈()〉

Where () is the deviation of intensity from the mean at time t. It is defined as () = () − 〈()〉.

The auto-correlation function is the cross-correlation between a fluorescence signal and itself, after displacing it by the time τ. The intensity of the auto- correlation curve can be described simplified as the chance of still being able to observe a particle that was seen at the time t, after the time t = t+τ.

The auto-correlation at τ=0, G(0), correspond to 1+1/ N, where N is the number of particles in the focal volume on average and τ at G(0)/2 equals to the diffusion time ( tD), i.e. a measure of how fast the particle moves. The diffusion time is related to the size of the particle.

Figure 12 Fluorescence correlation spectroscopy. (A) Representation of the focal volume with two fluorophores diffusing in and out of the focus. The fluorophore inside the focus is excited and the light is collected through the pinhole. (B) Theoret- ical scheme of two autocorrelation functions, corresponding to a faster (green trace) and a slower (purple trace) diffusing particles. From these curves, information can be derived regarding diffusion times (tD). (C) Schematic representation of three different time shifts (τ) of the intensity trace for the green autocorrelation curve. The autocorrelation function is a comparison of the original trace with the time-shifted traces.

When using a multi-channel confocal microscope, two proteins labeled with different fluorophores can be observed at the same time. By immobilizing giant liposomes and shifting the Z-plane in the confocal microscope to observe the surface on the top of the liposome, two-dimensional mapping of membrane protein diffusion can be made. By comparing their auto- correlation functions, the cross-correlation (FCCS) of the two different proteins can be determined and information on their interactions can be obtained. The FCCS curve is a result of an event when the two particles diffuse together 145 .

48 Main findings

In the work described in this thesis we studied the interplay between various components of the respiratory chain and the ATP synthesis process. We mainly used a bottom-up approach, i.e. using separate parts and assembling them together in a step-wise process to rebuild biological functions under controlled conditions with lower component complexity.

In Paper I we developed a method for co-reconstitution of CytcO / ubiquinol oxidase and ATP synthases into liposomes made with synthetic lipids and created a functional coupled system capable of oxidase-activated ATP production. We used ATP synthases from E. coli together with cytochrome bo 3 from E. coli and ATP synthase from spinach chloroplasts with Cyt cO from R. sphaeroides . Using this system we observed an ATP- production rate of up to 90 ATP / s / enzyme, much closer to the rates reported for e.g. inverted membrane vesicles (~ 180 ATP / s / enzyme 146 ) than observed in previous studies using reconstituted systems, where much lower rates were observed (5 ATP / s / enzyme 97,147 ).

Using this system we showed that the relatively high rates of ATP production could be maintained at steady state levels, even in the presence of low concentrations of uncouplers, despite abolishing the apparent ΔpH (e.g. mild uncoupling conditions). At higher concentrations of uncouplers, or mixtures of several different uncouplers, ATP synthesis was completely blocked. Previous studies have shown that the uncoupling process could be inhibited in mitochondria by addition of 6-ketocholestanol (kCh). It was suggested that during mild uncoupling, compounds like FCCP and SF6748 bind directly to Cyt cO, an action that is blocked by kCh 148,149 . While we could reproduce the effect of countering the uncoupling effect with addition of kCh, our data did not support any direct protein interaction of uncoupling agents with Cyt cO. Instead we suggested a general membrane mechanism where kCh prevents the anionic forms of the uncouplers from diffusing back to the acidic side of the membrane. This is a result of a membrane dipole potential created as kCh asymmetrically inserts, mainly into the outer leaflet.

49 In Paper II we used the method developed in Paper I to study the effects of different lipid compositions on ATP synthesis. In this study, we used the E. coli ubiquinol oxidase. The limitation to ubiquinol oxidase was mainly due to two reasons, based on observations discussed in Paper I . In short, ( i) As >97% of the ATP synthase is oriented with the F 1-domain on the outside of liposomes150 , the Cyt cO must be oriented with the cyt. c-binding site towards the interior of liposomes (pumping protons into the liposomes). (ii ) With cyt. c on the inside of the liposomes, electrons (supplied externally) must be transferred across the membrane via a mediator capable of reducing cyt. c. Phenazine methosulfate (PMS) acts as such a mediator. However, we observed in Paper I that PMS likely had an uncoupling effect when oxidized and could reduce O 2 directly when added at micromolar concentrations, needed to obtain sufficient ATP synthesis rates. Hence, we would have to prepare liposomes with cyt. c on the inside of the liposomes and remove all residual external cyt. c. Furthermore, we would have to add a membrane- penetrating reducing agent. While possible to do, the complexity of the system would increase, making interpretations of data more difficult.

The main focus in Paper II was an investigation of the effect of altering the lipid composition on ATP synthesis. We used zwitterionic lipid 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC)-containing liposomes as a starting point and then replaced DOPC with anionic lipids DOPA, DOPG or cardiolipin (with the same acyl chains as the other lipids). The synthetic DO lipids were used to avoid effects of differences in acyl chains. We also tried exchanging a fraction of DOPC with DOPE, another zwitterionic lipid, however the results were inconclusive as PE is not bilayer prone and the liposomes would either grow in size with increasing amounts of PE, or not form liposomes at all. The liposomes composed of a mixture of DOPC and the anionic lipids remained the same size. ATP synthesis rates dropped upon addition of the anionic lipids. At 40 % weight fraction of anionic lipids the rate was ~10 % of the DOPC liposome activity. The activity of each of the enzymes remained unaffected and it was concluded that the effect seen was either due to (i) changes in lateral transfer of protons along the membrane surface between the enzymes, or (ii ) changes in average distance between the ATP synthase and cytochrome bo 3 (I.e. co-migration / co-localization) as a result of changes in surface charge of the membrane. The conclusion about protein-protein distance was partially based on the observations that the lipid dependence effect was affected by the protein density.

In Paper III we showed that protein-protein interactions between ATP synthase and cytochrome bo 3 decreased in a mixture of DOPC and DOPG, compared to the pure DOPC membranes. We used fluorescence cross- correlation spectroscopy (FCCS) to determine protein-protein interactions. We observed lower co-migration of ATP synthase and cytochrome bo 3 in

50 membranes with DOPG than in pure DOPC membranes. We also showed that when forcing the enzymes to stick closer together, we could increase ATP synthesis rates as well as decrease the lipid dependence effect shown in Paper II . While we couldn’t exclude any effect on lateral proton movement, we showed that the lipid composition determined the extent of protein- protein interactions as a result lipid charge. The results described in Paper III thus support one of the two conclusions in Paper II .

In Paper IV we used the experimental tools developed in Paper I to address how Rcf1 directly affects the turnover of Cyt cO. We studied the effect of Rcf1 on the activity of Cyt cO from S. cerevisiae . We overexpressed the yeast peptide Rcf1 in E. coli and purified the peptide from inclusion bodies as described before 126 . The peptide was reconstituted into DOPC liposomes together with Cyt cO purified from the wild-type and rcf1 Δ strains of S. cerevisiae . Furthermore, we added the Rcf1 peptide to submitochondrial particles (SMPs) from both strains. Lack of Rcf1 resulted in a lower Cyt cO activity in the SMPs, however most of the activity was restored upon reconstitution of the peptide. We obtained similar results when co- reconstituting purified Cyt cO together with Rcf1 in DOPC liposomes.

To investigate whether the Cyt cO activation by Rcf1 was connected to the conserved Hig-domain or the fungi specific C-terminal region, we also reconstituted a truncated Rcf1 variant composed of only the first 112 residues that contain the domains homologous to the human Higd1a protein. This construct did not activate Cyt cO. We concluded that Rcf1 has an effect on both the activity of Cyt cO and on binding affinity for cyt. c, resulting in both a reversible effect (cyt. c binding) and an irreversible effect (alterations to Cyt cO during assembly).

In conclusion, the studies presented in this thesis show how co-reconstituting proteins into a membrane-mimetic system could be used to study details of protein-protein interactions within the respiratory chain and how the protein- protein interactions could be used as a means of regulation of oxidative phosphorylation. With the reduced complexity of the in vitro system it’s possible to change specific variables and study minute details. We showed that the membrane-mimetic methods used in Paper I-III could also be combined with more native-like systems, such as the SMPs (Paper IV ) to gain a greater insight into the workings of the cell.

However, even though current methods for membrane mimetic systems are very powerful, many of these methods still have some drawbacks. The lack of control over protein topology for reconstitution or lipid distribution between the leaflets sometimes makes results difficult to interpret. Methods for controlling these two features would not only make some experiments

51 easier to perform, but also allow for more diverse studies, as well as allowing for more close resemblance to in vivo situations. It is also currently difficult to form membranes in vitro with similar lipid and protein compositions as native membranes, as some non-bilayer prone lipids tend to aggregate rather than insert into membranes and protein insertion efficiency sometimes can be low. Low propensity for lipids and proteins to insert into membranes can sometimes make it difficult to form proteoliposomes and also makes it difficult to mimic some membrane features such as the dense packing of lipids and proteins.

52 Populärvetenskaplig sammanfattning

Alla våra apparater i vardagen drivs av elektricitet. Det är inte den ursprungliga källan av energi, utan en omvandlad, mer tillgänglig form som kan användas universiellt. På samma sätt har biologiska organismer en universiell energikälla i form av molekylen ATP. Denna molekyl produceras vid biologiska membran bestående av lipider och proteiner. Ett sådant exempel är det inre membranet i mitokondrierna hos oss människor. Där sitter det fyra proteinkomplex som i serie lämnar över elektroner till varandra, en process kallad ”andningskedjan”. Elektronerna är utvunna ur kemiska bindningar från näringsämnen som cellen har brutit ner. Några av proteinerna i andningskedjan använder energin från dessa elektroner för att pumpa positivt laddade vätejoner från ena sidan av membranet till den andra. Ett av dessa proteiner, det sista i andningskedjan, är cytokrom c oxidaset (Cyt cO). Cyt cO överlämnar dessutom elektronerna till syre, som omvandlas till vatten. Vätejonerna som ansamlas på ena sidan membranet, likt en damm vid ett vattenkraftverk, kan sedan flöda tillbaka via ett femte protein, ATP syntaset, som likt turbinen i vattenkraftverket roterar och använder energin för att driva den kemiska katalysen av ATP.

I den första studien utvecklade vi en metod för att sätta in ATP syntas och Cyt cO utvunna ur biologiska organismer i syntetiska membran. Vi visade hur denna metod kunde användas för att studera effekter på membranet samt de laddnings- och koncentrationsförskjutningar som uppstår när de båda proteinerna jobbade i samverkan, i en mer simplistisk miljö än deras naturliga membran.

I den andra studien använde vi denna metod för att studera hur ATP-syntas och CytcOs motsvarighet från bakterien E. coli , ubiquinol oxidas påverkades av att vi varierade lipiderna i membranet. Vi upptäckte att de två proteinerna var för sig inte påverkades, men att aktiviteten när de samverkade minskade om membranet bestod av ökande andel negativt laddade lipider, jämfört med s.k. zwitterjoniska lipider (neutrala lipider med både en positiv och en negativ laddning). Vi drog slutsatsen att denna effekt antingen berodde på hur snabbt vätejonerna kunde röra sig mellan de två proteinerna, eller att de två proteinernas avstånd från varandra ökade med mer negativa lipider i membranet.

53 I den tredje studien undersökte vi om ATP syntasets och ubiquinol oxidasets avstånd från varandra påverkades av de negativt laddade lipiderna. Vi märkte de båda proteinerna med fluorescerande färgämnen och kunde på så vis följa hur de rörde sig i membranet. Vi fann att de spenderade mindre tid tillsammans när membranet innehöll negativa lipider, vilket stärkte vår ena slutsats från studie två om att deras avstånd från varandra ökade.

I den fjärde studien satte vi in proteinet respiratorisk superkomplexfaktor 1 (Rcf1) från bagerijäst i membran innehållandes Cyt cO, även detta från jäst. Vi gjorde detta både i våra syntetiska membran och i membran från jäst- mitokondrier. Tidigare studier har visat att Rcf1 ökar aktiviteten hos Cyt cO i jäst. Våra resultat stämde överens med detta och vi kunde dra slutsatserna att Rcf1 hjälper till att binda substrated cytokrom c, som lämnar över elektroner till Cyt cO, samt att Rcf1 antagligen hjälper till vid ihopsättningen av Cyt cO i cellerna. Denna slutsats baseras på att vi kunde se att tillsats av Rcf1 ökade aktiviteten hos Cyt cO, men att en del permanent skada drabbade Cyt cO om Rcf1 togs bort genetiskt från jästen. Denna skada gick inte att reparera genom tillsats av Rcf1 efter att Cyt cO redan var ihopsatt av cellen.

Studierna i denna avhandling visar hur vi kunde efterlikna biologiska membran i ett provrörssystem och hur denna metod kunde användas för att studera flertalet effekter, både på protein och membran.

54 Acknowledgements

Peter for allowing me to work in your lab all these years and for being a good teacher and supervisor. Thank you for believing in me, allowing me so much freedom to pursue my ideas, but also for keeping me grounded when my mind drifts away too far.

Christoph for being my assistant supervisor and always bringing the encouraging perspective that sometimes things just don’t work and all we can do is laugh about it and keep going.

Pia for being so involved in my PhD studies and research, despite not being my supervisor. Your input and the discussions we’ve had over the years have been incredibly valuable.

Irina for all the knowledge you share, both in the lab and of science fiction. Whenever I was troubled by something in the lab, you knew a solution. I don’t know how I could have finished this fast without your help. Also, thanks for the long list of books I now have to read with all my newfound free time.

All my other colleagues in the Peter and Pia groups, past and present, in no particular order; Jacob , Johan , Markus , Finja , Linda , Camilla , Wataru , Sylwia , Agnes , Fede , Johanna , Ingrid , Olga , Max , Shu , Nathalie , Johannes , Gustav , Emelie , Davinia , Sylwia , Maria , Axel , Ann-Louise . Thank you all for making our workplace fun and interesting.

The DBB secretary and Matt , without you the department would fall apart. Thanks to all of you for all the help with both small and big things over the years. It made my time here both easier and more enjoyable.

My other coworkers at DBB for a fun and friendly working environment. Especially my fellows from the PhD council and the pub committee, Hannah , Theresa , Riccardo , Pascal , Joan ; for making life as a PhD student both serious and... not so serious.

My dear friends Emmy , Jaz , Åsa , Thomas , Fia , Sandra E, Pricken, Taco, Amanda , Johan , Suddis , Hrafn & Elaine for keeping me afloat even during my most stressful times, for letting me share and vent, for being involved in my life and for keeping me involved in yours.

The rest of KMS11 , though we may not always meet too often, every time we do is a reminder of how much I treasure our time together and how much it shaped me.

Last but not least, my Family for being patient with me during my absent years while studying at university and for keeping me humble.

56 References

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