Ammonium-dependent hydrogen peroxide production by mitochondria

NADH-supported generation of H2O2 by permeabilized rat heart mitochondria was partially prevented by the specific complex I-directed inhibitor, NADH-OH, and was significantly stimulated by ammonium. Ammonium did not affect H2O2 production by complex I in coupled submitochondrial particles. The soluble mitochondrial matrix protein fraction catalyzed NADH-dependent H2O2 production, which was greatly (approximately 10-fold) stimulated by ammonium. We conclude that complex I is not the major contributor to mitochondrial superoxide (hydrogen peroxide) generation and that there are specific ammonium-sensitive NADH:oxygen oxidoreductase(s) in the mitochondrial matrix which are responsible for mitochondrial H2O2 production.     

What are the sources of hydrogen peroxide production by heart mitochondria?

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4–State 3 transition) decreases the succinate- and glutamate plus malate-supported H₂O₂ production 8- and 1.3-times, respectively. NH₄⁺ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H₂O₂ production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H₂O₂ diffusion by alamethicin treatment are only partially (～ 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (～ 6-fold) stimulated by ammonium chloride. NAD⁺ inhibits both Complex I-mediated and ammonium-stimulated H₂O₂ production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).     

Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production

A homogeneous protein with a subunit apparent molecular mass of ∼50 kDa that catalyzes the previously described mitochondrial NADH-supported ammonium-stimulated hydrogen peroxide production (Grivennikova, V.G., Gecchini, G. and Vinogradov, A.D. (2008) FEBS Lett. 583, 1287–1291) was purified from the mitochondrial matrix of bovine heart. Chromatography of partially purified protein showed that the peaks of ammonium-stimulated NADH-dependent H₂O₂ production and that of NADH:lipoamide oxidoreductase activity coincided. The catalytic properties and mass spectrometry of the trypsin-digested protein revealed peptides that allowed identification of the protein as the Bos taurus dihydrolipoyl dehydrogenase.     

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.     

Long-chain 3-hydroxy fatty acids accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies uncouple oxidative phosphorylation in heart mitochondria

Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid β-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

Reversible glutathionylation of complex I increases mitochondrial superoxide formation

Increased production of reactive oxygen species (ROS) by mitochondria is involved in oxidative damage to the organelle and in committing cells to apoptosis or senescence, but the mechanisms of this increase are unknown. Here we show that ROS production by mitochondrial complex I increases in response to oxidation of the mitochondrial glutathione pool. This correlates with thiols on the 51- and 75-kDa subunits of complex I forming mixed disulfides with glutathione. Glutathionylation of complex I increases superoxide production by the complex, and when the mixed disulfides are reduced, superoxide production returns to basal levels. Within intact mitochondria oxidation of the glutathione pool to glutathione disulfide also leads to glutathionylation of complex I, which correlates with increased superoxide formation. In this case, most of this superoxide is converted to hydrogen peroxide, which can then diffuse into the cytoplasm. This mechanism of reversible mitochondrial ROS production suggests how mitochondria might regulate redox signaling and shows how oxidation of the mitochondrial glutathione pool could contribute to the pathological changes that occur to mitochondria during oxidative stress.     

Vitamin E regulates mitochondrial hydrogen peroxide generation.

The mitochondrial electron transport system consumes more than 85% of all oxygen used by the cells, and up to 5% of the oxygen consumed by mitochondria is converted to superoxide, hydrogen peroxide, and other reactive oxygen species (ROS) under normal physiologic conditions. Disruption of mitochondrial ultrastructure is one of the earliest pathologic events during vitamin E depletion. The present studies were undertaken to test whether a direct link exists between vitamin E and the production of hydrogen peroxide in the mitochondria. In the first experiment, mice were fed a vitamin E-deficient or-sufficient diet for 15 weeks, after which the mitochondria from liver and skeletal muscle were isolated to determine the rates of hydrogen peroxide production. Deprivation of vitamin E resulted in an approximately 5-fold increase of mitochondrial hydrogen peroxide production in skeletal muscle and a 1-fold increase in liver when compared with the vitamin E-supplemented group. To determine whether vitamin E can dose-dependently influence the production of hydrogen peroxide, four groups of male and female rats were fed diets containing 0, 20, 200, or 2000 lU/kg vitamin E for 90 d. Results showed that dietary vitamin E dose-dependently attenuated hydrogen peroxide production in mitochondria isolated from liver and skeletal muscle of male and female rats. Female rats, however, were more profoundly affected by dietary vitamin E than male rats in the suppression of mitochondrial hydrogen peroxide production in both organs studied. These results showed that vitamin E can directly regulate hydrogen peroxide production in mitochondria and suggest that the overproduction of mitochondrial ROS is the first event leading to the tissue damage observed in vitamin E-deficiency syndromes. Data further suggested that by regulating mitochondrial production of ROS, vitamin E modulates the expression and activation of signal transduction pathways and other redox-sensitive biologic modifiers, and thereby delays or prevents degenerative tissue changes.     

Intra-mitochondrial poly(ADP-ribosyl)ation: Potential role for alpha-ketoglutarate dehydrogenase

Poly(ADP-ribose) polymerase (PARP) is an intracellular enzyme involved in DNA repair and in building poly-ADP-ribose polymers on nuclear proteins using NAD⁺. While the majority of PARP resides in the nucleus, several studies indicated that PARP may also be located in the cytosol or in the mitochondrial matrix. In this study we found several poly-ADP-ribosylated proteins in isolated rat liver mitochondria following hydrogen peroxide (H₂O₂) or nitric oxide donor treatment. Protein poly-ADP-ribosylation was more intense in isolated mitochondria than in whole tissue homogenates and it was not associated with increased nuclear PARP activity. We identified five poly-ADP-ribose (PAR) positive mitochondrial bands by protein mass fingerprinting. All of the identified enzymes exhibited decreased activity or decreased levels following oxidative or nitrosative stress. One of the identified proteins is dihydrolipoamide dehydrogenase (DLDH), a component of the alpha-ketoglutarate dehydrogenase (KGDH) complex, which uses NAD⁺ as a substrate. This raised the possibility that KGDH may have a PARP-like enzymatic activity. The intrinsic PARP activity of KGDH and DLDH was confirmed using a colorimetric PARP assay kit and by the incubation of the recombinant enzymes with H₂O₂. The KGDH enzyme may, therefore, have a novel function as a PARP-like enzyme, which may play a role in regulating intramitochondrial NAD⁺ and poly(ADP-ribose) homeostasis, with possible roles in physiology and pathophysiology.     

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO₂ as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin‐mediated drug‐delivery of RGD‐modified DLDH (DLDH^RGD), leading to apoptotic cell death. In these experiments, DLDH^RGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA‐binding capability of this enzyme. To explore this interaction under cell‐free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel‐shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA‐binding. A Docking algorithm for prediction of protein‐DNA complexes, Paradoc, identified a putative DNA binding site at the C‐terminus of DLDH. Our finding that TiO₂‐bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.     

Localization of the Site of Oxygen Radical Generation inside the Complex I of Heart and Nonsynaptic Brain Mammalian Mitochondria

Mitochondrial production of oxygen radicals seems to be involved in many diseases and aging. Recent studies clearly showed that a substantial part of the free radical generation of rodent mitochondria comes from complex I. It is thus important to further localize the free radical generator site within this respiratory complex. In this study, superoxide production by heart and nonsynaptic brain submitochondrial particles from up to seven mammalian species, showing different longevities, were studied under different conditions. The results, taking together, show that rotenone stimulates NADH-supported superoxide generation, confirming that complex I is a source of oxygen radicals in mammals, in general. The rotenone-stimulated NADH-supported superoxide production of the heart and nonsynaptic brain mammalian submitochondrial particles was inhibited both by p-chloromercuribenzoate and by ethoxyformic anhydride. These results localize the complex I oxygen radical generator between the ferricyanide and the ubiquinone reduction site, making iron—sulfur centers possible candidates, although unstable semiquinones can not be discarded. The results also indicate that the previously described inverse correlation between rates of mitochondrial oxygen radical generation and mammalian longevity operates through mechanisms dependent on the presence of intact functional mitochondria.     

Effect of Auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

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.     

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.     

Glycerophosphate-dependent peroxide production by brown fat mitochondria from newborn rats

Glycerophosphate (GP)-dependent, ferricyanide-induced hydrogen peroxide production was studied in brown adipose tissue mitochondria from newborn rats. Relations between the rate of hydrogen peroxide production and total amount of hydrogen peroxide produced at different GP and ferricyanide concentrations were determined. It was found that the rate of hydrogen peroxide production increases with increasing GP concentration and decreases with increasing ferricyanide concentration. Total amount of hydrogen peroxide produced increases with increasing ferricyanide concentration, however, not proportionally, and the efficiency of this process (oxygen/ferricyanide ratio) strongly declines. Data presented provide further information on the character and kinetics of hydrogen peroxide production by mammalian mitochondrial glycerophosphate dehydrogenase.     

Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial fraction

1. The primary intermediate of catalase and hydrogen peroxide was identified and investigated in peroxisome-rich mitochondrial fractions of rat liver. On the basis of kinetic constants determined in vitro, it is possible to calculate with reasonable precision the molecular statistics of catalase action in the peroxisomes. 2. The endogenous hydrogen peroxide generation is adequate to sustain a concentration of the catalase intermediate (p(m)/e) of 60-70% of the hydrogen peroxide saturation value. Total amount of catalase corresponds to 0.12-0.15nmol of haem iron/mg of protein. In State 1 the rate of hydrogen peroxide generation corresponds to 0.9nmol/min per mg of protein or 5% of the mitochondrial respiratory rate in State 4. 3. Partial saturation of the catalase intermediate with hydrogen peroxide (p(m)/e) in the mitochondrial fraction suggests its significant peroxidatic activity towards its endogenous hydrogen donor. A variation of this value (p(m)/e) from 0.3 in State 4 to 0 under anaerobic conditions is observed. 4. For a particular preparation the hydrogen peroxide generation rate in the substrate-supplemented State 4 corresponds to 0.17s(-1) (eqn. 6), the hydrogen peroxide concentration to 2.5nm and the hydrogen-donor concentration (in terms of ethanol) to 0.12mm. The reaction is 70% peroxidatic and 30% catalatic. 5. A co-ordinated production of both oxidizing and reducing substrates for catalase in the mitochondrial fraction is suggested by a 2.2-fold increase of hydrogen peroxide generation and a threefold increase in hydrogen-donor generation in the State 1 to State 4 transition. 6. Additional hydrogen peroxide generation provided by the urate oxidase system of peroxisomes (8-12nmol of uric acid oxidized/min per mg of protein) permits saturation of the catalase with hydrogen peroxide to haem occupancy of 40% compared with values of 36% for a purified rat liver catalase ofk(1)=1.7x10(7)m(-1).s(-1) and k'(4)=2.6x10(7)m(-1). s(-1)(Chance, Greenstein & Roughton, 1952). 7. The turnover of the catalase ethyl hydrogen peroxide intermediate (k'(3)) in the peroxisomes is initially very rapid since endogenous hydrogen peroxide acts as a hydrogen donor. k'(3) decreases fivefold in the uncoupled state of the mitochondria.     

The contribution of thioredoxin-2 reductase and glutathione peroxidase to H(2)O(2) detoxification of rat brain mitochondria

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H(2)O(2)) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60±20% and 20±15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Dopamine modifies oxygen consumption and mitochondrial membrane potential in striatal mitochondria

Dopamine is a neurotransmitter that has been related to mitochondrial dysfunction. In this study, striatal intact mitochondria and submitochondrial membranes were incubated with different dopamine concentrations, and changes on mitochondrial function, hydrogen peroxide, and nitric oxide production were evaluated. A 35% decrease in state 3 oxygen uptake (active respiration state) was found after 1 mM dopamine incubation. In addition, mitochondrial respiratory control significantly decreased, indicating mitochondrial dysfunction. High dopamine concentrations induced mitochondrial depolarization. Also, evaluation of hydrogen peroxide production by intact striatal mitochondria showed a significant increase after 0.5 and 1 mM dopamine incubation. Incubation with 0.5 and 1 mM dopamine increased nitric oxide production in submitochondrial membranes by 28 and 49%, respectively, as compared with control values. This study provides evidence that high dopamine concentrations induce striatal mitochondrial dysfunction through a decrease in mitochondrial respiratory control and loss of membrane potential, probably mediated by free radical production.