Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.     

Hepatitis C virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ROS) production

Hepatitis C infection causes a state of chronic oxidative stress, which may contribute to fibrosis and carcinogenesis in the liver. Previous studies have shown that expression of the HCV core protein in hepatoma cells depolarized mitochondria and increased reactive oxygen species (ROS) production, but the mechanisms of these effects are unknown. In this study we examined the properties of liver mitochondria from transgenic mice expressing HCV core protein, and from normal liver mitochondria incubated with recombinant core protein. Liver mitochondria from transgenic mice expressing the HCV proteins core, E1 and E2 demonstrated oxidation of the glutathione pool and a decrease in NADPH content. In addition, there was reduced activity of electron transport complex I, and increased ROS production from complex I substrates. There were no abnormalities observed in complex II or complex III function. Incubation of control mitochondria in vitro with recombinant core protein also caused glutathione oxidation, selective complex I inhibition, and increased ROS production. Proteinase K digestion of either transgenic mitochondria or control mitochondria incubated with core protein showed that core protein associates strongly with mitochondria, remains associated with the outer membrane, and is not taken up across the outer membrane. Core protein also increased Ca(2+) uptake into isolated mitochondria. These results suggest that interaction of core protein with mitochondria and subsequent oxidation of the glutathione pool and complex I inhibition may be an important cause of the oxidative stress seen in chronic hepatitis C.     

The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex

Production of reactive oxygen species (ROS) by the mitochondrial respiratory chain is considered to be one of the major causes of degenerative processes associated with oxidative stress. Mitochondrial ROS has also been shown to be involved in cellular signaling. It is generally assumed that ubisemiquinone formed at the ubiquinol oxidation center of the cytochrome bc(1) complex is one of two sources of electrons for superoxide formation in mitochondria. Here we show that superoxide formation at the ubiquinol oxidation center of the membrane-bound or purified cytochrome bc(1) complex is stimulated by the presence of oxidized ubiquinone indicating that in a reverse reaction the electron is transferred onto oxygen from reduced cytochrome b(L) via ubiquinone rather than during the forward ubiquinone cycle reaction. In fact, from mechanistic studies it seems unlikely that during normal catalysis the ubisemiquinone intermediate reaches significant occupancies at the ubiquinol oxidation site. We conclude that cytochrome bc(1) complex-linked ROS production is primarily promoted by a partially oxidized rather than by a fully reduced ubiquinone pool. The resulting mechanism of ROS production offers a straightforward explanation of how the redox state of the ubiquinone pool could play a central role in mitochondrial redox signaling.     

Complex I within oxidatively stressed bovine heart mitochondria is glutathionylated on Cys-531 and Cys-704 of the 75-kDa subunit: potential role of CYS residues in decreasing oxidative damage

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. However, the Cys residues and the thiol modifications involved are not known. Here we investigate complex I thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. In mitochondria incubated with the thiol oxidant diamide, complex I is only glutathionylated on the 75-kDa subunit. Of the 17 Cys residues on the 75-kDa subunit, 6 are not involved in iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry of complex I from oxidatively stressed bovine heart mitochondria showed that only Cys-531 and Cys-704 were glutathionylated. The other four non-iron-sulfur center Cys residues remained as free thiols. Complex I glutathionylation also occurred in response to relatively mild oxidative stress caused by increased superoxide production from the respiratory chain. Although complex I glutathionylation within oxidatively stressed mitochondria correlated with loss of activity, it did not increase superoxide formation, and reversal of glutathionylation did not restore complex I activity. Comparison with the known structure of the 75-kDa ortholog Nqo3 from Thermus thermophilus complex I suggested that Cys-531 and Cys-704 are on the surface of mammalian complex I, exposed to the mitochondrial glutathione pool. These findings suggest that Cys-531 and Cys-704 may be important in preventing oxidative damage to complex I by reacting with free radicals and other damaging species, with subsequent glutathionylation recycling the thiyl radicals and sulfenic acids formed on the Cys residues back to free thiols.     

Mitochondrial catalase and oxidative injury

Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production.     

Low complex I content explains the low hydrogen peroxide production rate of heart mitochondria from the long-lived pigeon, Columba livia

Across a range of vertebrate species, it is known that there is a negative association between maximum lifespan and mitochondrial hydrogen peroxide production. In this report, we investigate the underlying biochemical basis of the low hydrogen peroxide production rate of heart mitochondria from a long-lived species (pigeon) compared with a short-lived species with similar body mass (rat). The difference in hydrogen peroxide efflux rate was not explained by differences in either superoxide dismutase activity or hydrogen peroxide removal capacity. During succinate oxidation, the difference in hydrogen peroxide production rate between the species was localized to the ΔpH-sensitive superoxide producing site within complex I. Mitochondrial ΔpH was significantly lower in pigeon mitochondria compared with rat, but this difference in ΔpH was not great enough to explain the lower hydrogen peroxide production rate. As judged by mitochondrial flavin mononucleotide content and blue native polyacrylamide gel electrophoresis, pigeon mitochondria contained less complex I than rat mitochondria. Recalculation revealed that the rates of hydrogen peroxide production per molecule of complex I were the same in rat and pigeon. We conclude that mitochondria from the long-lived pigeon display low rates of hydrogen peroxide production because they have low levels of complex I.     

Testing the vicious cycle theory of mitochondrial ROS production: effects of H2O2 and cumene hydroperoxide treatment on heart mitochondria

Vicious cycle theories of aging and oxidative stress propose that ROS produced by the mitochondrial electron transport chain damage the mitochondria leading exponentially to more ROS production and mitochondrial damage. Although this theory is widely discussed in the field of research on aging and oxidative stress, there is little supporting data. Therefore, in order to help clarify to what extent the vicious cycle theory of aging is correct, we have exposed mitochondria in vitro to different concentrations of hydrogen peroxide or cumene-hydroperoxide (0, 30, 100 and 500 μM). We have found that 30 μM hydrogen peroxide (or higher concentrations) inhibit oxygen consumption in state 3 and increase ROS production with pyruvate/malate but not with succinate as substrate, indicating that these effects occur specifically at complex I. Similar levels of cumene-OOH inhibit state 3 respiration with both kinds of substrates, and increase ROS production in both state 4 and state 3 with pyruvate/malate and with succinate. The effects of cumene-OOH on ROS generation are due to action of the peroxide in the complex III or in the complex III plus complex I ROS generators. In all cases, the increase in ROS production occurred at a threshold level of peroxide exposure without further exponential increase in ROS generation. These results are consistent with the idea that ROS production can contribute to increase oxidative stress in old animals, but the results do not fit with a vicious cycle theory in which peroxide generation leads exponentially to more and more ROS production with age.     

Topology of superoxide production from different sites in the mitochondrial electron transport chain

We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria.     

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.     

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging. To reconcile these findings, we used the mitochondria‐targeted mass spectrometry probe MitoB to measure hydrogen peroxide within mitochondria of living mice. Mitochondrial hydrogen peroxide was the same in young mutator and control mice, but as the mutator mice aged, hydrogen peroxide increased. This suggests that the prolonged presence of mtDNA mutations in vivo increases hydrogen peroxide that contributes to an accelerated aging phenotype, perhaps through the activation of pro‐apoptotic and pro‐inflammatory redox signaling pathways.     

Catalytic Coupling of Oxidative Phosphorylation,ATP Demand,and Reactive Oxygen Species Generation

Competing models of mitochondrial energy metabolism in the heart are highly disputed. In addition, the mechanisms of reactive oxygen species (ROS) production and scavenging are not well understood. To deepen our understanding of these processes, a computer model was developed to integrate the biophysical processes of oxidative phosphorylation and ROS generation. The model was calibrated with experimental data obtained from isolated rat heart mitochondria subjected to physiological conditions and workloads. Model simulations show that changes in the quinone pool redox state are responsible for the apparent inorganic phosphate activation of complex III. Model simulations predict that complex III is responsible for more ROS production during physiological working conditions relative to complex I. However, this relationship is reversed under pathological conditions. Finally, model analysis reveals how a highly reduced quinone pool caused by elevated levels of succinate is likely responsible for the burst of ROS seen during reperfusion after ischemia.     

Gender differences in free radical homeostasis during aging: shorter-lived female C57BL6 mice have increased oxidative stress

Gender is a profound determinant of aging and lifespan, but little is known about gender differences in free radical homeostasis. Free radicals are proposed as key elements in the multifactorial process of aging and it is predicted that the longer-lived gender should have lower levels of oxidative stress. While the majority of studies on aging have included a single gender, recent studies in rats compared genders and found that females, the longer-lived sex, had lower oxidative stress and mitochondrial dysfunction than males. We explored the association between oxidative stress and gender-specific aging in C57BL6 mice, in which females are the shorter-lived gender. Reactive oxygen species (ROS) were measured in young and old mice by confocal imaging of dihydroethidium (DHE) oxidation in the brain, and by electron paramagnetic resonance (EPR) spectrometry of isolated brain mitochondria. Both genders exhibited significant age-dependent increases in ROS. However, females had a greater increase with age than males in DHE oxidation but not mitochondrial EPR. Superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (GPx1) protein levels were lower in old females. To determine whether enhancing antioxidant defenses would eliminate gender differences in lifespan, mice were treated chronically with a superoxide dismutase mimetic. Treatment blocked the age-dependent increase in ROS, with a greater effect in females on DHE oxidation, but not mitochondrial EPR. Treatment also increased lifespan to a greater degree in females. Our results indicate that differences in ROS homeostasis contribute to gender divergence in survival, but also suggest that mitochondrial superoxide production may not be primarily responsible for gender differences in lifespan.     

Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production

Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.     

Effect of steroid hormones on production of reactive oxygen species in mitochondria

Among the targets of steroid hormones are mitochondria, which as the main source of reactive oxygen species (ROS) in the cell play a central role in the development of various pathologies. We studied the effect of progesterone and its synthetic analogs on mitochondrial ROS production. It was found that progesterone promoted formation of superoxide anion and hydrogen peroxide in mitochondria oxidizing the substrates of complex I of the respiratory chain but did not influence the production of ROS during oxidation of succinate, respiratory chain complex II substrate. Progesterone derivatives—Medroxyprogesterone acetate, Buterol, Acetomepregenol, Megestrol acetate—had different effects on ROS production, depending on their chemical structure. By the stimulation of ROS production in mitochondria (during oxidation of pyruvate + malate), the tested steroids can be arranged in decreasing order as follows: progesterone > Buterol ≈ Acetomepregenol > Medroxyprogesterone acetate = Megestrol acetate. Activation of ROS production by progesterone and by Buterol involves different mechanisms: progesterone acts as an inhibitor of NAD-dependent respiration, while Buterol and Acetomepregenol perhaps form noncovalent complexes by hydrogen bonding of the ester carbonyl at C3 to the SH groups of the respective targets.     

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.     

Mitochondrial adaptations to obesity-related oxidant stress

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.     

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates

The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.     

Complex III releases superoxide to both sides of the inner mitochondrial membrane

Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for approximately 50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining approximately 50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.