Aims: The mitochondrial respiratory chain is recognized today to be arranged in supramolecular assemblies (supercomplexes). Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. In the present study, we have directly addressed this issue by testing the ROS generation by Complex I in two experimental systems in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. Results: The results of our investigation provide experimental evidence that the production of ROS is strongly increased in either model, supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I. Innovation: Dissociation of supercomplexes may link oxidative stress and energy failure in a vicious circle. Conclusion: Our findings support a central role of mitochondrial supramolecular structure in the development of the aging process and in the etiology and pathogenesis of most major chronic diseases.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production and found by combined use of specific inhibitors of Complex I that the one-electron donor in the Complex to oxygen is a redox center located prior to the sites where three different types of coenzyme Q (CoQ) competitors bind, to be identified with an Fe-S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain coenzyme Q analogues enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analogue idebenone is particularly effective, raising doubts about its safety as a drug. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by ROS as the primary cause of energy decline; in rat liver mitochondria, Complex I appears to be most affected by aging and to become strongly rate limiting for electron transfer. Mitochondrial energetics is also deranged in human platelets upon aging, as demonstrated by the decreased Pasteur effect (enhancement of lactate production by respiratory inhibitors). Cells counteract oxidative stress by antioxidants: CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems: likewise, they are overexpressed under oxidative stress conditions.

The enzymatic complexes of the mitochondrial respiratory chain have been extensively investigated in their structural and functional properties. A clear distinction is possible today between three complexes in which the difference in redox potential allows proton translocation (complexes I, III, and IV) and those having the mere function to convey electrons to the respiratory chain. We also have a clearer understanding of the structure and function of most respiratory complexes, of their biogenesis and regulation, and of their capacity to generate reactive oxygen species. Past investigations led to the conclusion that the complexes are randomly dispersed and functionally connected by diffusion of smaller redox components, coenzyme Q and cytochrome c. More-recent investigations by native gel electrophoresis and single-particle image processing showed the existence of supramolecular associations. Flux-control analysis demonstrated that complexes I and III in mammals and I, III, and IV in plants kinetically behave as single units, suggesting the existence of substrate channeling. This review discusses conditions affecting the formation of supercomplexes that, besides kinetic advantage, have a role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Disruption of supercomplex organization may lead to functional derangements responsible for pathologic changes.

Recent experimental evidence has replaced the random diffusion model of electron transfer with a model of supramolecular organisation based upon specific interactions between individual respiratory complexes. These supercomplexes were found to be functionally relevant by flux control analysis and to confer a kinetic advantage to NAD-linked respiration (channelling). However, the Coenzyme Q pool is still required for FAD-linked oxidations and for the proper equilibrium with Coenzyme Q bound in the supercomplex. Channelling in the cytochrome c region probably also occurs but does not seem to confer a particular kinetic advantage. The supramolecular association of individual complexes strongly depends on membrane lipid amount and composition and is affected by lipid peroxidation; it also seems to be modulated by membrane potential and protein phosphorylation. Additional properties of supercomplexes are stabilisation of Complex I, as evidenced by the destabilising effect on Complex I of mutations in either Complex III or IV, and prevention of excessive generation of reactive oxygen species. The dynamic character of the supercomplexes allows their involvement in metabolic adaptations and in control of cellular signalling pathways. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.

The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV.Considerable information exists on the structure at atomic resolution of most of the transmembrane protein complexes forming the mitochondrial respiratory chain; there is, however, still little direct information on the supramolecular organization of the enzymatic complexes in the inner mitochondrial membrane. Two extreme models for their arrangement in the membrane are conceivable: the model of a random organization of the individual respiratory complexes and that of a supercomplex assembly formed by stable association between proteins.The original solid-state model of Chance and Williams (1) changed gradually because the oxidative phosphorylation enzymes were found functionally active when isolated as individual complexes (2) Despite the acceptance of the idea that electron transfer in mitochondrial membranes depends on random collisions between small diffusing molecules (coenzyme Q and cytochrome c) and complexes