Nitric oxide degradation by potato tuber mitochondria: Evidence for the involvement of external NAD(P)H dehydrogenases

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O₂⁻). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O₂⁻ generation, besides complex III, stimulated NO degradation. Larger amounts of O₂⁻ were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O₂⁻ production were stimulated by free Ca²⁺ concentration. Together, these results indicate that Ca²⁺-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O₂⁻ production that favors NO degradation in potato tuber mitochondria.     

Myxothiazol induces H(2)O(2) production from mitochondrial respiratory chain

Interruption of electron flow at the quinone-reducing center (Q(i)) of complex III of the mitochondrial respiratory chain results in superoxide production. Unstable semiquinone bound in quinol-oxidizing center (Q(o)) of complex III is thought to be the sole source of electrons for oxygen reduction; however, the unambiguous evidence is lacking. We investigated the effects of complex III inhibitors antimycin, myxothiazol, and stigmatellin on generation of H(2)O(2) in rat heart and brain mitochondria. In the absence of antimycin A, myxothiazol stimulated H(2)O(2) production by mitochondria oxidizing malate, succinate, or alpha-glycerophosphate. Stigmatellin inhibited H(2)O(2) production induced by myxothiazol. Myxothiazol-induced H(2)O(2) production was dependent on the succinate/fumarate ratio but in a manner different from H(2)O(2) generation induced by antimycin A. We conclude that myxothiazol-induced H(2)O(2) originates from a site located in the complex III Q(o) center but different from the site of H(2)O(2) production inducible by antimycin A.     

Peroxynitrite formed by mitochondrial NO synthase promotes mitochondrial Ca2+ release

Mitochondria contribute to the maintenance of the intracellular Ca2+ homeostasis by taking up and releasing the cation via separate and specific pathways. The molecular details of the release pathway are elusive but its stimulation by the cross-linking of some vicinal thiols and consequently NAD+ hydrolysis are known. Thiol cross-linking and NAD+ hydrolysis can be achieved by addition of peroxynitrite (ONOO-), the product of the reaction between superoxide (O2-) and nitric oxide (nitrogen monoxide, NO*) to mitochondria. Mitochondria contain an NO synthase (mtNOS), which is stimulated by Ca2+, and are a copious source of O2-. We show here that intramitochondrially formed ONOO- stimulates the specific, NAD+-linked Ca2+ release from mitochondria. Our findings that upon Ca2+ uptake mtNOS is stimulated, that ONOO- is formed, and that Ca2+ is subsequently released from intact mitochondria suggest the existence of a feedback loop, which prevents overloading of mitochondria with Ca2+.     

Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.     

Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.     

Substrate-dependent effect of phenolic antioxidants on Ca2+ accumulation by rat liver mitochondria

The effect of different phenolic antioxidants on mitochondrial Ca2+ capacity (maximum amount of Ca2+ mitochondria can accumulate) was studied. Butylated hydroxytoluene substantially enhanced the Ca2+ capacity in mitochondria oxidizing succinate, butylated hydroxyanisole had a moderate effect while 2,5-di-(t-butyl)- 1,4 benzohydroquinone did not affect Ca2+ capacity at all. The analysis of Ca2+ accumulation in mitochondria oxidizing succinate in the presence of 2,5-di-(t-butyl)-1,4 benzohydroquinone revealed inhibition of the rate of Ca2+ accumulation. This effect was absent when ATP hydrolysis or NAD+-dependent substrate oxidation supported Ca2+ transport. Direct measurements of oxygen consumption revealed the concentration-dependent inhibition of succinate oxidation by increasing concentrations of 2,5-di-(t-butyl)- 1,4 benzohydroquinone. When succinate was substituted by NAD+-dependent respiratory substrates, the Ca2+ capacity of mitochondria with 2,5-di-(t-butyl)-1,4 benzohydroquinone was even higher than in the presence of butylated hydroxytoluene.     

Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: Influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H₂O₂ production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.     

Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2*- production in the intact coronary endothelium

The present study tested the hypothesis that endostatin stimulates superoxide (O2*-) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]i was unchanged. With the conversion rate of L-[3H]arginine to L-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2*-. production was enhanced in endothelial cells. O2*-. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2*-. production through activation of NAD(P)H oxidase. This ceramide-O2*-. signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

The effect of superoxide generation on the ability of mitochondria to take up and retain Ca2+

When heart or liver mitochondria are exposed to superoxide radicals generated from xanthine + xanthine oxidase their ability to take up and to retain Ca2+ is impaired. The rate of oxidation of pyruvate + malate as substrates is diminished and the appearance of thiol groups when the mitochondria are supplied with these substrates is abolished. These inhibitory effects are offset if respiration is supported by succinate in presence of rotenone provided that a substrate (beta-hydroxybutyrate) is provided to maintain the reduction of NADH. The data agree with the thesis that a generation of thiol groups is essential to maintain membrane integrity and that the generation depends on provision of reduced NAD(P)H.     

Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes

The fumigant insecticide phosphine (PH3) is known to inhibit cytochrome c oxidase in vitro. Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O2-), which dismutate to form hydrogen peroxide (H2O2). This study was performed in order to investigate the production of H2O2 by mitochondria isolated from granary weevil (Sitophilus granarius) and mouse liver on exposure to PH3. Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H2O2 after inhibition by PH3. Insect organelles released threefold more H2O2 than did mouse organelles, when exposed to PH3. Production of H2O2 by PH3-treated insect mitochondria was increased significantly on addition of the substrate alpha-glycerophosphate. Succinate did not enhance H2O2 production, however, indicating that the H2O2 did not result from the autoxidation of ubiquinone. NAD(+)-linked substrates, malate and pyruvate also had no effect on H2O2 production, suggesting that NADH-dehydrogenase was not the source of H2O2. Data obtained using antimycin and myxothiazol, both of which stimulated the release of H2O2 from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H2O2. The effect of combining PH3, antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH3 reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH3. The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H2O2 from the sites proposed previously.     

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.     

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase

Ca(2+)-activated K(+) channels (K(Ca)) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of K(Ca) with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with N(ω)-nitro-L-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100 nM ACh or 1 μM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance K(Ca) (SK(Ca)) channels with apamin and intermediate-conductance K(Ca) (IK(Ca)) channels with triarylmethane-34 (TRAM-34). Apamin-TRAM-34 treatment also abolished 100 nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca(2+) buffering with BAPTA elicited a similar increment in superoxide. Apamin-TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOS(Thr495)). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOS(Thr495) observed during apamin and TRAM-34 application. Our results indicate that blockade of SK(Ca) and IK(Ca) activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOS(Thr495). These findings disclose a novel mechanism involved in the control of NO production.     

Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca2+-sensitive K+ channels

Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O(2) concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH(2)-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O(2)(*-)); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H(2)O(2) production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca(2+)-sensitive K(+) channel (mtBK(Ca)) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O(2) consumption, membrane potential (DeltaPsi(m)), H(2)O(2) release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 microM) increased state 2 and state 4 respiration by 26 +/- 4% and 14 +/- 4%, respectively; this increase was abolished by the BK(Ca) channel blocker paxilline (5 microM). Paxilline alone had no effect on respiration. NS-1619 did not alter DeltaPsi(m) or redox state but decreased H(2)O(2) production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K(+) influx through mtBK(Ca) channels reduces mitochondrial H(2)O(2) production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H(2)O(2) production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K(+) influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.     

Characterization of superoxide-producing sites in isolated brain mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.     

Hypoxia-adaptation involves mitochondrial metabolic depression and decreased ROS leakage

Through long-term laboratory selection, we have generated a Drosophila melanogaster population that tolerates severe, normally lethal, level of hypoxia. This strain lives perpetually under severe hypoxic conditions (4% O(2)). In order to shed light on the mechanisms involved in this adaptation, we studied the respiratory function of isolated mitochondria from the thorax of hypoxia-adapted flies (AF) using polarographic oxygen consumption while monitoring superoxide generation by electron paramagnetic resonance (EPR) techniques. AF mitochondria exhibited a significant 30% decrease in respiratory rate during state 3, while enhancing the resting respiratory rate during State 4-oligo by 220%. The activity of individual electron transport complexes I, II and III were 107%, 65%, and 120% in AF mitochondria as compared to those isolated from control flies. The sharp decrease in complex II activity and modest increase in complexes I and III resulted in >60% reduction in superoxide leakage from AF mitochondria during both NAD(+)-linked state 3 and State 4-oligo respirations. These results provide evidence that flies with mitochondria exhibiting decreased succinate dehydrogenase activity and reduced superoxide leakage give flies an advantage for survival in long-term hypoxia.     

Menadione- (2-methyl-1,4-naphthoquinone-) dependent enzymatic redox cycling and calcium release by mitochondria

The results presented in this paper reveal the existence of three distinct menadione (2-methyl-1,4-naphthoquinone) reductases in mitochondria: NAD(P)H:(quinone-acceptor) oxidoreductase (D,T-diaphorase), NADPH:(quinone-acceptor) oxidoreductase, and NADH:(quinone-acceptor) oxidoreductase. All three enzymes reduce menadione in a two-electron step directly to the hydroquinone form. NADH-ubiquinone oxidoreductase (NADH dehydrogenase) and NAD(P)H azoreductase do not participate significantly in menadione reduction. In mitochondrial extracts, the menadione-induced NAD(P)H oxidation occurs beyond stoichiometric reduction of the quinone and is accompanied by O2 consumption. Benzoquinone is reduced more rapidly than menadione but does not undergo redox cycling. In intact mitochondria, menadione triggers oxidation of intramitochondrial pyridine nucleotides, cyanide-insensitive O2 consumption, and a transient decrease of delta psi. In the presence of intramitochondrial Ca2+, the menadione-induced oxidation of pyridine nucleotides is accompanied by their hydrolysis, and Ca2+ is released from mitochondria. The menadione-induced Ca2+ release leaves mitochondria intact, provided excessive Ca2+ cycling is prevented. In both selenium-deficient and selenium-adequate mitochondria, menadione is equally effective in inducing oxidation of pyridine nucleotides and Ca2+ release. Thus, menadione-induced Ca2+ release is mediated predominantly by enzymatic two-electron reduction of menadione, and not by H2O2 generated by menadione-dependent redox cycling. Our findings argue against D,T-diaphorase being a control device that prevents quinone-dependent oxygen toxicity in mitochondria.     

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2- generation in mitochondria respiring on the complex I substrates pyruvate+malate, an effect fully inhibited by rotenone. Antimycin A increased O2- production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2- production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2- formation driven with the complex II substrate succinate. At 0.6 microM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2- formation; however, at 40 microM myxothiazol (which completely inhibits both complexes I and III) eliminated O2- production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2- from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II.