Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers

We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III₂-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.     

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.     

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.     

Supercomplex organization of the mitochondrial respiratory chain and the role of the Coenzyme Q pool: pathophysiological implications

In this review we examine early and recent evidence for an aggregated organization of the mitochondrial respiratory chain. Blue Native Electrophoresis suggests that in several types of mitochondria Complexes I, III and IV are aggregated as fixed supramolecular units having stoichiometric proportions of each individual complex. Kinetic evidence by flux control analysis agrees with this view, however the presence of Complex IV in bovine mitochondria cannot be demonstrated, presumably due to high levels of free Complex. Since most Coenzyme Q appears to be largely free in the lipid bilayer of the inner membrane, binding of Coenzyme Q molecules to the Complex I-III aggregate is forced by its dissociation equilibrium; furthermore free Coenzyme Q is required for succinate-supported respiration and reverse electron transfer. The advantage of the supercomplex organization is in a more efficient electron transfer by channelling of the redox intermediates and in the requirement of a supramolecular structure for the correct assembly of the individual complexes. Preliminary evidence suggests that dilution of the membrane proteins with extra phospholipids and lipid peroxidation may disrupt the supercomplex organization. This finding has pathophysiological implications, in view of the role of oxidative stress in the pathogenesis of many diseases.     

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.     

Structural organization of the mitochondrial respiratory chain

Two models exist of the mitochondrial respiratory chain: the model of a random organization of the individual respiratory enzyme complexes and that of a super-complex assembly formed by stable association between the individual complexes. Recently Sch?gger, using digitonin solubilization and Blue Native PAGE produced new evidence of preferential associations, in particular a Complex I monomer with a Complex III dimer, and suggested a model of the respiratory chain (the respirasome) based on direct electron channelling between complexes. Discrimination between the two models is amenable to kinetic testing using flux control analysis. Experimental evidence obtained in beef heart SMP, according to the extension of the Metabolic Control Theory for pathways with metabolic channelling, showed that enzyme associations involving Complex I and Complex III take place in the respiratory chain while Complex IV seems to be randomly distributed, with cytochrome c behaving as a mobile component. Flux control analysis at anyone of the respiratory complexes involved in aerobic succinate oxidation indicated that Complex II and III are not functionally associated in a stable supercomplex. A critical appraisal of the solid-state model of the mitochondrial respiratory chain requires its reconciliation with previous biophysical and kinetic evidence that CoQ behaves as a homogeneous diffusible pool between all reducing enzyme and all oxidizing enzymes: the hypothesis can be advanced that both models (CoQ pool and supercomplexes) are true, by postulating that supercomplexes physiologically exist in equilibrium with isolated complexes depending on metabolic conditions of the cell.     

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.     

Structural and functional organization of Complex I in the mitochondrial respiratory chain

Metabolic flux control analysis of NADH oxidation in bovine heart submitochondrial particles revealed high flux control coefficients for both Complex I and Complex III, suggesting that the two enzymes are functionally associated as a single enzyme, with channelling of the common substrate, Coenzyme Q. This is in contrast with the more accepted view of a mobile diffusable Coenzyme Q pool between these enzymes. Dilution with phospholipids of a mitochondrial fraction enriched in Complexes I and III, with consequent increased theoretical distance between complexes, determines adherence to pool behavior for Coenzyme Q, but only at dilution higher than 1:5 (protein:phospholipids), whereas, at lower phospholipid content, the turnover of NADH cytochrome c reductase is higher than expected by the pool equation.     

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 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.     

Threshold Effects and Control of Oxidative Phosphorylation in Nonsynaptic Rat Brain Mitochondria

Abstract: The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by ∼72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by ∼70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.     

Failure of exogenous NADH and cytochrome c to support energy-dependent swelling of mitochondria

The possibility of direct oxidation of external NADH in rat liver mitochondria and of the inner membrane potential generation in this process is still not clear. In the present work, the energy-dependent swelling of mitochondria in the medium containing valinomycin and potassium acetate was measured as one of the main criteria of the proton-motive force generation by complex III, complex IV, and both complexes III and IV of the respiratory chain. Mitochondria swelling induced by external NADH oxidation was compared with that induced by succinate or ferrocyanide oxidation, or by electron transport from succinate to ferricyanide. Mitochondria swelling, nearly equal to that promoted by ferrocyanide oxidation, was observed under external NADH oxidation, but only after the outer mitochondrial membrane was ruptured as a result of the swelling-contraction cycle, caused by succinate oxidation and its subsequent inhibition. In this case, significantly accelerated intermembrane electron transport and well-detected inner membrane potential generation, in addition to mitochondria swelling, were also observed. Presented results suggest that exogenous NADH and cytochrome c do not support the inner membrane potential generation in intact rat liver mitochondria, because the external NADH-cytochrome c reductase system, oriented in the outer mitochondrial membrane toward the cytoplasm, is inaccessible for endogenous cytochrome c reduction; as well, the inner membrane cytochrome c oxidase is inaccessible for exogenous cytochrome c oxidation.     

Inactivation of complex I of the respiratory chain of maize mitochondria incubated in vitro by elevated temperature

The functional stability of the ‘external’ NADH dehydrogenase and complexes I–IV of the respiratory chain of maize mitochondria was studied during mitochondria incubation in vitro at elevated temperatures. The increase in the incubation temperature from 0°C to 37°C significantly changed the stability of the respiratory chain. At 27°C and higher, the rate of oxidation of NAD-depended substrates decreased drastically, which is related to inactivation of complex I. Complexes II, III and IV of the respiratory chain and the ‘external’ NADH dehydrogenase were functionally stable at elevated temperatures. Moreover, the possibility of electron transport during oxidation of NAD-dependent substrates, in particular malate, bypasses complex I using rotenon insensitive NADH dehydrogenase.     

Mitochondrial fatty acid oxidation and oxidative stress: Lack of reverse electron transfer-associated production of reactive oxygen species

Reverse electron transfer (RET) from succinate to NAD⁺ is known to be accompanied by high generation of reactive oxygen species (ROS). In contrast, oxidation of fatty acids by mitochondria, despite being a powerful source of FADH₂, does not lead to RET-associated high ROS generation. Here we show that oxidation of carnitine esters of medium- and long-chain fatty acids by rat heart mitochondria is accompanied by neither high level of NADH/NAD⁺ nor intramitochondrial reduction of acetoacetate to β-hydroxybutyrate, comparable to those accompanying succinate oxidation, although it produces the same or higher energization of mitochondria as evidenced by high transmembrane potential. Also in contrast to the oxidation of succinate, where conversion of the pH difference between the mitochondrial matrix and the medium into the transmembrane electric potential by addition of nigericin results in a decrease of ROS generation, the same treatment during oxidation of octanoylcarnitine produces a large increase of ROS. Analysis of respiratory chain complexes by Blue Native polyacrylamide gel electrophoresis revealed bands that could tentatively point to supercomplex formation between complexes II and I and complexes II and III. However, no such association could be found between complex I and the electron transferring flavoprotein that participates in fatty acid oxidation. It is speculated that structural association between respective respiratory chain components may facilitate effective reverse electron transfer.     

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.     

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.     

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.     

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.     

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.     

Association of ferrochelatase with Complex I in bovine heart mitochondria

The location of ferrochelatase in bovine heart mitochondria has been studied. When the mitochondria were fractionated into Complexes I, II and III, ferrochelatase activity was only found in Complex I. Complex I also showed heme synthesis from ferric ion in the presence of NADH as an electron donor. Immunoblot experiments confirmed the presence of ferrochelatase in Complex I, but not in Complexes II or III. Some phospholipids, including phosphatidylserine and cardiolipin, stimulated NADH-dependent heme synthesis from ferric ion. When purified ferrochelatase was incubated with the low molecular weight form of NADH dehydrogenase prepared from Complex I, heme synthesis from ferric ion occurred by the addition of NADH. FMN markedly elevated the synthesis. These results indicate that ferrous ion is produced by NADH oxidation in Complex I and is then utilized for heme synthesis by ferrochelatase.