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.     

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.     

The role of Coenzyme Q in mitochondrial electron transport

In mitochondria, most Coenzyme Q is free in the lipid bilayer; the question as to whether tightly bound, non-exchangeable Coenzyme Q molecules exist in mitochondrial complexes is still an open question.We review the mechanism of inter-complex electron transfer mediated by ubiquinone and discuss the kinetic consequences of the supramolecular organization of the respiratory complexes (randomly dispersed vs. super-complexes) in terms of Coenzyme Q pool behavior vs. metabolic channeling, respectively, both in physiological and in some pathological conditions. As an example of intra-complex electron transfer, we discuss in particular Complex I, a topic that is still under active investigation.     

Mitochondrial Complex I: structural and functional aspects

This review examines two aspects of the structure and function of mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) that have become matter of recent debate. The supramolecular organization of Complex I and its structural relation with the remainder of the respiratory chain are uncertain. Although the random diffusion model [C.R. Hackenbrock, B. Chazotte, S.S. Gupte, The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembranes 18 (1986) 331-368] has been widely accepted, recent evidence suggests the presence of supramolecular aggregates. In particular, evidence for a Complex I-Complex III supercomplex stems from both structural and kinetic studies. Electron transfer in the supercomplex may occur by electron channelling through bound Coenzyme Q in equilibrium with the pool in the membrane lipids. The amount and nature of the lipids modify the aggregation state and there is evidence that lipid peroxidation induces supercomplex disaggregation. Another important aspect in Complex I is its capacity to reduce oxygen with formation of superoxide anion. The site of escape of the single electron is debated and either FMN, iron-sulphur clusters, and ubisemiquinone have been suggested. The finding in our laboratory that two classes of hydrophobic inhibitors have opposite effects on superoxide production favours an iron-sulphur cluster (presumably N2) is the direct oxygen reductant. The implications in human pathology of better knowledge on these aspects of Complex I structure and function are briefly discussed.     

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.     

Mitochondrial respiratory chain super-complex I–III in physiology and pathology

Recent investigations by native gel electrophoresis showed the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis demonstrated that Complex I and Complex III in mammalian mitochondria kinetically behave as a single unit with control coefficients approaching unity for each component, suggesting the existence of substrate channeling within the super-complex. The formation of this supramolecular unit largely depends on the lipid content and composition of the inner mitochondrial membrane. The function of the super-complexes 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, particularly Complex I, and in preventing excess oxygen radical formation. There is increasing evidence that disruption of the super-complex organization leads to functional derangements responsible for pathological changes, as we have found in K-ras-transformed fibroblasts.     

Mitochondrial Complex I defects in aging

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed.The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase.A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats.In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases. (Mol Cell Biochem 174: 329–333, 1997)     

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.     

Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling

Recent evidence, mainly based on native electrophoresis, has suggested that the mitochondrial respiratory chain is organized in the form of supercomplexes, due to the aggregation of the main respiratory chain enzymatic complexes. This evidence strongly contrasts the previously accepted model, the Random Diffusion Model, largely based on kinetic studies, stating that the complexes are randomly distributed in the lipid bilayer of the inner membrane and functionally connected by lateral diffusion of small redox molecules, i.e., coenzyme Q and cytochrome c. This review critically examines the experimental evidence, both structural and functional, pertaining to the two models and attempts to provide an updated view of the organization of the respiratory chain and of its kinetic consequences. The conclusion that structural respiratory assemblies exist is overwhelming, whereas the expected functional consequence of substrate channeling between the assembled enzymes is controversial. Examination of the available evidence suggests that, although the supercomplexes are structurally stable, their kinetic competence in substrate channeling is more labile and may depend on the system under investigation and the assay conditions. mitochondria; supercomplexes; ubiquinone; complex I (NADH-ubiquinone oxidoreductase)     

Mitochondrial Complex I: structure, function, and implications in neurodegeneration

Mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) is the least understood of respiratory complexes. In this review we emphasize some novel findings on this enzyme that are of relevance to the pathogenesis of neurodegenerative diseases. Besides Coenzyme Q (CoQ), also oxygen may be an electron acceptor from the enzyme, with generation of superoxide radical in the mitochondrial matrix. The site of superoxide generation is debated: we present evidence based on the rational use of several inhibitors that the one-electron donor to oxygen is an iron-sulphur cluster, presumably N2. On this assumption we present a novel mechanism of electron transfer to the acceptor, CoQ. Strong evidence is accumulating that electron transfer from Complex I to Complex III via CoQ is not performed by operation of the CoQ pool but by direct channelling within a super-complex including Complex I, Complex III and bound CoQ. Besides structural evidence of a Complex I -Complex III aggregate obtained by native electrophoresis, we have obtained kinetic evidence based on metabolic flux analysis, demonstrating that Complexes I and III behave as an individual enzyme. Quantitative and qualitative changes of phospholipids, including peroxidation, may affect the supercomplex formation. Complex I is deeply involved in pathological changes, including neurodegeneration. Maternally inherited mutations in mitochondrial DNA genes encoding for Complex I subunits are at the basis of Leber's Hereditary Optic Neuropathy; a decrease of electron transfer in the complex, due to the mutations, is not sufficient per se to explain the clinical phenotype, and other factors including proton translocation and oxygen radical generation have been considered of importance. Complex I changes are also involved in more common neurological diseases of the adult and old ages. In this review we discuss Parkinson's disease, where the pathogenic involvement of Complex I is better understood; the accumulated evidence on the mode of action of Complex I inhibitors and their effect on oxygen radical generation is discussed in terms of the aetiology and pathogenesis of the disease.     

Two‐dimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica

Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2‐D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue‐native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2‐D native systems are useful means to identify the constituent proteins and their copy numbers in detergent‐labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.     

Supramolecular organization of protein complexes in the mitochondrial inner membrane

The liquid state model that envisions respiratory chain complexes diffusing freely in the membrane is increasingly challenged by reports of supramolecular organization of the complexes in the mitochondrial inner membrane. Supercomplexes of complex III with complex I and/or IV can be isolated after solubilisation with mild detergents like digitonin. Electron microscopic studies have shown that these have a distinct architecture and are not random aggregates. A 3D reconstruction of a I1III2IV1 supercomplex shows that the ubiquinone and cytochrome c binding sites of the individual complexes are facing each other, suggesting a role in substrate channelling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain. Furthermore, a supramolecular organisation of ATP synthases has been observed in mitochondria, where ATP synthase is organised in dimer rows. Dimers can be isolated by mild detergent extraction and recent electron microscopic studies have shown that the membrane domains of the two partners in the dimer are at an angle to each other, indicating that in vivo the dimers would cause the membrane to bend. The suggested role in crista formation is supported by the observation of rows of ATP synthase dimers in the most curved parts of the cristae. Together these observations show that the mitochondrial inner membrane is highly organised and that the molecular events leading to ATP synthesis are carefully coordinated.     

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.     

Generation of Reactive Oxygen Species by Mitochondrial Complex I: Implications in Neurodegeneration

Mitochondrial Complex I [NADH Coenzyme Q (CoQ) oxidoreductase] is the least understood of respiratory complexes. In this review we emphasize some novel findings on this enzyme that are of relevance to the pathogenesis of neurodegenerative diseases. Besides CoQ, also oxygen may be an electron acceptor from the enzyme, with generation of superoxide radical in the mitochondrial matrix. The site of superoxide generation is debated: we present evidence based on the rational use of several inhibitors that the one-electron donor to oxygen is an iron-sulphur cluster, presumably N2. On this assumption we present a novel mechanism of electron transfer to the acceptor, CoQ. Complex I is deeply involved in pathological changes, including neurodegeneration. Complex I changes are involved in common neurological diseases of the adult and old ages. Mitochondrial cytopathies due to mutations of either nuclear or mitochondrial DNA may represent a useful model of neurodegeneration. In this review we discuss Parkinson’s disease, where the pathogenic involvement of Complex I is better understood; the accumulated evidence on the mode of action of Complex I inhibitors and their effect on oxygen radical generation is discussed in terms of the aetiology and pathogenesis of the disease. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

The higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes

The organization of the oxidative phosphorylation (OXPHOS) system within the inner mitochondrial membrane appears to be far more complicated than previously thought. In particular, the individual protein complexes of the OXPHOS system (complexes I to V) were found to specifically interact forming defined supramolecular structures. Blue-native polyacrylamide gel electrophoresis and single particle electron microscopy proved to be especially valuable in studying the so-called "respiratory supercomplexes". Based on these procedures, increasing evidence was presented supporting a "solid state" organization of the OXPHOS system. Here, we summarize results on the formation, organisation and function of the various types of mitochondrial OXPHOS supercomplexes.     

Attraction within the membrane. Forces behind transmembrane protein folding and supramolecular complex assembly

Biological membranes are fascinating two-dimensional microenvironments that exhibit unique solvent behaviours due to their varying lipid composition. Although many important bioenergetic and signalling events involve the transient or permanent assembly of membrane protein complexes, the characterization of the thermodynamic and kinetic properties behind this assembly is just beginning. In particular, the molecular forces that govern protein association within these structures remain poorly understood. An understanding of the docking of transmembrane proteins to supramolecular complexes, which will make possible the development of predictive computational tools, will require detailed knowledge of interaction forces at the atomistic or residue level. Here, I review current data on supramolecular complexes in membrane environments and make a tentative comparison between assembly processes in membranes and those driven by the hydrophobic effect in water. This comparison suggests that, in addition to being controlled by specific characteristics of the lipid molecules themselves, molecular assembly in the membrane milieu also depends more generally on the entropy of the lipid fraction.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance

Respiratory chains are crucial for cellular energy conversion and consist of multi‐subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high‐resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.     

A critical appraisal of the mitochondrial coenzyme Q pool.

The function of the coenzyme Q (CoQ) pool in the inner mitochondrial membrane is reviewed in view of recent findings suggesting a supramolecular organization of the mitochondrial respiratory complexes. In spite of the structural evidence for preferential aggregations of the inner membrane components, most kinetic evidence is in favor of a dispersed organization based on random collisions of the small connecting redox components, in particular CoQ, with the individual complexes. The shape of the CoQ molecule in the pool, suggested to be a folded one, is in agreement with its very rapid lateral diffusion mobility in the membrane midplane. Since the structural evidence in favor of specific supercomplexes is rather strong, it cannot be excluded that electron transfer may follow either pool behavior or preferential channeling depending on the physiological conditions. Another function ascribed to the CoQ pool is the antioxidant action of the reduced CoQ molecules; although it cannot be excluded that protein-bound ubisemiquinones may be a source of oxygen radicals, particularly at the level of complex III, the available evidence suggests that the mitochondrial pool only behaves as an antioxidant under physiological conditions.