Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.     

Inhibition of mitochondrial respiratory complex I by nitric oxide, peroxynitrite and S-nitrosothiols

NO or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial complex I by several different mechanisms that are not well characterised. There is an inactivation by NO, peroxynitrite and S-nitrosothiols that is reversible by light or reduced thiols, and therefore may be due to S-nitrosation or Fe-nitrosylation of the complex. There is also an irreversible inhibition by peroxynitrite, other oxidants and high levels of NO, which may be due to tyrosine nitration, oxidation of residues or damage of iron sulfur centres. Inactivation of complex I by NO or RNS is seen in cells or tissues expressing iNOS, and may be relevant to inflammatory pathologies, such as septic shock and Parkinson's disease.     

Lack of Oxygen Deactivates Mitochondrial Complex I: IMPLICATIONS FOR ISCHEMIC INJURY?

For S-nitrosothiols and peroxynitrite to interfere with the activity of mitochondrial complex I, prior transition of the enzyme from its active (A) to its deactive, dormant (D) state is necessary. We now demonstrate accumulation of the D-form of complex I in human epithelial kidney cells after prolonged hypoxia. Upon reoxygenation after hypoxia there was an initial delay in the return of the respiration rate to normal. This was due to the accumulation of the D-form and its slow, substrate-dependent reconversion to the A-form. Reconversion to the A-form could be prevented by prolonged incubation with endogenously generated NO. We propose that the hypoxic transition from the A-form to the D-form of complex I may be protective, because it would act to reduce the electron burst and the formation of free radicals during reoxygenation. However, this may become an early pathophysiological event when NO-dependent formation of S-nitrosothiols or peroxynitrite structurally modifies complex I in its D-form and impedes its return to the active state. These observations provide a mechanism to account for the severe cell injury that follows hypoxia and reoxygenation when accompanied by NO generation.     

Nitric oxide-induced persistent inhibition and nitrosylation of active site cysteine residues of mitochondrial cytochrome-c oxidase in lung endothelial cells

Persistent inhibition of cytochrome-c oxidase, a terminal enzyme of the mitochondrial electron transport chain, by excessive nitric oxide (NO) derived from inflammation, polluted air, and tobacco smoke contributes to enhanced oxidant production and programmed cell death or apoptosis of lung cells. We sought to determine whether the long-term exposure of pulmonary artery endothelial cells (PAEC) to pathophysiological concentrations of NO causes persistent inhibition of complex IV through redox modification of its key cysteine residues located in a putative NO-sensitive motif. Prolonged exposure of porcine PAEC to 1 mM 2,2'-(hydroxynitrosohydrazino)-bis-ethanamine (NOC-18; slow-releasing NO donor, equivalent to 1–5 µM NO) resulted in a gradual, persistent inhibition of complex IV concomitant with a reduction in ratios of mitochondrial GSH and GSSG. Overexpression of thioredoxin in mitochondria of PAEC attenuated NO-induced loss of complex IV activities, suggesting redox regulation of complex IV activity. Sequence analysis of complex IV subunits revealed a novel putative NO-sensitive motif in subunit II (S2). There are only two cysteine residues in porcine complex IV S2, located in the putative motif. Immunoprecipitation and Western blot analysis and "biotin switch" assay demonstrated that exposure of PAEC to 1 mM NOC-18 increased S-nitrosylation of complex IV S2 by 200%. Site-directed mutagenesis of these two cysteines of complex IV S2 attenuated NO-increased nitrosylation of complex IV S2. These results demonstrate for the first time that NO nitrosylates active site cysteines of complex IV, which is associated with persistent inhibition of complex IV. NO inhibition of complex IV via nitrosylation of NO-sensitive cysteine residues can be a novel upstream event in NO-complex IV signaling for NO toxicity in lung endothelial cells. S-nitrosylation; redox regulation     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Opposing effects of nitric oxide and prostaglandin inhibition on muscle mitochondrial Vo(2) during exercise

Nitric oxide (NO) and prostaglandins (PG) together play a role in regulating blood flow during exercise. NO also regulates mitochondrial oxygen consumption through competitive binding to cytochrome-c oxidase. Indomethacin uncouples and inhibits the electron transport chain in a concentration-dependent manner, and thus, inhibition of NO and PG synthesis may regulate both muscle oxygen delivery and utilization. The purpose of this study was to examine the independent and combined effects of NO and PG synthesis blockade (L-NMMA and indomethacin, respectively) on mitochondrial respiration in human muscle following knee extension exercise (KEE). Specifically, this study examined the physiological effect of NO, and the pharmacological effect of indomethacin, on muscle mitochondrial function. Consistent with their mechanism of action, we hypothesized that inhibition of nitric oxide synthase (NOS) and PG synthesis would have opposite effects on muscle mitochondrial respiration. Mitochondrial respiration was measured ex vivo by high-resolution respirometry in saponin-permeabilized fibers following 6 min KEE in control (CON; n = 8), arterial infusion of N(G)-monomethyl-L-arginine (L-NMMA; n = 4) and Indo (n = 4) followed by combined inhibition of NOS and PG synthesis (L-NMMA + Indo, n = 8). ADP-stimulated state 3 respiration (OXPHOS) with substrates for complex I (glutamate, malate) was reduced 50% by Indo. State 3 O(2) flux with complex I and II substrates was reduced less with both Indo (20%) and L-NMMA + Indo (15%) compared with CON. The results indicate that indomethacin reduces state 3 mitochondrial respiration primarily at complex I of the respiratory chain, while blockade of NOS by L-NMMA counteracts the inhibition by Indo. This effect on muscle mitochondria, in concert with a reduction of blood flow accounts for in vivo changes in muscle O(2) consumption during combined blockade of NOS and PG synthesis.     

Glutamoyl diester of the dietary polyphenol curcumin offers improved protection against peroxynitrite-mediated nitrosative stress and damage of brain mitochondria in vitro: implications for Parkinson��s disease

Oxidative/nitrosative stress plays a crucial role in Parkinson's disease (PD) by triggering mitochondrial dysfunction. Nitrosative stress is mediated by reactive species such as peroxynitrite (PN) which could damage biomolecules thereby impinging on the cellular machinery. We observed that PN (0-1000 μM) inhibited brain mitochondrial complex I (CI) activity in a dose-dependent manner with concomitant tyrosine nitration of proteins. We also observed that exposure to PN at low concentrations (62.5-125 μM) significantly decreased the mitochondrial membrane potential and affected the mitochondrial integrity at higher doses (500-750 μM) as indicated by the mitochondrial swelling experiment. Therefore, it could be surmised that compounds that prevent such mitochondrial damage might have therapeutic value in neurological conditions such as PD. We previously showed that curcumin could detoxify PN and protect against CI inhibition and protein nitration. However, the therapeutic potential of curcumin is constrained by limited bioavailability. To address this issue and obtain improved antioxidants, three bioconjugates of curcumin (Di-demethylenated piperoyl, di-valinoyl and di-glutamoyl esters) were generated and tested against PN-mediated nitrosative stress and mitochondrial damage. We found that among the bioconjugates, the glutamoyl diester of curcumin showed improved protection against PN-dependent CI inhibition and protein nitration compared to other conjugates. Di-glutamoyl curcumin protected dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP(+))-mediated neuronal death. These effects were improved compared to curcumin alone suggesting that di-glutamoyl curcumin could be a better neuroprotective agent in neurodegenerative diseases such as PD.     

Critical role of mitochondrial glutathione in the survival of hepatocytes during hypoxia

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.     

Biomarker signatures of mitochondrial NDUFS3 in invasive breast carcinoma

We present evidence for potential biomarker utility of a mitochondrial complex I subunit, (NDUFS3) in discriminating normal and highly invasive breast carcinoma specimens obtained from clinical patients. Besides being a robust indicator of breast cancer aggressiveness, NDUFS3 also shows clear signatures of a hypoxia/necrosis marker in invasive ductal carcinoma specimens. Statistically significant positive correlation was observed between nuclear grade and NDUFS3 expression level in the tumor specimens analyzed. We support these findings with a plausible mechanism involving mitochondrial complex I assembly defects and/or redox buffering induced mitochondrial dysfunction during the process of cancer cell transformation. From a clinical standpoint, this novel observation adds value in augmenting the current receptor-based biomarkers for better accuracy in diagnosis and predicting survival rate in patients with breast carcinoma.     

Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease

Mitochondria are the specialized organelles for energy metabolism but also participate in the production of O(2) active species, cell cycle regulation, apoptosis and thermogenesis. Classically, regulation of mitochondrial energy functions was based on the ADP/ATP ratio, which dynamically stimulates the transition between resting and maximal O(2) uptake. However, in the last years, NO was identified as a physiologic regulator of electron transfer and ATP synthesis by inhibiting cytochrome oxidase. Additionally, NO stimulates the mitochondrial production of O(2) active species, primarily O(2)(-) and H(2)O(2), and, depending on NO matrix concentration, of ONOO(-), which is responsible for the nitrosylation and nitration of mitochondrial components. By this means, alteration in mitochondrial complexes restricts energy output, further increases O(2) active species and changes cell signaling for proliferation and apoptosis through redox effects on specific pathways. These mechanisms are prototypically operating in prevalent generalized diseases like sepsis with multiorgan failure or limited neurodegenerative disorders like Parkinson's disease. Complex I appears to be highly susceptible to ONOO(-) effects and nitration, which defines an acquired group of mitochondrial disorders, in addition to the genetically induced syndromes. Increase of mitochondrial NO may follow over-expression of nNOS, induction and translocation of iNOS, and activation and/or increased content of the newly described mtNOS. Likewise, mtNOS is important in the modulation of O(2) uptake and cell signaling, and in mitochondrial pathology, including the effects of aging, dystrophin deficiency, hypoxia, inflammation and cancer.     

Aspects, mechanism, and biological relevance of mitochondrial protein nitration sustained by mitochondrial nitric oxide synthase

The goal of this study was to explore the occurrence of nitrated proteins in mitochondria given that these organelles are endowed with a mitochondrial nitric oxide (NO.-) synthase and considering the important role that mitochondria have in energy metabolism. Our hypothesis is that nitration of proteins constitutes a posttranslational modification by which NO.- exhibits long-term effects above and beyond those bioregulatory ones mediated through the interaction with cytochrome c oxidase. Our studies are aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such a process in the cellular milieu. On promoting a sustained NO.- production by mitochondria, we investigated various aspects of protein nitration. Among them, the localization of nitrated proteins in mitochondrial subfractions, the identification of nitrated proteins through proteomic approaches, the characterization of affected pathways, and depiction of a target sequence. The biological relevance was analyzed by considering the turnover of native and nitrated proteins. In this regard, mitochondrial dysfunction, ensuing nitrative stress, may be envisioned as the result of accumulation of nitrated proteins, resulting from an overproduction of endogenous NO.- (this study), a failure in the proteolytic system to catabolize modified proteins, or a combination of both. Finally, this study allows one to gain understanding on the mechanism and nitrating species underlying mitochondrial protein nitration.     

The composition of plant mitochondrial supercomplexes changes with oxygen availability

Respiratory supercomplexes are large protein structures formed by various enzyme complexes of the mitochondrial electron transport chain. Using native gel electrophoresis and activity staining, differential regulation of complex activity within the supercomplexes was investigated. During prolonged hypoxia, complex I activity within supercomplexes diminished, whereas the activity of the individual complex I-monomer increased. Concomitantly, an increased activity was observed during hypoxia for complex IV in the smaller supercomplexes that do not contain complex I. These changes in complex activity within supercomplexes reverted again during recovery from the hypoxic treatment. Acidification of the mitochondrial matrix induced similar changes in complex activity within the supercomplexes. It is suggested that the increased activity of the small supercomplex III(2)+IV can be explained by the dissociation of complex I from the large supercomplexes. This is discussed to be part of a mechanism regulating the involvement of the alternative NADH dehydrogenases, known to be activated by low pH, and complex I, which is inhibited by low pH. It is concluded that the activity of complexes within supercomplexes can be regulated depending on the oxygen status and the pH of the mitochondrial matrix.     

Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia

Cortical nitric oxide (NO) production increases during hypoxia/ischemia in the immature brain and is associated with both neurotoxicity and mitochondrial dysfunction. Mitochondrial redistribution within the cell is critical to normal neuronal function, however, the effects of hypoxia on mitochondrial dynamics are not known. This study tested the hypothesis that hypoxia impairs mitochondrial movement via NO-mediated pathways. Fluorescently labeled mitochondria were studied using time-lapse digital video microscopy in cultured cortical neurons exposed either to hypoxia/re-oxygenation or to diethyleneamine/nitric oxide adduct, DETA-NO (100-500 microm). Two NO synthase inhibitors, were used to determine NO specificity. Mitochondrial mean velocity, the percentage of movement (i.e. the time spent moving) and mitochondrial morphology were analyzed. Exposure to hypoxia reduced mitochondrial movement to 10.4 +/- 1.3% at 0 h and 7.4 +/- 1.7% at 1 h of re-oxygenation, versus 25.6 +/- 1.4% in controls (p < 0.05). Mean mitochondrial velocity (microm s(-1)) decreased from 0.374 +/- 0.01 in controls to 0.146 +/- 0.01 at 0 h and 0.177 +/- 0.02 at 1 h of re-oxygenation (p < 0.001). Exposure to DETA-NO resulted in a significant decrease in mean mitochondrial velocity at all tested time points. Treatment with NG-nitro-L-arginine methyl ester (L-NAME) prevented the hypoxia-induced decrease in mitochondrial movement at 0 h (30.1 +/- 1.6%) and at 1 h (26.1 +/- 9%) of re-oxygenation. Exposure to either hypoxia/re-oxygenation or NO also resulted in the rapid decrease in mitochondrial size. Both hypoxia and NO exposure result in impaired mitochondrial movement and morphology in cultured cortical neurons. As the effect of hypoxia on mitochondrial movement and morphology can be partially prevented by a nitric oxide synthase (NOS) inhibitor, these data suggest that an NO-mediated pathway is at least partially involved.     

Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's disease

An early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH + GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss, and cell death. We have demonstrated using dopaminergic PC12 cell lines genetically engineered to inducibly down-regulate glutathione synthesis that total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Here, we demonstrate that inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitrite-mediated inhibition of complex I or through H2O2-mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme alpha-ketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex; activity of both enzymes are reduced in PD. While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. Our data suggest that NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells. This may have major implications in terms of understanding mechanisms of dopamine cell death associated with PD especially as they relate to complex I inhibition.     

Nitric oxide and neuronal death

NO and its derivatives can have multiple effects, which impact on neuronal death in different ways. High levels of NO induces energy depletion-induced necrosis, due to: (i) rapid inhibition of mitochondrial respiration, (ii) slow inhibition of glycolysis, (iii) induction of mitochondrial permeability transition, and/or (iv) activation of poly-ADP-ribose polymerase. Alternatively, if energy levels are maintained, NO can induce apoptosis, via oxidant activation of: p53, p38 MAPK pathway or endoplasmic reticulum stress. Low levels of NO can block cell death via cGMP-mediated: vasodilation, Akt activation or block of mitochondrial permeability transition. High NO may protect by killing pathogens, activating NF-κB or S-nitro(sy)lation of caspases and the NMDA receptor. GAPDH, Drp1, mitochondrial complex I, matrix metalloprotease-9, Parkin, XIAP and protein-disulphide isomerase can also be S-nitro(sy)lated, but the contribution of these reactions to neurodegeneration remains unclear. Neurons are sensitive to NO-induced excitotoxicity because NO rapidly induces both depolarization and glutamate release, which together activate the NMDA receptor. nNOS activation (as a result of NMDA receptor activation) may contribute to excitotoxicity, probably via peroxynitrite activation of poly-ADP-ribose polymerase and/or mitochondrial permeability transition. iNOS is induced in glia by inflammation, and may protect; however, if there is also hypoxia or the NADPH oxidase is active, it can induce neuronal death. Microglial phagocytosis may contribute actively to neuronal loss.     

Nitric oxide produced by cytochrome c oxidase helps stabilize HIF-1α in hypoxic mammalian cells

The mitochondrial respiratory chain has been reported to play a role in the stabilization of HIF-1α when mammalian cells experience hypoxia, most likely through the generation of free radicals. Although previous studies have suggested the involvement of superoxide catalyzed by complex III more recent studies raise the possibility that nitric oxide (NO) catalyzed by cytochrome c oxidase (Cco/NO), which functions in hypoxic signaling in yeast, may also be involved. Herein, we have found that HEK293 cells, which do not express a NOS isoform, possess Cco/NO activity and that this activity is responsible for an increase in intracellular NO levels when these cells are exposed to hypoxia. By using PTIO, a NO scavenger, we have also found that the increased NO levels in hypoxic HEK293 cells help stabilize HIF-1α. These findings suggest a new mechanism for mitochondrial involvement in hypoxic signaling in mammalian cells.     

Reduced L‐arginine transport contributes to the pathogenesis of myocardial ischemia‐reperfusion injury

Myocardial injury due to ischemia‐reperfusion (I‐R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I‐R process induces a state of insufficient L ‐arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I‐R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia‐reoxygenation significantly decreased L ‐arginine uptake and NO production (42 ± 2% and 71 ± 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L ‐arginine transporter, CAT1, and NVCMs supplemented with L ‐arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia‐reoxygenation. In contrast, L ‐arginine deprived NVCM had significantly worsened responses to hypoxia‐reoxygenation. In isolated perfused mouse hearts, L ‐arginine infusion during reperfusion significantly improved left ventricular function after I‐R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L ‐arginine availability as a key factor in the pathogenesis of I‐R injury. Increasing L ‐arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I‐R. Restoration of L ‐arginine availability may therefore be a valuable strategy to ameliorate I‐R injury. J. Cell. Biochem. 108: 156–168, 2009. © 2009 Wiley‐Liss, Inc.