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Under aerobic conditions, mitochondrial oxidative phosphorylation (OXPHOS) converts the energy released by nutrient oxidation into ATP, the currency of living organisms. The whole biochemical machinery is hosted by the inner mitochondrial membrane (mtIM) where the protonmotive force built by respiratory complexes, dynamically assembled as super-complexes, allows the F1FO-ATP synthase to make ATP from ADP + Pi. Recently mitochondria emerged not only as cell powerhouses, but also as signaling hubs by way of reactive oxygen species (ROS) production. However, when ROS removal systems and/or OXPHOS constituents are defective, the physiological ROS generation can cause ROS imbalance and oxidative stress, which in turn damages cell components. Moreover, the morphology of mitochondria rules cell fate and the formation of the mitochondrial permeability transition pore in the mtIM, which, most likely with the F1FO-ATP synthase contribution, permeabilizes mitochondria and leads to cell death. As the multiple mitochondrial functions are mutually interconnected, changes in protein composition by mutations or in supercomplex assembly and/or in membrane structures often generate a dysfunctional cascade and lead to life-incompatible diseases or severe syndromes. The known structural/functional changes in mitochondrial proteins and structures, which impact mitochondrial bioenergetics because of an impaired or defective energy transduction system, here reviewed, constitute the main biochemical damage in a variety of genetic and age-related diseases.

In this chapter we provide a review with a focus on the function of Coenzyme Q (CoQ, ubiquinone) in mitochondria. The notion of a mobile pool of CoQ in the lipid bilayer as the vehicle of electrons from respiratory complexes has somewhat changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising Complexes I and III; in such assembly the electron transfer is thought to be mediated by direct channelling, and we provide evidence for a kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound CoQ, besides being required for electron transfer from other dehydrogenases to Complex III. CoQ bound to Complex I and to Complex III is also involved in proton translocation; although the mechanism of the Q-cycle is well established for Complex III, the involvement of CoQ in proton translocation by Complex I is still debated. This review also briefly examines some additional roles of CoQ, such as the antioxidant effect of its reduced form and its postulated action at the transcriptional level.

It was discovered over 60 years ago that the mitochondrial respiratory chain is constituted of a series of protein complexes imbedded in the inner mitochondrial membrane. Experimental evidence has more recently ascertained that the major respiratory complexes involved in energy conservation are assembled as supramolecular units (supercomplexes, SCs) in stoichiometric ratios. The functional role of SCs is less well defined, and still open to discussion. Several lines of evidence favour the concept that electron transfer from Complex I to Complex III operates by channelling of electrons through Coenzyme Q molecules bound to the SC I1III2IV n , in contrast with the previously accepted hypothesis that the transfer of reducing equivalents from Complex I to Complex III occurs via random diffusion of the Coenzyme Q molecules in the lipid bilayer. On the contrary, electron transfer from Complex III to Complex IV seems to operate, at least in mammals, by random diffusion of cytochrome c molecules between the respiratory complexes even if assembled in SCs. Furthermore, another property provided by the supercomplex assembly is the control of generation of reactive oxygen species by Complex I, that might be important in the regulation of signal transduction from mitochondria. This review discusses physiological and pathological implications of the supercomplex assembly of the respiratory chain.

Table of contents 12.1. INTRODUCTION 12.1.1. The respiratory chain of mitochondria 12.1.2. Organization of the respiratory chain: historical outline 12.2. DISTRIBUTION AND COMPOSITION OF RESPIRATORY SUPERCOMPLEXES 12.2.1. Distribution in different organisms 12.2.2. Composition of respiratory supercomplexes 12.3. SUPERCOMPLEX ASSOCIATION PROVIDES A KINETIC ADVANTAGE 12.3.1. Structural evidence 12.3.1.1. Molecular structure of supercomplexes 12.3.1.2. Dynamic nature of supercomplexes: the plasticity model 12.3.1.3. The role of lipids: cardiolipin in supercomplexes 12.3.1.4. Standing uncertainties 12.3.2. Evidence for channelling in the Coenzyme Q region 12.3.2.1. Rate advantage in the Coenzyme Q region 12.3.2.2. Evidence for channelling by metabolic flux control analysis 12.3.2.3. Separate compartments of Coenzyme Q? 12.3.2.4. The function of the Coenzyme Q pool 12.3.2.4.1. Dissociation equilibrium of bound Coenzyme Q 12.3.2.4.2. Electron transfer between individual complexes not involved in supercomplex organization 12.3.2.5. Concluding evidence about channelling in the Coenzyme Q region 12.3.3. Electron transfer through cytochrome c 12.4. SUPERCOMPLEXES AND REACTIVE OXYGEN SPECIES 12.5. PHYSIOLOGICAL AND PATHOLOGICAL IMPLICATIONS 12.5.1. Supercomplexes and regulation of metabolic fluxes 12.5.2. Supercomplexes and ROS signalling 12.5.3. Supercomplexes in pathology and aging

Familial aggregation is a significant risk factor for the development of thyroid cancer and familial non-medullary thyroid cancer (FNMTC) accounts for 5-7% of all NMTC. Whole exome sequencing analysis in the family affected by FNMTC with oncocytic features where our group previously identified a predisposing locus on chromosome 19p13.2, revealed a novel heterozygous mutation (c.400G > A, NM_012335; p.Gly134Ser) in exon 5 of MYO1F, mapping to the linkage locus. In the thyroid FRTL-5 cell model stably expressing the mutant MYO1F p.Gly134Ser protein, we observed an altered mitochondrial network, with increased mitochondrial mass and a significant increase in both intracellular and extracellular reactive oxygen species, compared to cells expressing the wild-type (wt) protein or carrying the empty vector. The mutation conferred a significant advantage in colony formation, invasion and anchorage-independent growth. These data were corroborated by in vivo studies in zebrafish, since we demonstrated that the mutant MYO1F p.Gly134Ser, when overexpressed, can induce proliferation in whole vertebrate embryos, compared to the wt one. MYO1F screening in additional 192 FNMTC families identified another variant in exon 7, which leads to exon skipping, and is predicted to alter the ATP-binding domain in MYO1F. Our study identified for the first time a role for MYO1F in NMTC.

Two alternative models of organization of the mitochondrial electron transport chain (mETC) have been alternatively favored or questioned by the accumulation evidences of different sources, the solid model or the random collision model. Both agree in the number of respiratory complexes (I-IV) that participate in the mETC, but while the random collision model proposes that Complexes I-IV do not interact physically and that electrons are transferred between them by coenzyme Q and cytochrome c, the solid model proposes that all complexes super-assemble in the so-called respirasome. Recently, the plasticity model has been developed to incorporate the solid and the random collision model as extreme situations of a dynamic organization, allowing super-assembly free movement of the respiratory complexes. In this review, we evaluate the supporting evidences of each model and the implications of the super-assembly in the physiological role of coenzyme Q.

Despite more than 50 years of investigations into the free radical theory, the direct role of oxidative stress (OS) in aging and age-related diseases remains unproven. Little progress in identifying antioxidant drugs promoting longevity has been made, likely due to selectivity toward one or few radical species, variable efficacy in vivo, inherent pro-oxidant behavior of such drugs, or lack of synergism with metabolic redox homeostasis. Silencing the wide range of reactive free radicals has a great impact on OS-linked outcomes and age-related disorders. Here we show that an innovative, redox-active, multi-radical-scavenger catalytic drug delays the age-associated decline in physiological processes and markedly prolongs the mean lifespan of the adult freshwater annelids Aeolosoma viride by 170%. This unprecedented extension is associated with a decreased OS status. Consistently, treatment of annelids increases their natural resistance to oxygen-derived damage without affecting mitochondrial respiration or reproductive activity. Conversely, the superoxide dismutase (SOD)-mimetic EUK 134 that we selected as a positive control led to an increase in lifespan of ~50%, the same increase previously observed in nematodes. Our results show that reduction of the global network of OS has a profound impact on aging, prompting the development of a possible redox-based therapeutic intervention to counteract the progression of aging.

The discovery of respiratory supercomplexes (SCs) led to the proposal that electron transfer between complexes I and III (CI, CIII) is mediated by channelling of Coenzyme Q (Q), with a kinetic advantage on the transfer based on random collisions, whereas electron transfer from CII to CIII obeys to the random collision model. The evidence for Q channelling, however, is highly controversial [1, 2]. We have approached the problem in bovine heart submitochondrial particles and in reconstituted proteoliposomes in which CI and CIII are preserved as SC I1III2. We restricted electron transfer to the Q area by studying NADH and succinate oxidation by exogenous cytochrome c (cyt. c) as acceptor, thus avoiding the bottleneck of endogenous cyt. c. Using this system we found the rates of NADH and succinate oxidation by cyt. c to be almost completely additive. The rate obtained by simultaneous addition of NADH and succinate was much higher than that predicted for a homogeneous Q pool [3], thus suggesting that NADH and succinate oxidation by cyt. c follow two different routes. The NADH route presumably operates through Q channelling in the SC I1III2. However Qpool molecules may exchange with Qbound in SC, approaching the rates predicted for a single pool, when the reducing pressure increases by strong CIII inhibition or when detergents destabilize the SCs. The accessibility of Qpool to SC I1III2 may be a physiological device to control electron fluxes from different substrates and implies a dissociation equilibrium of Qbound with the Q pool, by which the size of the pool determines saturation of the binding site(s) in the SC. Thus bulk Qpool has a role also in oxidation of NAD-linked substrates, providing a rationale for the beneficial effect of exogenous Q supplementation on mitochondrial bioenergetics. References 1. JN Blaza et al. Proc Natl Acad Sci USA 111 (2014) 15735-40. 2. G Lenaz et al. BBA Bioenerg. (2016) Epub ahead of print. 3. A Kröger, M Klingenberg. Eur J Biochem. 34 (1973) 358-68.

This review discusses the functional properties of mitochondrial Complex I originating from its presence in an assembled form as a supercomplex comprising Complex III and Complex IV in stoichiometric ratios. In particular several lines of evidence are presented favouring the concept that electron transfer from Complex I to Complex III is operated by channelling of electrons through Coenzyme Q molecules bound to the supercomplex, in contrast with the hypothesis that the transfer of reducing equivalents from Complex I to Complex III occurs via random diffusion of the Coenzyme Q molecules in the lipid bilayer. Furthermore, another property provided by the supercomplex assembly is the control of generation of reactive oxygen species by Complex I. This article is part of a Special Issue entitled Respiratory Complex I, edited by Volker Zickermann and Ulrich Brandt.

Evidence from several investigations demonstrates the existence of supramolecular units of Complex I, Complex III, and multiple copies of Complex IV in mitochondria and indicates that specific respiratory complexes may preferentially associate to form cytochrome-containing supercomplexes in the native membrane. There are now indications that cardiolipin, a distinctive mitochondrial lipid, stabilizes the respiratory assemblies. The isolated supercomplexes are active with respect to both their component individual complexes and the entire respiratory function that relies on Coenzyme Q and cytochrome c as intermediate substrates. The latter finding argues against previous models of a random distribution of the respiratory complexes in mitochondria. The supercomplex organization is compatible with electron transfer, but experimental evidence is scant for an effective mechanism via substrate channeling compared to free diffusion of substrates in accordance with the random collision model. The finding that Complex I is almost totally associated in a supercomplex with Complex III seems to exclude a role for the ubiquinone pool in physiological electron transfer between these two complexes, whereas it is certainly required for electron transfer from Complex II or from other dehydrogenases to Complex III; likely, only a small fraction of Complex IV forms a functional supercomplex with channeling of cytochrome c. Nevertheless, the supercomplexes may physiologically exist in equilibrium with free complexes (plasticity model). The supercomplex organization appears to prevent excessive generation of reactive oxygen species from the respiratory chain; accordingly, many pathological conditions and the mitochondrial aging phenotype characterized by a loss of supercomplex assembly correlate with mitochondrial dysfunction and increased oxidative stress. Specific metabolic signals may also arise in the cell in response to a tuned production of reactive oxygen species as a consequence of the controlled dynamics of supercomplex assembling/ disassembling at different physio-pathological conditions. The present review paper provides an updated and extensive discussion on the subject.

Liver ischemia-reperfusion injury is still an open problem in many clinical circumstances, including surgery and transplantation. This study investigates how mitochondrial structure, mass and oxidative phosphorylation change and may be preserved during a brief period of ischemia followed by a long period of reperfusion, an experimental model that mimics the condition to which a liver is exposed during transplantation. Livers were explanted from rats and exposed for 24 h to three different oxygen availability conditions at 4 °C. Mitochondrial mass, respiration, oxidative phosphorylation (OXPHOS), and levels of OXPHOS complexes were all significantly altered in livers stored under the currently used preservation condition of normoxia. Remarkably, liver perfusion with hyperoxic solutions fully preserved mitochondrial morphology and function, suggesting that perfusion of the graft with hyperoxic solution should be considered in human transplantation.

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.

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 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 channelling. The present review discusses conditions affecting formation of supercomplexes which, besides kinetic advantage, have a role in 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 pathological changes.

We have investigated by immuno-electron microscopy the presence of phosphotyrosine in cells as a whole and in different cell districts (nucleus, cytoplasm, plasma membrane, and mitochondria) in peripheral blood lymphocytes of IDDM (insulindependent diabetes mellitns) patients and agematched controls. Immuno-gold particle density was highest in mitochondria and decreased in cytoplasm, nucleus and plasma membrane. The time dependence of phosphotyrosine labelling after cell isolation was very strong in all subcellular populations, with a fall in immunogold staining after 30 min. Staining levels at zero time were similar in controls and IDDM patients; the loss of phosphotyrosine labelling was much stronger in controls, except in the plasma membrane. Plasma membrane NADH oxidoreductase activity, studied using cytosolic NADH as substrate and assayed with DCIP as acceptor, was significantly reduced in IDDM patients, suggesting a response to a deficient mitochondrial energetic activity. The fact that NADH oxidoreductase is a growth factor related to tyrosine phosphorylation pathways raises intriguing questions on the cellular derangement occurring in peripheral lymphocytes in IDDM, although the relationships among the immunocytochemical and biochemical changes is still obscure.

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.

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.

Mitochondrial reactive oxygen species (ROS) are mainly produced by the respiratory chain enzymes. The sites for ROS production in mitochondrial respiratory chain are normally ascribed to the activity of Complex I and III. The presence of specific inhibitors modulates reactive oxygen species production in Complex I: inhibitors such as rotenone induce a strong ROS increase, while inhibitors such as stigmatellin prevent it. We have investigated the effect of hydrophilic quinones on Complex I ROS production in presence of different inhibitors. Some short chain quinones are Complex I inhibitors (CoQ2, idebenone and its derivatives), while CoQ1, decylubiquinone~ (DB) and duroquinone (DQ) are good electron acceptors from Complex I. Our results show that the ability of short chain quinones to induce an oxidative stress depends on the site of interaction with Complex I and on their physical-chemical characteristics. We can conclude that hydrophilic quinones may enhance oxidative stress by interaction with the electron escape sites on Complex I while more hydrophobic quinones can be reduced only at the physiological quinone reducing site without reacting with molecular oxygen.

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.

Background and Methodology Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. Principal Findings A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Conclusions and Significance Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands., Author Summary The multitarget approach can represent a promising strategy for the discovery of innovative drug candidates against neglected tropical diseases. However, multitarget drug discovery can be very demanding, because of the highly time-consuming step related to the fine balancing of the biological activities against selected targets. An innovative workflow for discovering multitarget drugs can be envisioned: i) design and synthesis of natural-like compounds; ii) test them using phenotypic cell-based assays; iii) fishing potential targets by means of chemical proteomics. This workflow might rapidly provide new hit candidates that can be further progressed to the hit-to-lead and lead optimization steps of the drug discovery process. The two latter steps can benefit from information on the molecular target(s), which may be identified by chemical proteomics. Herein, we report on the elucidation of the mode of action of a new series of anti-trypanosomal naphthoquinone compounds, previously tested using cell-based assays, by means of chemical proteomics, classical biochemistry, molecular and system biology.

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. Antioxid. Redox Signal. 19, 1469–1480.

Substantial evidence exists that the mitochondrial respiratory chain is organized in supramolecular units called supercomplexes or respirasomes. While the structural evidence of the supercomplexes is overwhelming, fewer studies have focused on their functional relevance. Although the presence of coenzyme Q channeling between complexes I and III has been ascertained, no such clear demonstration has been carried out for cytochrome c between complexes III and IV, at least in mammalian mitochondria. This review also discusses the implications concerning the number of respiratory complexes organized in supercomplexes and the possibility that they represent associations in dynamic equilibrium with the individual complexes.

Significance: The molecular mechanism of aging is still vigorously debated, although a general consensus exists that mitochondria are significantly involved in this process. However, the previously postulated role of mitochondrial-derived reactive oxygen species (ROS) as the damaging agents inducing functional loss in aging has fallen out of favor in the recent past. In this review, we critically examine the role of ROS in aging in the light of recent advances on the relationship between mitochondrial structure and function. Recent Advances: The functional mitochondrial respiratory chain is now recognized as a reflection of the dynamic association of respiratory complexes in the form of supercomplexes (SCs). Besides providing kinetic advantage (channeling), SCs control ROS generation by the respiratory chain, thus providing a means to regulate ROS levels in the cell. Depending on their concentration, these ROS are either physiological signals essential for the life of the cell or toxic species that damage cell structure and functions. Critical Issues: We propose that under physiological conditions the dynamic nature of SCs reversibly controls the generation of ROS as signals involved in mitochondrial- nuclear communication. During aging, there is a progressive loss of control of ROS generation so that their production is irreversibly enhanced, inducing a vicious circle in which signaling is altered and structural damage takes place. Future Directions: A better understanding on the forces affecting SC association would allow the manipulation of ROS generation, directing these species to their physiological signaling role.

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.

The established protective effect of coenzyme Q (CoQ) analogs is dependent on the location of reactive oxygen species (ROS) generation. One of these analogs--idebenone (hydroxydecyl-ubiquinone) is used as an antioxidative therapeutic drug. We tested its scavenging effect on the glycerophosphate (GP)-dependent ROS production as this enzyme was shown as a new site in the mitochondrial respiratory chain where ROS can be generated. We observed that idebenone inhibits both GP- and succinate-dependent ROS production. Idebenone and CoQ1 were found to be more efficient in the scavenging activity (IC50: 0.052 and 0.075 microM, respectively) than CoQ3 (IC50: 45.8 microM). Idebenone also inhibited ferricyanide (FeCN)-activated, GP-dependent ROS production. Our data thus extend previous findings on the scavenging effect of idebenone and show that it can also eliminate GP-dependent ROS generation.

Coenzyme Q (Q) is reduced in plasma membrane and mitochondria by NAD(P)H-dependent reductases providing reducing equivalents to maintain both respiratory chain and antioxidant protection. Reactive oxygen species (ROS) are accumulated in the aging process originating mainly in mitochondria but also in other membranes, such as plasma membrane partially by the loss of electrons from the semiquinone. The reduction of Q by NAD(P)H-dependent reductases in plasma membrane is responsible for providing its antioxidant capacity, preventing both the lipid peroxidation chain and the activation of the ceramide-dependent apoptosis pathway. Both Q content and its reductases are decreased in plasma membrane of aging mammals. Calorie restriction, which extends mammal life span, increases the content of Q in the plasma membrane and also activates Q reductases in this membrane. Both lipid peroxidation and ceramide production are decreased in the plasma membrane in calorie-restricted animals. Plasma membrane is, then, an important cellular component to control the aging process through its concentration and redox state of Q.

In this work we describe the synthesis of a series of imidazo[2,1- b ]thiazoles and 2,3-dihydroimidazo[2,1- b ]thiazoles connected by means of a methylene bridge to CoQ 0 . These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC 50 for NADH-Q 1 activity ranging between 0.25 and 0.96 μM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ 0 , which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.

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.

Background/Aims As the impairment of the cellular energy metabolism contributes to the failure of fatty liver grafts after transplantation, we aimed to determine whether steatosis affects the oxidative phosphorylation activity during preservation. Methods Rat normal and fatty livers were preserved for 18 h and then reperfused with warm oxygenated solution. The oxidative phosphorylation, the F 0 F 1 -ATPase and the Complex I activities were assessed in isolated mitochondria before and after preservation, and during reperfusion. The ALT release and portal pressure were monitored during reperfusion. Results The baseline phosphorylation activity was similar in normal and steatotic mitochondria. After cold preservation, the respiratory control index and state 3 respiration decreased significantly only in steatotic livers. Reperfusion induced a further deterioration in either group. Contrary to normal liver, uncoupling of fatty liver mitochondria allowed the recovery of the maximal respiration rate only using succinate (Complex II-dependent substrate), but not glutamate-malate (Complex I-dependent). Complex I dysfunction was confirmed spectrophotometrically. The ATPase activity was also significantly lower in fatty livers. Finally, ALT release and portal pressure were greater in steatotic livers. Conclusions The alteration of the oxidative phosphorylation activity during preservation is greatly exacerbated by fatty infiltration likely resulting from damage of the respiratory chain Complex I and of the F 0 F 1 -ATP synthase.

The bioenergetic function of mitochondria can be investigated in intact cells by a variety of methods. A simple biochemical method to compare mitochondrial oxidative phosphorylation with glycolytic ATP synthesis takes advantage of the Pasteur effect, since the amount of lactate produced under basal conditions is an indication of glycolytic ATP, while the Delta-lactate (the difference between excess lactate produced after inhibition of respiration and basal lactate) represents ATP produced anaerobically in order to compensate for decreased oxidative phosphorylation after respiratory chain inhibition. The system has been validated in a series of cells, including human platelets and lymphocytes and lines cultured in vitro. Measurement of KCN-sensitive oxygen consumption by the cells and its sensitivity to uncouplers can be good supplementary indication of mitochondrial phosphorylative capacity.

A T8993G point mutation in the mtDNA results in a Leu156Arg substitution in the MTATP6 subunit of the mitochondrial F1F0-ATPase. The T8993G mutation causes impaired oxidative phosphorylation (OXPHOS) in two mitochondrial disorders, NARP (neuropathy, ataxia and retinitis pigmentosa) and MILS (maternally inherited Leigh's syndrome). It has been reported, in some studies, that the T8993G mutation results in loss of assembled F1F0-ATPase. Others reported that the mutation causes impairment of proton flow through F0. In addition, it was shown that fibroblasts from NARP subjects have a tendency to undergo apoptotic cell death, perhaps as a result of increased free radical production. Here, we show that the T8993G mutation inhibits oxidative phosphorylation and results in enhanced free radical production. We suggest that free radical-mediated inhibition of OXPHOS contributes to the loss of ATP synthesis. Importantly, we show that antioxidants restore respiration and partially rescue ATP synthesis in cells harboring the T8993G mutation. Our results indicate that free radicals might play an important role in the pathogenesis of NARP/MILS and that this can be prevented by antioxidants. The effectiveness of antioxidant agents in cultured NARP/MILS cells suggests that they might have a potential beneficial role in the treatment of patients with NARP.

In this work we describe the synthesis and the biological activity of 2-[(E)-3-(6chloroimidazo[2,1-b]thiazol-5-yl)prop-2-enyl]-5,6-dimethoxy-3-methyl-1,4-benzoquinone i.e. an imidazothiazole derivative connected to the benzoquinone ring of Q0. This compound was tested as specific inhibitor of the NADH:ubiquinone (UBQ) reductase activity of NADH dehydrogenase in mitochondrial membranes. Binding of the imidazothiazole moiety to the quinone site normally occupied by the isoprenoid lateral side chain increases the inhibitory effect (with an IC50 for NADH-Q1 activity of 0.24 μM) whereas the benzoquinone moiety seems to lose the capability as electron acceptor from Complex I. The new compound was also tested as potential antitumor agent at the National Cancer Institute.

Dichlorophenol indophenol (DCIP) reduction by intracellualr pyridine nucleotides was investigated in two different lines of cultured cells characterized by enhanced production of reacive oxygen species (ROS) with respect to suitable controls. The first line denominated XTC-UC1 was derived from a metastasis of an oxyphilic thyroid tumor characterized by mitochondrial hyperplasia and compared with a line (B-CPAP) derived from a papillary thyroid carcinoma with normal mitochondrial mass. The second line (170 MN) was a cybrid line derived from ρ0 cells from an osteosarcoma line (143B) fused with platelets from a patient with a nucleotide 9957 mutation in mitochondrial DNA (encoding for cytochrome c oxidase subunit III) in comparison with the parent 143B line. The experimental lines had no major decreases of electron transfer activities with respect to the controls; both of them, however, exhibited an increased peroxide production. The XTC-UC1 cell line exhibited enhanced activity with respect to control of dicoumarol-sensitive DCIP reduction, identified with membrane bound DT-diaphorase, whereas dicoumarol insensitive DCIP reduction was not significantly changed. On the other hand the mtDNA mutated cybrids exhibited a strong increase of both dicoumarol sensitive and insensitive DCIP reduction. The results suggest that enhanced oxidative stress and not deficient respiratory activity per se is the stimulus triggering over-expression of plasma membrane oxidative enzymes.

Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F0. As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching. N,N′-dicyclohexylcarbodiimide (DCCD) behaved similarly to oligomycin in increasing the rate of quenching. These findings indicate that RH-123 fluorescence quenching kinetics give reliable and sensitive evaluation of mitochondrial membrane potential, complementing steady-state fluorescence measurements, and provide a mean to study proton flow from the mitochondrial intermembrane space to the matrix through the F0 channel.

Data presented in this paper show that the size of the endogenous coenzyme Q (CoQ) pool is not a limiting factor in the activation of mitochondrial glycerophosphate-dependent respiration by exogenous CoQ(3), since successive additions of succinate and NADH to brown adipose tissue mitochondria further increase the rate of oxygen uptake. Because the inhibition of glycerophosphate-dependent respiration by oleate was eliminated by added CoQ(3), our data indicate that the activating effect of CoQ(3) is related to the release of the inhibitory effect of endogenous free fatty acids (FFA). Both the inhibitory effect of FFA and the activating effect of CoQ(3) could be demonstrated only for glycerophosphate-dependent respiration, while succinate- or NADH-dependent respiration was not affected. The presented data suggest differences between mitochondrial glycerophosphate dehydrogenase and succinate or NADH dehydrogenases in the transfer of reducing equivalents to the CoQ pool.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is 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 the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex 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 analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. 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 onset of resistance to drug-induced apoptosis of tumour cells is a major problem in cancer therapy. We studied a drug-selected clone of promyelocytic HL-60 cells, called HCW-2, which display a complex resistance to a wide variety of apoptosis-inducing agents and we found that these cells show a dramatic increase in the expression of heat shock proteins (Hsps) 70 and 27, while the parental cell line does not. It is known that stress proteins such as Hsps can confer resistance to a variety of damaging agents other than heat shock, such as TNF-alpha, monocyte-induced cytotoxicity, and also play a role in resistance to chemotherapy. This elevated expression of Hsps is paralleled by an increased activity of mitochondrial metabolism and pentose phosphate pathway, this latter leading to high levels of glucose-6-phosphate dehydrogenase and, consequently, of glutathione. Thus, the apoptotic-deficient phenotype is likely because of the presence of high levels of stress response proteins and GSH, which may confer resistance to apoptotic agents, including chemotherapy drugs. Moreover, the fact that in HCW-2 cells Hsp70 are mainly localised in mitochondria may account for the increased performances of mitochondrial metabolism. These observations could have some implications for the therapy of cancer, and for the design of combined strategies that act on antioxidant defences of the neoplastic cell.

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one-electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe–S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.

To seek some specific biochemical markers of age-related macular degeneration (AMD), coenzyme Q10 (CoQ10) levels were determined in plasma and platelets from 19 exudative AMD patients and 19 age-matched controls. Lipid peroxidation was followed in plasma in vitro after the addition of a free radical initiator. Most patients had lower plasma CoQ10 content than most controls. Plasma from controls showed greater capacity to oppose the oxidative damage. These results support the concept that free radicals play a pathogenic role in AMD and that CoQ10 may have a protective effect.

We have investigated the effect of ageing and of adriamycin treatment on the bioenergetics of isolated rat hepatocytes. Ageing per se, whilst being associated with a striking increase of hydrogen peroxide in the cells, induces only minor changes on mitochondrial functions. The adriamycin treatment induces a decrease of the mitochondrial membrane potential in situ and a consistent increase of the superoxide anion cellular content independently of the donor’s age, whilst the hydrogen peroxide is significantly higher in aged than in adult rat hepatocytes. Kinetic studies in isolated mitochondria show that the mitochondrial respiratory chain activity (NADH→O 2 ) of 50 μM adriamycin-treated hepatocytes is lowered both in adult and aged rats. The same adriamycin concentration induces a slight decrease of the maximal rate of ATP hydrolysis in both young and aged rats, without affecting the K m for the substrate. However, at drug concentrations lower than 50 μM, both ATPase and NADH oxidation activities decrease significantly in aged rats only. The results suggest that free radicals increase during ageing in rat hepatocytes but are unable to induce major modifications of mitochondrial bioenergetics. This contrasts with the damaging effect of adriamycin, suggesting that some effects of the drug may be due to other reasons besides oxidative stress.

Starting from the potent inhibitory effect of the previously described 2-methyl-6-(2-thienyl)imidazo[2,1-b]thiazole on mitochondrial complex I, we prepared a series of derivatives in order to study the effect of a different substitution at the positions 2, 5 and 6. The replacement of the thienyl group at position 6 with a phenyl group does not modify the biological behaviour of the lead compound, whereas the shift of the methyl group from position 2 to position 5 yields a compound devoid of inhibitory effects. In both the 6-thienyl and 6-phenyl series, the lengthening of the chain at position 2 has provided useful information to outline the structural determinants of the ubiquinone antagonist action in imidazothiazole derivatives.

Mitochondria are strongly involved in production of reactive oxygen species, considered today as the main pathogenic agent of many diseases. A vicious circle of oxidative stress and damage to cellular structures can lead to either cell death by apoptosis or to a cellular energetic decline and ageing. The early involvement of mitochondria in apoptosis includes expression of pro-apoptotic factors, release of cytochrome c from the inter-membrane space and opening of the permeability transition pore: cytochrome c release appears to precede pore opening. The mitochondrial theory of ageing considers somatic mutations (deletions) of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, Complex I appears to be mostly affected. We have developed the Pasteur effect (enhancement of lactate production by mitochondrial inhibition) as a bio-marker of mitochondrial bioenergetics in human platelets, and found it to be decreased in aged individuals. Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants coenzyme Q is the only one of endogenous biosynthesis; exogenous coenzyme Q, however, may protect cells from oxidative stress in vivo.

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94 %. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70 % in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme. © 2006 Biochemical Society.