Nitration of solvent-exposed tyrosine 74 on cytochrome c triggers heme iron-methionine 80 bond disruption. Nuclear magnetic resonance and optical spectroscopy studies

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.     

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.     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.     

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.     

Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.     

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.     

Role of calcium and superoxide dismutase in sensitizing mitochondria to peroxynitrite-induced permeability transition

The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca(2+) and oxidants such as peroxynitrite (ONOO(-)). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca(2+) in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca(2+) was a critical factor in determining the impact of ONOO(-) on PTP. Specifically, addition of Ca(2+) alone, or ONOO(-) and then Ca(2+), elicited similar low levels of PTP opening, whereas ONOO(-) alone was ineffective. In contrast, addition of Ca(2+) and then ONOO(-) induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca(2+) reveals a molecular target that is now reactive with ONOO(-). As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO(-) alone or to Ca(2+) and then ONOO(-), and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca(2+) include dehydrogenases and CoA - containing enzymes. These data are discussed in the context of the role of mitochondria, Ca(2+), and ONOO(-) in apoptotic signaling.     

Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.     

The impact of metal catalysis on protein tyrosine nitration by peroxynitrite

In a series of heme and non-heme proteins the nitration of tyrosine residues was assessed by complete pronase digestion and subsequent HPLC-based separation of 3-nitrotyrosine. Bolus addition of peroxynitrite caused comparable nitration levels in all tested proteins. Nitration mainly depended on the total amount of tyrosine residues as well as on surface exposition. In contrast, when superoxide and nitrogen monoxide (NO) were generated at equal rates to yield low steady-state concentrations of peroxynitrite, metal catalysis seemed to play a dominant role in determining the sensitivity and selectivity of peroxynitrite-mediated tyrosine nitration in proteins. Especially, the heme-thiolate containing proteins cytochrome P450(BM-3) (wild type and F87Y variant) and prostacyclin synthase were nitrated with high efficacy. Nitration by co-generated NO/O(2)(-) was inhibited in the presence of superoxide dismutase. The NO source alone only yielded background nitration levels. Upon changing the NO/O(2)(-) ratio to an excess of NO, a decrease in nitration in agreement with trapping of peroxynitrite and derived radicals by NO was observed. These results clearly identify peroxynitrite as the nitrating species even at low steady-state concentrations and demonstrate that metal catalysis plays an important role in nitration of protein-bound tyrosine.     

Nitration of annexin II tetramer.

Annexin II tetramer (AII(t)) is a member of the Ca(2+)- and phospholipid-binding protein family and is implicated in membrane fusion during surfactant secretion. It had previously been shown that high concentrations of nitric oxide (NO) inhibit surfactant secretion from lung type II cells. NO reacts with superoxide (O(2)(-)) to form peroxynitrite (ONOO(-)), a tyrosine nitrating agent, which is found in lungs under certain pathological conditions. It is therefore hypothesized that nitration of AII(t) by ONOO(-) may be a mechanism for the NO inhibition of regulated exocytosis. We therefore performed in vitro studies to test effects of ONOO(-) on AII(t). Western blot analysis using anti-nitrotyrosine antibodies showed a dose-dependent nitration of tyrosine residues in AII(t) treated with ONOO(-). Nitration occurred on the core domain of the p36 subunit, as well as on the p11 subunit. ONOO(-) also caused the formation of dimers between p36 and p11 subunits which were stable in the presence of heating, SDS, and beta-mercaptoethanol. AII(t)-mediated liposome aggregation was inhibited by ONOO(-) with an IC(50) of approximately 30 microM. The inhibition was abolished by urate (a scavenger of ONOO(-) and *OH), but not by mannitol (a scavenger of *OH) or superoxide dismutase (a scavenger of O(2)(-)) and appeared to be specific to AII(t), since ONOO(-) only slightly influenced annexin I-mediated liposome aggregation. The conformational change of AII(t) induced by Ca(2+) had no effect on the inhibition. Furthermore, ONOO(-) only partially inhibited the binding of AII(t) to membranes. Nitration of AII(t) also occurred in intact A549 cells, a lung epithelial cell line, treated with ONOO(-). The results of this study suggest that AII(t)-mediated liposome aggregation was inhibited by nitration of the protein.     

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H(2) synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO(-)) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO(-) exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO(-), heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO(-). Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO(-) only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO(-) and the demise of PGHS-1.     

Biochemical properties of cytochrome c nitrated by peroxynitrite

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.     

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.     

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging

Excess superoxide (O(2)(-)) and nitric oxide (NO) forms peroxynitrite (ONOO(-)) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO(-). Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O(2)(-)/ONOO(-) during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC-MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO(-). We also found that ONOO(-) directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO(-) formed oligomers. Resveratrol (RES), a scavenger of O(2)(-)/ONOO(-), reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O(2)(-) and ONOO(-) levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO(-) during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.     

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.     

Chemical modification of human alpha 1-proteinase inhibitor by tetranitromethane. Structure-function relationship.

Nitration of tyrosine residues of alpha 1-proteinase inhibitor (alpha 1-PI) by tetranitromethane yielded a product that maintained its inhibitory activity against trypsin but lost most of its inhibitory activity against elastase. Chemical analysis of the product showed that four out of the six tyrosine residues in alpha 1-PI had been nitrated to various degrees: Tyr-38 and Tyr-297 were not nitrated, whereas Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were nitrated to extents in the range 40-80%. We interpreted these data to mean that modification of these tyrosine residues decreased the association constant between alpha 1-PI and the proteinases and that the decrease differs from one proteinase to the other. When either alpha 1-PI-trypsin or alpha 1-PI-elastase complex was nitrated, nitration took place only to a very slight extent at these latter four tyrosine residues. On the other hand, Tyr-38 and Tyr-297 underwent nitration to about 20%. We concluded that Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were located on the surface of alpha 1-PI that interacts with either trypsin or elastase in the formation of complexes, and were therefore protected from nitration.     

Mechanism for the peroxynitrite scavenging activity by anthocyanins

We show that anthocyanins can function as potent inhibitors of the formation of nitrated tyrosine in vitro, and clarified how pelargonidin (Pel), which has a mono-hydroxyl group on the B-ring, can scavenge peroxynitrite (ONOO(-)) by detection of the reaction products. Pel was reacted with ONOO(-), then the reaction mixture was analyzed using high-performance liquid chromatography (HPLC). The HPLC analyses showed two novel peaks assumed to be the reaction products. Based on the instrumental analyses, the reaction products were identified as p-hydroxybenzoic acid and 4-hydroxy-3-nitrobenzoic acid. Pel can protect tyrosine from undergoing nitration through the formation of p-hydroxybenzoic acid and 4-hydroxy-3-nitrobenzoic acid.     

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.     

Mitochondrial nitric oxide synthase, oxidative stress and apoptosis

Nitric oxide (NO) exerts a wide range of its biological properties via its interaction with mitochondria. By competing with O(2), physiologically relevant concentrations of NO reversibly inhibit cytochrome oxidase and decrease O(2) consumption, in a manner resembling a pharmacological competitive antagonism. The inhibition regulates many cellular functions, by e.g., regulating the synthesis of ATP and the formation of mitochondrial transmembrane potential (Delta Psi). NO regulates the oxygen consumption of both the NO-producing and the neighboring cells; thus, it can serve as autoregulator and paracrine modulator of the respiration. On the other hand, NO reacts avidly with superoxide anion (O(2)(-)) to produce the powerful oxidizing agent, peroxynitrite (ONOO(-)) which affects mitochondrial functions mostly in an irreversible manner. How mitochondria and cells harmonize the reversible effects of NO versus the irreversible effects of ONOO(-) will be discussed in this review article. The exciting recent finding of mitochondrial NO synthase will also be discussed.     

Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death.

Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) together with hydrogen peroxide (H(2)O(2)) caused rapid breakdown of nitric oxide (NO) and production of peroxynitrite (ONOO(-)) indicated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. The breakdown of NO by this reaction was inhibited by cyanide (CN(-)) or by diethyldithiocarbamate (DETC), both Cu/Zn-SOD inhibitors, and the conversion of DHR to rhodamine-1,2,3 was inhibited by incubating Cu/Zn-SOD with either CN(-) or with high levels of H(2)O(2) or by including urate, a potent scavenger of ONOO(-). In the presence of phenol, the reaction of SOD, H(2)O(2) and NO caused nitration of phenol, which is known to be a footprint of ONOO(-) formation. H(2)O(2) addition to macrophages (cell line J774) expressing the inducible form of NO synthase (i-NOS) caused rapid breakdown of the NO they produced and this was also inhibited by CN(-) and by DETC. Subsequent ONOO(-) production by the macrophages, via this reaction, was inhibited by CN(-), high levels of H(2)O(2) or by urate. H(2)O(2) addition to i-NOS macrophages also caused cell death which was, in part, prevented by DETC or urate. We also found inhibition of mitochondrial respiration with malate and pyruvate as substrates, when isolated liver mitochondria were incubated with Cu/Zn-SOD, H(2)O(2) and NO. Inhibition of mitochondrial respiration was partly prevented by urate. The production of ONOO(-) by SOD may be of significant importance pathologically under conditions of elevated H(2)O(2) and NO levels, and might contribute to cell death in inflammatory and neurodegenerative diseases, as well as in macrophage-mediated host defence.