Mitochondrial complex I,aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.     

Down-regulation of iron regulatory protein 1 activities and expression in superoxide dismutase 1 knock-out mice is not associated with alterations in iron metabolism

Iron and oxygen (O2) are intimately associated in many well characterized patho-physiological processes. These include oxidation of the [4Fe-4S] cluster of mitochondrial aconitase and inactivation of this Krebs cycle enzyme by the superoxide anion (O2*-), a product of the one-electron of reduction O2. In contrast to the apparent toxicity of this reaction, the biological consequences of O2*- -mediated inactivation of the cytosolic counterpart of mitochondrial aconitase, commonly known as iron regulatory protein 1 (IRP1), are not clear. Apart from its ability to convert citrate to iso-citrate, IRP1 in its apo-form binds to iron-responsive elements in the untranslated regions of mRNAs coding for proteins involved in iron metabolism, to regulate their synthesis and thus control the cellular homeostasis of this metal. Here, we show that in superoxide dismutase 1 (SOD1) knock-out mice, lacking Cu,Zn-SOD, an enzyme that acts to reduce the concentration of O2*- mainly in cytosol, not only is aconitase activity of IRP1 inhibited but the level of IRP1 is also strongly decreased. Despite such an evident alteration in IRP1 status, SOD1-deficient mice display a normal iron metabolism phenotype. Our findings clearly show that under conditions of O2*- -mediated oxidative stress, IRP1 is not essential for the maintenance of iron metabolism in mammals.     

Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase.

The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain. Growth of these strains on a gluconate minimal medium was also impaired under these conditions. The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation. The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E. coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1. The dehydratase was much less sensitive to O2 or H2O2 than to O2-. The virtual substrate, alpha-glycerophosphate, provided partial protection. Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme. Fluoride ions stabilized the dehydratase and blocked the effect of oxidants. The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.     

Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation

Using the inactivation of mitochondrial and cytosolic aconitases as markers of compartment-specific superoxide (O2(-)) production, we show that oxygen-glucose deprivation (OGD) or excitotoxin exposure produce a time-dependent inactivation of mitochondrial, but not cytosolic, aconitase in cortical cultures. To determine if mitochondrial O2(-) production was an important determinant in neuronal death resulting from OGD, metalloporphyrins with varying superoxide dismutase (SOD) activity were tested for their ability to protect against mitochondrial aconitase inactivation and cell death. OGD-induced mitochondrial aconitase inactivation and cell death was inhibited by manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP), manganese tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) and NMDA receptor antagonists. By contrast, NMDA- or kainate (KA)-induced mitochondrial aconitase inactivation and cell death was inhibited by MnTBAP, but not MnTE-2-PyP. Moreover, both MnTBAP and MnTE-2-PyP penetrated mitochondrial fractions of cortical cells. These data suggest that mitochondrial aconitase inactivation closely correlates with subsequent neuronal death following excitotoxicity produced by OGD or NMDA/KA exposure. Assessment of biological rather biochemical antioxidant activities better predicted neuroprotection by metalloporphyrins. Moreover, antioxidants that protect oxidant-sensitive mitochondrial targets such as aconitase may be useful as therapies for disease states involving excitotoxicity.     

Quinolinate synthetase: the oxygen-sensitive site of de novo NAD(P)+ biosynthesis.

The ability of niacin to relieve the growth-inhibiting effect of hyperoxia on Escherichia coli can be attributed to the dioxygen sensitivity of quinolinate synthetase. The activity of this enzyme within E. coli was diminished by exposure of the cells to 4.2 atm O2, while the activity in extracts was rapidly decreased by 0.2 atm O2. Neither catalase nor superoxide dismutase afforded detectable protection against the inactivating effect of O2, indicating that H2O2 and O2- were not significant intermediates in this process. Nevertheless, H2O2 at 1.0 mM did inactivate quinolinate synthetase, even under anaerobic conditions and in the absence of catalatic activity which might have generated O2. Addition of paraquat to aerobic cultures of E. coli caused an inactivation of quinolinate synthetase, which may be explained in terms of an increase in the production of H2O2. The O2-dependent inactivation of quinolinate synthetase in extracts was gradually reversed during anaerobic incubation and this reactivation was blocked by alpha, alpha'-dipyridyl or by 1,10-phenanthroline. The sequence of the quinolinate synthetase "A" protein contains a--cys-w-x-cys-y-z-cys--sequence, which is characteristic of (Fe-S)4-containing proteins. This sequence, together with the effect of the Fe(II)-chelating agents, suggests that the O2-sensitive site of quinolinate synthetase is an iron-sulfur cluster which is essential for the dehydration reaction catalyzed by the A protein.     

Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation

Red blood cells (RBC) are thought to be well protected against oxidative stress by the antioxidant, cu-pro-zinc enzyme superoxide dismutase (CuZn SOD) which dismutates O2- to H2O2. CuZn SOD, however, is irreversibly inactivated by its product H2O2. Exposure of intact RBC to H2O2 resulted in the inactivation (up to 50%) of endogenous SOD in a concentration-dependent manner. When RBC were exposed to O2- and H2O2, generated by xanthine + xanthine oxidase, an even greater loss of SOD activity (approximately 75%) was observed. Intracellular proteolysis was markedly increased by exposure to these same oxidants; up to a 12-fold increase with H2O2 and a 50-fold increase with xanthine oxidase plus xanthine. When purified SOD was treated with H2O2, inactivation of the enzyme also occurred in a concentration-dependent manner. Accompanying the loss of SOD activity, the binding of the copper ligand to the active site of the enzyme diminished with H2O2 exposure, as evidenced by an increase in accessible copper. Significant direct fragmentation of SOD was evident only under conditions of prolonged exposure (20 h) to relatively high concentrations of H2O2. Gel electrophoresis studies indicated that under most experimental conditions (i.e. 1-h incubation) H2O2, O2-, and H2O2 + O2- treated SOD experienced charge changes and partial denaturation, rather than fragmentation. The proteolytic susceptibility of H2O2-modified SOD, during subsequent incubation with (rabbit, bovine or human) red cell extracts also increased as a function of pretreatment with H2O2. Both enzyme inactivation and altered copper binding appeared to precede the increase in proteolytic susceptibility (whether measured as an effect of H2O2 concentration or as a function of the duration of H2O2 exposure). These results suggest that SOD inactivation and modification of copper binding are prerequisites for increased protein degradation. Proteolytic susceptibility was further enhanced by H2O2 exposure under alkaline conditions, suggesting that the hydroperoxide anion is the damaging species rather than H2O2 itself. In RBC extracts, the proteolysis of H2O2-modified SOD was inhibited by sulfhydryl reagents, serine reagents, transition metal chelators, and ATP; suggesting the existence of an ATP-independent proteolytic pathway of sulfhydryl, serine, and metalloproteases, and peptidases. The proteolytic activity was conserved in a "Fraction II" of both human and rabbit RBC, and was purified from rabbit reticulocytes and erythrocytes to a 670-kDa proteinase complex, for which we have suggested the trivial name macroxyproteinase. In erythrocytes macroxyproteinase may prevent the accumulation of H2O2-modified SOD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Redox-dependent modulation of aconitase activity in intact mitochondria

It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O(2)(-)(*) or hydrogen peroxide (H(2)O(2)) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H(2)O(2). Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H(2)O(2) does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H(2)O(2). Nevertheless, prolonged exposure of mitochondria to steady-state levels of H(2)O(2) or O(2)(-)(*) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.     

Oxidative inactivation of angiotensin-converting enzyme]

Hydrogen peroxide inactivates the purified human angiotensin-converting enzyme (ACE) in vitro; the inactivating effect of H2O2 is eliminated by an addition of catalase. The lung and kidney ACE are equally sensitive to the effect of hydrogen peroxide. After addition of oxidants (H2O2 alone or H2O2 + ascorbate or H2O2 + Fe2+ mixtures) to the membranes or homogenates of the lung, the inactivation of membrane-bound ACE is far less pronounced despite the large-scale accumulation of lipid peroxidation products. The marked inactivation of ACE in the membrane fraction (up to 55% of original activity) was observed during ACE incubation with a glucose:glucose oxidase:Fe2+ mixture. Presumably the oxidative potential of H2O2 in tissues in consumed, predominantly, for the oxidation of other components of the membrane (e.g., lipids) rather than for ACE inactivation.     

The oxidative inactivation of mitochondrial electron transport chain components and ATPase

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rotenone-like action of the branched-chain phytanic acid induces oxidative stress in mitochondria

Phytanic acid (Phyt) increase is associated with the hereditary neurodegenerative Refsum disease. To elucidate the still unclear toxicity of Phyt, mitochondria from brain and heart of adult rats were exposed to free Phyt. Phyt at low micromolar concentrations (maximally: 100 nmol/mg of protein) enhances superoxide (O(2)(.))(2) generation. Phyt induces O(2)(.) in state 3 (phosphorylating), as well as in state 4 (resting). Phyt stimulates O(2)(.) generation when the respiratory chain is fed with electrons derived from oxidation of glutamate/malate, pyruvate/malate, or succinate in the presence of rotenone. With succinate alone, Phyt suppresses O(2)(.) generation caused by reverse electron transport from succinate to complex I. The enhanced O(2)(.) generation by Phyt in state 4 is in contrast to the mild uncoupling concept. In this concept uncoupling by nonesterified fatty acids should abolish O(2)(.) generation. Stimulation of O(2)(.) generation by Phyt is paralleled by inhibition of the electron transport within the respiratory chain or electron leakage from the respiratory chain. The interference of Phyt with the electron transport was demonstrated by inhibition of state 3- and p-trifluoromethoxyphenylhydrazone (FCCP)-dependent respiration, inactivation of the NADH-ubiquinone oxidoreductase complex in permeabilized mitochondria, decrease in reduction of the synthetic electron acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in state 4, and increase of the mitochondrial NAD(P)H level in FCCP-uncoupled mitochondria. Thus, we suggest that complex I is the main site of Phyt-stimulated O(2)(.) generation. Furthermore, inactivation of aconitase and oxidation of the mitochondrial glutathione pool show that enhanced O(2)(.) generation with chronic exposure to Phyt causes oxidative damage.     

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts.

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.     

Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27

The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1(+/+)) and HSF-1 knockout (HSF-1(-/-)) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1(-/-) cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1(-/-) cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1(-/-), were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O(2)*(-), by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.     

Mechanism of the oxidative stress-mediated increase in lipid accumulation by the bacterium,R. opacus PD630: Experimental analysis and genome-scale metabolic modeling

Appropriate species of oleaginous bacteria, with their high growth rates and lipid accumulation capabilities, can be good contenders for industrial triacylglycerol (TAG) production, compared to microalgae. Further, oxidative stress (OS) can be used to significantly increase TAG yields in oleaginous microbes, but the mechanism is unexplored. In a first, this study explored the mechanism behind OS-mediated increase in TAG accumulation by the bacterium, Rhodococccus opacus PD630, through experimental analysis and metabolic modelling. Two mechanisms that could increase acetyl-CoA (TAG-precursor) levels were hypothesized based on literature information. One was OS-mediated inactivation of the aconitase (TCA cycle), and another was the inactivation of the triosephosphate isomerase (TPI; glycolysis). The results negated the involvement of aconitase in increased acetyl-CoA levels. Analysis of the metabolic model showed that inactivation of TPI, re-routed the flux through the pentose phosphate pathway (PPP), supplying both NADPH and acetyl-CoA for TAG synthesis. Additionally, inactivation of TPI increased TAG flux by 143%, whereas, inactivating both TPI and aconitase, increased it by 152%. We present experimental evidence for OS-mediated decrease in TPI activity and increase in activity of glucose-6-phosphate dehydrogenase (PPP enzyme). The findings indicate that increased flux through PPP can be explored to improve TAG accumulation on a large-scale.     

Growth on ethanol results in co-ordinated Saccharomyces cerevisiae response to inactivation of genes encoding superoxide dismutases

Superoxide dismutase (SOD) is an essential enzyme protecting cells against oxidative stress. However, its specific role under different conditions is not clear. To study the possible role of SOD in the cell during respiration, Saccharomyces cerevisiae single and double mutants with inactivated SOD1 and/or SOD2 genes growing on ethanol as an energy and carbon source were used. Activities of antioxidant and associated enzymes as well as the level of protein carbonyls were measured. SOD activity was significantly higher in a Mn-SOD deficient strain than that in the wild-type parental strain, but significantly lower in a Cu, Zn-SOD mutant. A strong positive correlation between SOD and catalase activities (R(2) = 0.99) shows possible protection of catalase by SOD from inactivation in vivo and/or decrease in catalase activity because of lower H(2)O(2) formation in the mutant cells. SOD deficiency resulted in a malate dehydrogenase activity increase, whereas glucose-6-phosphate dehydrogenase (G6PDH) activity was lower in SOD-deficient strains. Linear and non-linear positive correlations between SOD and isocitrate dehydrogenase activities are discussed. No changes in the activity of glutathione reductase and protein carbonyl levels support the idea that SOD-deficient cells are not exposed to strong oxidative stress during exponential growth of yeast cultures on ethanol.     

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H2O2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H2O2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H2O2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H2O2 concentration and/or time of exposure to H2O2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H2O2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H2O2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo protein synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H2O2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of mitochondrial iron homeostasis on cofactor specificity of superoxide dismutase 2

Many metalloproteins have the capacity to bind diverse metals, but in living cells connect only with their cognate metal cofactor. In eukaryotes, this metal specificity can be achieved through metal-specific metallochaperone proteins. Herein, we describe a mechanism whereby Saccharomyces cerevisiae manganese superoxide dismutase (SOD2) preferentially binds manganese over iron based on the differential bioavailability of these ions within mitochondria. The bulk of mitochondrial iron is normally unavailable to SOD2, but when mitochondrial iron homeostasis is disrupted, for example, by mutations in S. cerevisiae mtm1, ssq1 and grx5, iron accumulates in a reactive form that potently competes with manganese for binding to SOD2, inactivating the enzyme. Studies in mtm1 mutants indicate that iron inactivation of SOD2 involves the Mrs3p/Mrs4p mitochondrial carriers and iron-binding frataxin (Yfh1p). A small pool of SOD2-reactive iron also exists under normal iron homeostasis conditions and binds SOD2 when mitochondrial manganese is low. The ability to control this reactive pool of iron is critical to maintaining SOD2 activity and has important potential implications for oxidative stress in disorders of iron overload.     

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.     

Mitochondrial O2*- and H2O2 mediate glucose deprivation-induced stress in human cancer cells

The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial O2*- and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial O2*- and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2-8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho0) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial O2*- and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells.     

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.