Purification of Active Respiratory Supercomplex from Bovine Heart Mitochondria Enables Functional Studies

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8–35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q₁₀ (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q₁₀, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I₁, III₂, and IV₁ using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.     

Knockdown of Human COX17 Affects Assembly and Supramolecular Organization of Cytochrome c Oxidase

Assembly of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the insertion of subunits, accessory proteins, cofactors and prosthetic groups. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so-called supercomplexes. Here, we investigate the role of COX17 in the biogenesis of the respiratory chain in HeLa cells. In accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper centre of cytochrome c oxidase, COX17 siRNA knockdown affects activity and assembly of cytochrome c oxidase. While the abundance of cytochrome c oxidase dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. We observe the accumulation of a novel ∼ 150 kDa complex that contains Cox1, but not Cox2. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. We suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. An interdependent assembly scenario for the formation of supercomplexes that rather requires the coordinated synthesis and association of individual complexes, is proposed.     

Aim24 stabilizes respiratory chain supercomplexes and is required for efficient respiration

The mitochondrial respiratory chain is essential for the conversion of energy derived from the oxidation of metabolites into the membrane potential, which drives the synthesis of ATP. The electron transporting complexes bc₁ complex and the cytochrome c oxidase assemble into large supercomplexes, allowing efficient energy transduction. Currently, we have only limited information about what determines the structure of the supercomplex. Here, we characterize Aim24 in baker’s yeast as a protein, which is integrated in the mitochondrial inner membrane and is required for the structural integrity of the supercomplex. Deletion of AIM24 strongly affects activity of the respiratory chain and induces a growth defect on non-fermentable medium. Our data indicate that Aim24 has a function in stabilizing the respiratory chain supercomplexes.     

A unique respiratory adaptation in Drosophila independent of supercomplex formation

Large assemblies of respiratory chain complexes, known as supercomplexes, are present in the mitochondrial membrane in mammals and yeast, as well as in some bacterial membranes. The formation of supercomplexes is thought to contribute to efficient electron transfer, stabilization of each enzyme complex, and inhibition of reactive oxygen species (ROS) generation. In this study, mitochondria from various organisms were solubilized with digitonin, and then the solubilized complexes were separated by blue native PAGE (BN-PAGE). The results revealed a supercomplex consisting of complexes I, III, and IV in mitochondria from bovine and porcine heart, and a supercomplex consisting primarily of complexes I and III in mitochondria from mouse heart and liver. However, supercomplexes were barely detectable in Drosophila flight-muscle mitochondria, and only dimeric complex V was present. Drosophila mitochondria exhibited the highest rates of oxygen consumption and NADH oxidation, and the concentrations of the electron carriers, cytochrome c and quinone were higher than in other species. Respiratory chain complexes were tightly packed in the mitochondrial membrane containing abundant phosphatidylethanolamine with the fatty acid palmitoleic acid (C16:1), which is relatively high oxidation-resistant as compared to poly-unsaturated fatty acid. These properties presumably allow efficient electron transfer in Drosophila. These findings reveal the existence of a new mechanism of biological adaptation independent of supercomplex formation.     

Evidence for Physical Association of Mitochondrial Fatty Acid Oxidation and Oxidative Phosphorylation Complexes

Fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Reducing equivalents from FAO enter OXPHOS at the level of complexes I and III. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway. In this study, the physical and functional interactions between these pathways were examined. Extracts of isolated rat liver mitochondria were subjected to blue native polyacrylamide gel electrophoresis (BNGE) to separate OXPHOS complexes and supercomplexes followed by Western blotting using antisera to various FAO enzymes. Extracts were also subjected to sucrose density centrifugation and fractions analyzed by BNGE or enzymatic assays. Several FAO enzymes co-migrated with OXPHOS supercomplexes in different patterns in the gels. When palmitoyl-CoA was added to the sucrose gradient fractions containing OXPHOS supercomplexes in the presence of potassium cyanide, cytochrome c was reduced. Cytochrome c reduction was completely blocked by myxothiazol (a complex III inhibitor) and 3-mercaptopropionate (an inhibitor of the first step of FAO), but was only partially inhibited by rotenone (a complex I inhibitor). Although palmitoyl-CoA and octanoyl-CoA provided reducing equivalents to OXPHOS-containing supercomplex fractions, no accumulation of their intermediates was detected. In contrast, short branched acyl-CoA substrates were not metabolized by OXPHOS-containing supercomplex fractions. These data provide evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.     

Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability of Mitochondrial Respiratory Chain Supercomplexes

The mitochondrial inner membrane contains two non-bilayer‐forming phospholipids, phosphatidylethanolamine (PE) and cardiolipin (CL). Lack of CL leads to destabilization of respiratory chain supercomplexes, a reduced activity of cytochrome c oxidase, and a reduced inner membrane potential Δψ. Although PE is more abundant than CL in the mitochondrial inner membrane, its role in biogenesis and assembly of inner membrane complexes is unknown. We report that similar to the lack of CL, PE depletion resulted in a decrease of Δψ and thus in an impaired import of preproteins into and across the inner membrane. The respiratory capacity and in particular the activity of cytochrome c oxidase were impaired in PE-depleted mitochondria, leading to the decrease of Δψ. In contrast to depletion of CL, depletion of PE did not destabilize respiratory chain supercomplexes but favored the formation of larger supercomplexes (megacomplexes) between the cytochrome bc₁ complex and the cytochrome c oxidase. We conclude that both PE and CL are required for a full activity of the mitochondrial respiratory chain and the efficient generation of the inner membrane potential. The mechanisms, however, are different since these non-bilayer‐forming phospholipids exert opposite effects on the stability of respiratory chain supercomplexes.     

Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc₁ and aa₃-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O₂ reduction activity of the cyt. bc₁-aa₃ supercomplex. The potential role of the membrane-anchored cyt. c_y as a component in supercomplexes was also investigated.     

Kinetic advantage of forming respiratory supercomplexes

Components of respiratory chains in mitochondria and some aerobic bacteria assemble into larger, multiprotein membrane-bound supercomplexes. Here, we address the functional significance of supercomplexes composed of respiratory-chain complexes III and IV. Complex III catalyzes oxidation of quinol and reduction of water-soluble cytochrome c (cyt c), while complex IV catalyzes oxidation of the reduced cyt c and reduction of dioxygen to water. We focus on two questions: (i) under which conditions does diffusion of cyt c become rate limiting for electron transfer between these two complexes? (ii) is there a kinetic advantage of forming a supercomplex composed of complexes III and IV? To answer these questions, we use a theoretical approach and assume that cyt c diffuses in the water phase while complexes III and IV either diffuse independently in the two dimensions of the membrane or form supercomplexes. The analysis shows that the electron flux between complexes III and IV is determined by the equilibration time of cyt c within the volume of the intermembrane space, rather than the cyt c diffusion time constant. Assuming realistic relative concentrations of membrane-bound components and cyt c and that all components diffuse independently, the data indicate that electron transfer between complexes III and IV can become rate limiting. Hence, there is a kinetic advantage of bringing complexes III and IV together in the membrane to form supercomplexes.     

Structural and functional organization of the mitochondrial respiratory chain: A dynamic super-assembly

The structural organization of the mitochondrial oxidative phosphorylation (OXPHOS) system has received large attention in the past and most investigations led to the conclusion that the respiratory enzymatic complexes are randomly dispersed in the lipid bilayer of the inner membrane and functionally connected by fast diffusion of smaller redox components, Coenzyme Q and cytochrome c. More recent investigations by native gel electrophoresis, however, have shown the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis has demonstrated that Complexes I and III in mammalian mitochondria and Complexes I, III, and IV in plant mitochondria kinetically behave as single units with control coefficients approaching unity for each single component, suggesting the existence of substrate channelling within the supercomplexes. The reasons why the presence of substrate channelling for Coenzyme Q and cytochrome c was overlooked in the past are analytically discussed. The review also discusses the forces and the conditions responsible for the formation of the supramolecular units. The function of the supercomplexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Finally, there is increasing evidence that disruption of the supercomplex organization leads to functional derangements responsible for pathological changes.     

Cardiolipin-dependent Reconstitution of Respiratory Supercomplexes from Purified Saccharomyces cerevisiae Complexes III and IV

Here, we report for the first time in vitro reconstitution of the respiratory supercomplexes from individual complexes III and IV. Complexes III and IV were purified from Saccharomyces cerevisiae mitochondria. Complex III contained eight molecules of cardiolipin, and complex IV contained two molecules of cardiolipin, as determined by electrospray ionization-mass spectrometry. Complex IV also contained Rcf1p. No supercomplexes were formed upon mixing of the purified complexes, and low amounts of the supercomplex trimer III₂IV₁ were formed after reconstitution into proteoliposomes containing only phosphatidylcholine and phosphatidylethanolamine. Further addition of cardiolipin to the proteoliposome reconstitution mixture resulted in distinct formation of both the III₂IV₁ supercomplex trimer and III₂IV₂ supercomplex tetramer. No other anionic phospholipid was as effective as cardiolipin in supporting tetramer formation. Phospholipase treatment of complex IV prevented trimer formation in the absence of cardiolipin. Both trimer and tetramer formations were restored by cardiolipin. Analysis of the reconstituted tetramer by single particle electron microscopy confirmed native organization of individual complexes within the supercomplex. In conclusion, although some trimer formation occurred dependent only on tightly bound cardiolipin, tetramer formation required additional cardiolipin. This is consistent with the high cardiolipin content in the native tetramer. The dependence on cardiolipin for supercomplex formation suggests that changes in cardiolipin levels resulting from changes in physiological conditions may control the equilibrium between individual respiratory complexes and supercomplexes in vivo.     

Cytochrome c1 exhibits two binding sites for cytochrome c in plants

In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c₁, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c₁ and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a “floating boat bridge” of cytochrome c molecules (between complexes III and IV) in plant respirasome.     

Modulation of the Respiratory Supercomplexes in Yeast: ENHANCED FORMATION OF CYTOCHROME OXIDASE INCREASES THE STABILITY AND ABUNDANCE OF RESPIRATORY SUPERCOMPLEXES*

Yeast cells deficient in the Rieske iron-sulfur subunit (Rip1) of ubiquinol-cytochrome c reductase (bc₁) accumulate a late core assembly intermediate, which weakly associates with cytochrome oxidase (CcO) in a respiratory supercomplex. Expression of the N-terminal half of Rip1, which lacks the C-terminal FeS-containing globular domain (designated N-Rip1), results in a marked stabilization of trimeric and tetrameric bc₁-CcO supercomplexes. Another bc₁ mutant (qcr9Δ) stalled at the same assembly intermediate is likewise converted to stable supercomplex species by the expression of N-Rip1, but not by expression of intact Rip1. The N-Rip1-induced stabilization of bc₁-CcO supercomplexes is independent of the Bcs1 translocase, which mediates Rip1 translocation during bc₁ biogenesis. N-Rip1 induces the stabilization of bc₁-CcO supercomplexes through an enhanced formation of CcO. The association of N-Rip1 with the late core bc₁ assembly intermediate appears to confer stabilization of a CcO assembly intermediate. This induced stabilization of CcO is dependent on the Rcf1 supercomplex stabilization factor and only partially dependent on the presence of cardiolipin. N-Rip1 exerts a related induction of CcO stabilization in WT yeast, resulting in enhanced respiration. Additionally, the impact of CcO stabilization on supercomplexes was observed by means other than expression of N-Rip1 (via overexpression of CcO subunits Cox4 and Cox5a), demonstrating that this is a general phenomenon. This study presents the first evidence showing that supercomplexes can be stabilized by the stimulated formation of CcO.     

Respiratory chain supercomplexes in plant mitochondria

Supercomplexes are defined associations of protein complexes, which are important for several cellular functions. This "quintenary" organization level of protein structure recently was also described for the respiratory chain of plant mitochondria. Except succinate dehydrogenase (complex II), all complexes of the oxidative phosphorylation (OXPOS) system (complexes I, III, IV and V) were found to form part of supercomplexes. Compositions of these supramolecular structures were systematically investigated using digitonin solubilizations of mitochondrial fractions and two-dimensional Blue-native (BN) polyacrylamide gel electrophoresis. The most abundant supercomplex of plant mitochondria includes complexes I and III at a 1:2 ratio (I1 + III2 supercomplex). Furthermore, some supercomplexes of lower abundance could be described, which have I2 + III4, V2, III2 + IV(1-2), and I1 + III2 + IV(1-4) compositions. Supercomplexes consisting of complexes I plus III plus IV were proposed to be called "respirasome", because they autonomously can carry out respiration in the presence of ubiquinone and cytochrome c. Plant specific alternative oxidoreductases of the respiratory chain were not associated with supercomplexes under all experimental conditions tested. However, formation of supercomplexes possibly indirectly regulates alternative respiratory pathways in plant mitochondria on the basis of electron channeling. In this review, procedures to characterize the supermolecular organization of the plant respiratory chain and results concerning supercomplex structure and function are summarized and discussed.     

Architecture of active mammalian respiratory chain supercomplexes

In the inner mitochondrial membrane, the respiratory chain complexes generate an electrochemical proton gradient, which is utilized to synthesize most of the cellular ATP. According to an increasing number of biochemical studies, these complexes are assembled into supercomplexes. However, little is known about the architecture of the proposed multicomplex assemblies. Here, we report the electron microscopic characterization of the two respiratory chain supercomplexes I1III2 and I1III2IV1 in bovine heart mitochondria, which are also two major supercomplexes in human mitochondria. After purification and demonstration of enzymatic activity, their structures in projection were determined by single particle image analysis. A difference map between the supercomplexes I1III2 and I1III2IV1 closely fits the x-ray structure of monocomplex IV and shows its location in the assembly. By comparing different views of supercomplex I1III2IV1, the location and mutual arrangement of complex I and the complex III dimer are discussed. Detailed knowledge of the architecture of the active supercomplexes is a prerequisite for a deeper understanding of energy conversion by mitochondria in mammals.     

Supramolecular structure of the OXPHOS system in highly thermogenic tissue of Arum maculatum

The protein complexes of the mitochondrial respiratory chain associate in defined ways forming supramolecular structures called respiratory supercomplexes or respirasomes. In plants, additional oxidoreductases participate in respiratory electron transport, e.g. the so-called “alternative NAD(P)H dehydrogenases” or an extra terminal oxidase called “alternative oxidase” (AOX). These additional enzymes were previously reported not to form part of respiratory supercomplexes. However, formation of respiratory supercomplexes might indirectly affect “alternative respiration” because electrons can be channeled within the supercomplexes which reduces access of the alternative enzymes towards their electron donating substrates. Here we report an investigation on the supramolecular organization of the respiratory chain in thermogenic Arum maculatum appendix mitochondria, which are known to have a highly active AOX for heat production. Investigations based on mild membrane solubilization by digitonin and protein separation by blue native PAGE revealed a very special organization of the respiratory chain in A. maculatum, which strikingly differs to the one described for the model plant Arabidopsis thaliana: (i) complex I is not present in monomeric form but exclusively forms part of a I + III₂ supercomplex, (ii) the III₂ + IV and I + III₂ + IV supercomplexes are detectable but of low abundance, (iii) complex II has fewer subunits than in A. thaliana, and (iv) complex IV is mainly present as a monomer in a larger form termed “complex IVa”. Since thermogenic tissue of A. maculatum at the same time has high AOX and I + III₂ supercomplex abundance and activity, negative regulation of the alternative oxidase by supercomplex formation seems not to occur. Functional implications are discussed.     

Respiratory Complexes III and IV Are Not Essential for the Assembly/Stability of Complex I in Fungi

The functional relevance of respiratory supercomplexes in various eukaryotes including mammals, plants, and fungi is hitherto poorly elucidated. However, substantial evidence indicates as a major role the assembly and/or stabilization of mammalian complex I by supercomplex formation with complexes III and IV. Here, we demonstrate by using native electrophoresis that the long-lived Podospora anserina mutant Cyc1-1, respiring exclusively via the alternative oxidase (AOX), lacks an assembled complex III and possesses complex I partially assembled with complex IV into a supercomplex. This resembles the situation in complex-IV-deficient mutants displaying a corresponding phenotype but possessing I-III supercomplexes instead, suggesting that either complex III or complex IV is in a redundant manner necessary for assembly/stabilization of complex I as previously shown in mammals. To corroborate this notion, we constructed the double mutant Cyc1-1,Cox5::ble. Surprisingly, this mutant lacking both complexes III and IV is viable and essentially a phenocopy of mutant Cyc1-1 including the reversal of the phenotype towards wild-type-like characteristics by the several-fold overexpression of the AOX in mutant Cyc1-1,Cox5::ble(Gpd-Aox). Fungal specific features (not found in mammals) that must be responsible for assembly/stabilization of fungal complex I when complexes III and IV are absent, such as the presence of the AOX and complex I dimerization, are addressed and discussed. These intriguing results unequivocally prove that complexes III and IV are dispensable for assembly/stability of complex I in fungi contrary to the situation in mammals, thus highlighting the imperative to unravel the biogenesis of complex I as well as the true supramolecular organization of the respiratory chain and its functional significance in a variety of model eukaryotes. In summary, we present the first obligatorily aerobic eukaryote with an artificial, simultaneous lack of the respiratory complexes III and IV.     

A structural model of the cytochrome C reductase/oxidase supercomplex from yeast mitochondria

Mitochondrial respiratory chain complexes are arranged in supercomplexes within the inner membrane. Interaction of cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) was investigated in Saccharomyces cerevisiae. Projection maps at 15 A resolution of supercomplexes III(2) + IV(1) and III(2) + IV(2) were obtained by electron microscopy. Based on a comparison of our maps with atomic x-ray structures for complexes III and IV we present a pseudo-atomic model of their precise interaction. Two complex IV monomers are specifically attached to dimeric complex III with their convex sides. The opposite sides, which represent the complex IV dimer interface in the x-ray structure, are open for complex IV-complex IV interactions. This could lead to oligomerization of III(2) + IV(2) supercomplexes, but this was not detected. Instead, binding of cytochrome c to the supercomplexes was revealed. It was calculated that cytochrome c has to move less than 40 A at the surface of the supercomplex for electron transport between complex III(2) and complex IV. Hence, the prime function of the supercomplex III(2) + IV(2) is proposed to be a scaffold for effective electron transport between complexes III and IV.     

Role of phospholipids in respiratory cytochrome bc1 complex catalysis and supercomplex formation

Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc₁ complex and how disruption of a specific cardiolipin binding site in cytochrome c₁ alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc₁ complex was treated with phospholipase A₂. The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from − 52 to − 82 mV (heme b_L) and from + 113 to − 2 mV (heme b_H). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL_i) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc₁ complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.