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.     

New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex II

A project to systematically investigate respiratory supercomplexes in plant mitochondria was initiated. Mitochondrial fractions from Arabidopsis, potato (Solanum tuberosum), bean (Phaseolus vulgaris), and barley (Hordeum vulgare) were carefully treated with various concentrations of the nonionic detergents dodecylmaltoside, Triton X-100, or digitonin, and proteins were subsequently separated by (a) Blue-native polyacrylamide gel electrophoresis (PAGE), (b) two-dimensional Blue-native/sodium dodecyl sulfate-PAGE, and (c) two-dimensional Blue-native/Blue-native PAGE. Three high molecular mass complexes of 1,100, 1,500, and 3,000 kD are visible on one-dimensional Blue native gels, which were identified by separations on second gel dimensions and protein analyses by mass spectrometry. The 1,100-kD complex represents dimeric ATP synthase and is only stable under very low concentrations of detergents. In contrast, the 1,500-kD complex is stable at medium and even high concentrations of detergents and includes the complexes I and III(2). Depending on the investigated organism, 50% to 90% of complex I forms part of this supercomplex if solubilized with digitonin. The 3,000-kD complex, which also includes the complexes I and III, is of low abundance and most likely has a III(4)I(2) structure. The complexes IV, II, and the alternative oxidase were not part of supercomplexes under all conditions applied. Digitonin proved to be the ideal detergent for supercomplex stabilization and also allows optimal visualization of the complexes II and IV on Blue-native gels. Complex II unexpectedly was found to be composed of seven subunits, and complex IV is present in two different forms on the Blue-native gels, the larger of which comprises additional subunits including a 32-kD protein resembling COX VIb from other organisms. We speculate that supercomplex formation between the complexes I and III limits access of alternative oxidase to its substrate ubiquinol and possibly regulates alternative respiration. The data of this investigation are available at http://www.gartenbau.uni-hannover.de/genetik/braun/AMPP.     

Cherry fruit development: Indoleacetic acid oxidase isoenzymes in the seed

Two anionic indoleacetic acid oxidase isoenzymes were separated by polyacrylamide gel electrophoresis from an acetate buffer (0.2 M, pH 4.0) extract of sour cherry ( Prunus cerasus L. cv. Montmorency) seed. One isoenzyme migrated to Rf 0.25 (I1) and the other to Rf 0.78 (I₂). Isoenzyme I, exhibited hyperbolic kinetics and was found during all three stages of fruit development with the highest levels during early stage II. The isoenzyme I₂ showed sigmoidal kinetics and was found only during stages II and III of fruit growth with highest levels during stage III. The activities of both isoenzymes were markedly enhanced by addition of Mn2+ and 2,4-dichloro-phenol to the reaction mixture. Isoenzyme I, showed higher affinity for indoleacetic acid than isoenzyme I₂. The significance of these isoenzymes in cherry fruit growth is discussed.     

Gluing the respiratory chain together. Cardiolipin is required for supercomplex formation in the inner mitochondrial membrane

Cytochrome bc(1) complex (complex III) and cytochrome c oxidase complex (complex IV) are multisubunit homodimers that are essential components of the mitochondrial respiratory chain. Complexes III and IV associate to form a supercomplex that can be displayed using blue native polyacrylamide gel electrophoresis. Both homodimeric complexes contain tightly associated cardiolipin (CL) required for function. We report here that in a crd1Delta strain of yeast (null in expression of CL synthase) approximately 90% of complexes III and IV were observed as individual homodimers; only the supercomplex was observed with CRD1 wild type cells. Introduction of a plasmid born copy of the CRD1 gene under exogenous regulation by doxycycline made possible controlled variation in the in vivo CL levels. At an intermediate level of CL, a mixture of individual homodimers (30%) and supercomplex (70%) was observed. These results strongly indicate that CL plays a central role in higher order organization of components of the respiratory chain of mitochondria.     

Respiratory chain supercomplexes of mitochondria and bacteria

Respiratory chain complexes are fragments of larger structural and functional units, the respiratory chain supercomplexes or "respirasomes", which exist in bacterial and mitochondrial membranes. Supercomplexes of mitochondria and bacteria contain complexes III, IV, and complex I, with the notable exception of Saccharomyces cerevisiae, which does not possess complex I. These supercomplexes often are stable to sonication but sensitive to most detergents except digitonin. In S. cerevisiae, a major component linking complexes III and IV together is cardiolipin.In Paracoccus denitrificans, complex I itself is rather detergent-sensitive and thus could not be obtained in detergent-solubilized form so far. However, it can be isolated as part of a supercomplex. Stabilization of complex I by binding to complex III was also found in human mitochondria. Further functional roles of the organization in a supercomplex are catalytic enhancement by reducing diffusion distances of substrates or, depending on the organism, channelling of the substrates quinone and cytochrome c. This makes redox reactions less dependent of midpoint potentials of substrates, and permits electron flow at low degree of substrate reduction.A dimeric state of ATP synthase seems to be specific for mitochondria. Exclusively, monomeric ATP synthase was found in Acetobacterium woodii, in P. denitrificans, and in spinach chloroplasts.     

Identification and characterization of respirasomes in potato mitochondria

Plant mitochondria were previously shown to comprise respiratory supercomplexes containing cytochrome c reductase (complex III) and NADH dehydrogenase (complex I) of I(1)III(2) and I(2)III(4) composition. Here we report the discovery of additional supercomplexes in potato (Solanum tuberosum) mitochondria, which are of lower abundance and include cytochrome c oxidase (complex IV). Highly active mitochondria were isolated from potato tubers and stems, solubilized by digitonin, and subsequently analyzed by Blue-native (BN) polyacrylamide gel electrophoresis (PAGE). Visualization of supercomplexes by in-gel activity stains for complex IV revealed five novel supercomplexes of 850, 1,200, 1,850, 2,200, and 3,000 kD in potato tuber mitochondria. These supercomplexes have III(2)IV(1), III(2)IV(2), I(1)III(2)IV(1), I(1)III(2)IV(2), and I(1)III(2)IV(4) compositions as shown by two-dimensional BN/sodium dodecyl sulfate (SDS)-PAGE and BN/BN-PAGE in combination with activity stains for cytochrome c oxidase. Potato stem mitochondria include similar supercomplexes, but complex IV is partially present in a smaller version that lacks the Cox6b protein and possibly other subunits. However, in mitochondria from potato tubers and stems, about 90% of complex IV was present in monomeric form. It was suggested that the I(1)III(2)IV(4) supercomplex represents a basic unit for respiration in mammalian mitochondria termed respirasome. Respirasomes also occur in potato mitochondria but were of low concentrations under all conditions applied. We speculate that respirasomes are more abundant under in vivo conditions.     

Three-dimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.     

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.     

Respiration, cyanide-insensitive oxygen uptake and oxidative phosphorylation in cyanobacteria

Cyanide-insensitive oxygen uptake in the dark of 9 species of cyanobacteria was 6–20% of the total oxygen uptake of intact cells. In Phormidium , no cyanideinsensitive oxygen uptake was observed. In intact cells, the I₅₀ value for cyanide was significantly lower in cyanobacteria of the taxonomic sections I to III (1–9 μ M ) than in those from section IV and V (10–60 μ M ). Cyanide-insensitive oxygen uptake in the cell-free system of Anabaena variabilis was not affected by typical inhibitors of the alternative pathway of plants. Cell-free oxidation of cytochrome c was completely inhibited by cyanide with an I₅₀ value of 0.5–1 μ M . Electron transport of intact cells without cyanide present yielded P/O ratios of 0.7–3.0. The data on oxidative phosphorylation using intact cells and the cell-free system, indicate that cyanide-insensitive oxygen uptake is not coupled to ATP formation.     

Changes of Respiratory Chain Activity in Mitochondrial and Synaptosomal Fractions Isolated from the Gerbil Brain After Graded Ischaemia

Abstract: In this study we have examined (1) the integrated function of the mitochondrial respiratory chain by polarographic measurements and (2) the activities of the respiratory chain complexes I, II–III, and IV as well as the ATP synthase (complex V) in free mitochondria and synaptosomes isolated from gerbil brain, after a 30-min period of graded cerebral ischaemia. These data have been correlated with cerebral blood flow (CBF) values as measured by the hydrogen clearance technique. Integrated functioning of the mitochondrial respiratory chain, using both NAD-linked and FAD-linked substrates, was initially affected at CBF values of ∼35 ml 100 g⁻¹ min⁻¹, and declined further as the CBF was reduced. The individual mitochondrial respiratory chain complexes, however, showed differences in sensitivity to graded cerebral ischaemia. Complex I activities decreased sharply at blood flows below ∼30 ml 100 g⁻¹ min⁻¹ (mitochondria and synaptosomes) and complex II–III activities decreased at blood flows below 20 ml 100 g⁻¹ min⁻¹ (mitochondria) and 35–30 ml 100 g⁻¹ min⁻¹ (synaptosomes). Activities declined further as CBF was reduced below these levels. Complex V activity was significantly affected only when the blood flow was reduced below 15–10 ml 100 g⁻¹ min⁻¹ (mitochondria and synaptosomes). In contrast, complex IV activity was unaffected by graded cerebral ischaemia, even at very low CBF levels.     

Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp. strain 7

Abstract The role of cytochrome b ₅₆₂, a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b ₅₆₂ did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form ( b ₅₆₂) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b ₅₆₂ was upstream of both the ' g _y= 1.89' Rieske FeS cluster and the a -type cytochromes. These results show that the b -type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.     

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.     

Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans

Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.     

Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients

Mutations in the human TAZ gene are associated with Barth Syndrome, an often fatal X-linked disorder that presents with cardiomyopathy and neutropenia. The TAZ gene encodes Tafazzin, a putative phospholipid acyltranferase that is involved in the remodeling of cardiolipin, a phospholipid unique to the inner mitochondrial membrane. It has been shown that the disruption of the Tafazzin gene in yeast (Taz1) affects the assembly and stability of respiratory chain Complex IV and its supercomplex forms. However, the implications of these results for Barth Syndrome are restricted due to the additional presence of Complex I in humans that forms a supercomplex with Complexes III and IV. Here, we investigated the effects of Tafazzin, and hence cardiolipin deficiency in lymphoblasts from patients with Barth Syndrome, using blue-native polyacrylamide gel electrophoresis. Digitonin extraction revealed a more labile Complex I/III(2)/IV supercomplex in mitochondria from Barth Syndrome cells, with Complex IV dissociating more readily from the supercomplex. The interaction between Complexes I and III was also less stable, with decreased levels of the Complex I/III(2) supercomplex. Reduction of Complex I holoenzyme levels was observed also in the Barth Syndrome patients, with a corresponding decrease in steady-state subunit levels. We propose that the loss of mature cardiolipin species in Barth Syndrome results in unstable respiratory chain supercomplexes, thereby affecting Complex I biogenesis, respiratory activities and subsequent pathology.     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

Preparation of Porcine Neurophysin Proteins by High Performance Liquid Chromatography

Abstract: The crude neurophysin containing extract from posterior lobes of porcine pituitaries was roughly purified by gel chromatography. 15 mg of the lyophilized neurophysin complex were completely separated by HPLC yielding in neurophysin I₁ (3.6 mg), I₂ (4.0 mg), II (4.6 mg) and III (1.9 mg). All of the neurophysins were homogenous by PAGE and SDS-electrophoresis, isoelectrofocussing, amino-acid composition and N- and C-terminal amino acid analysis. In conclusion, HPLC is a reliable and quick method for the preparation of pure neurophysins.     

Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria

Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Delta mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872-4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria.     

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.     

Effect of mitochondrial genome rearrangement on respiratory activity, photosynthesis, photorespiration and energy status of MSC16 cucumber (Cucumis sativus) mutant

The effects of changes in mitochondrial DNA in cucumber ( Cucumis sativus L.) mosaic mutant (MSC16) on respiration, photosynthesis and photorespiration were analyzed under non-stressed conditions. Decreased respiratory capacity of complex I in MSC16 mitochondria was indicated by lower respiration rates of intact mitochondria with malate and by rotenone-inhibited NADH or malate oxidation in the presence of alamethicin. Moreover, blue native PAGE indicated decreased intensity of protein bands of respiratory chain complex I in MSC16 leaves. Concerning the redox state, complex I impairment could be compensated to some extent by increased external NADH dehydrogenases (ND_exNADH) and alternative oxidase (AOX) capacity, the latter presenting differential expression in the light and in the dark. Although MSC16 mitochondria have a higher AOX protein level and an increased capacity, the AOX activity measured in the dark conditions by oxygen discrimination technique is similar to that in wild-type (WT) plants. Photosynthesis induction by light followed different patterns in WT and MSC16, suggesting changes in feedback chloroplast ΔpH caused by different adenylate levels. At steady-state, net photosynthesis was only slightly impaired in MSC16 mutants, while photorespiration rate (PR) was significantly increased. This was the result of large decreases in both stomatal and mesophyll conductance to CO₂, which resulted in a lower CO₂ concentration in the chloroplasts. The observed changes on CO₂ diffusion caused by mitochondrial mutations open a whole new view of interaction between organelle metabolism and whole tissue physiology. The sum of all the described changes in photosynthetic and respiratory metabolism resulted in a lower ATP availability and a slower plant growth.     

Mutations in mitochondrial complex III uniquely affect complex I in Caenorhabditis elegans

Mitochondrial supercomplexes containing complexes I, III, and IV of the electron transport chain are now regarded as an established entity. Supercomplex I·III·IV has been theorized to improve respiratory chain function by allowing quinone channeling between complexes I and III. Here, we show that the role of the supercomplexes extends beyond channeling. Mutant analysis in Caenorhabditis elegans reveals that complex III affects supercomplex I·III·IV formation by acting as an assembly or stabilizing factor. Also, a complex III mtDNA mutation, ctb-1, inhibits complex I function by weakening the interaction of complex IV in supercomplex I·III·IV. Other complex III mutations inhibit complex I function either by decreasing the amount of complex I (isp-1), or decreasing the amount of complex I in its most active form, the I·III·IV supercomplex (isp-1;ctb-1). ctb-1 suppresses a nuclear encoded complex III defect, isp-1, without improving complex III function. Allosteric interactions involve all three complexes within the supercomplex and are necessary for maximal enzymatic activities.