Formation of 3-nitrotyrosine by riboflavin photosensitized oxidation of tyrosine in the presence of nitrite

The results of the present investigation show the susceptibility of tyrosine to undergo visible light-induced photomodification to 3-nitrotyrosine in the presence of nitrite and riboflavin, as sensitizer. By changing H₂O by D₂O, it could be established that singlet oxygen has a minor role in the reaction. The finding that nitration of tyrosine still occurred to a large extent under anaerobic conditions indicates that the process proceeds mainly through a type I mechanism, which involves the direct interaction of the excited state of riboflavin with tyrosine and nitrite to give tyrosyl radical and nitrogen dioxide radical, respectively. The tyrosyl radicals can either dimerize to yield 3,3′-dityrosine or combine with nitrogen dioxide radical to form 3-nitrotyrosine. The formation of 3-nitrotyrosine was found to increase with the concentration of nitrite added and was accompanied by a decrease in the recovery of 3,3′-dityrosine, suggesting that tyrosine nitration competes with dimerization reaction. The riboflavin photosensitizing reaction in the presence of nitrite was also able to induce nitration of tyrosine residues in proteins as revealed by the spectral changes at 430 nm, a characteristic absorbance of 3-nitrotyrosine, and by immunoreactivity using 3-nitrotyrosine antibodies. Since riboflavin and nitrite are both present endogenously in living organism, it is suggested that this pathway of tyrosine nitration may potentially occur in tissues and organs exposed to sunlight such as skin and eye.     

Tempol diverts peroxynitrite/carbon dioxide reactivity toward albumin and cells from protein-tyrosine nitration to protein-cysteine nitrosation

Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10-100 microM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5-0.7 cys/mol) and reductively denatured BSA (7-19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein-tyrosine nitration (70-90%) and protein-cysteine oxidation (20-50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein-cysteine nitrosation (200-400%). Tempol consumption was attributed mainly to its reaction with protein-cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA-cysteinyl and BSA-tyrosyl/tryptophanyl radicals, as well their decay products such as protein-3-nitrotyrosine. The parallel increase in protein-nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein-nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein-cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation.     

Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution.

It has been reported that peroxynitrite will initiate both oxidation and nitration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydrophobic tyrosyl probe located in a lipid bilayer was significantly greater than its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyrosyl probe was greater than that of tyrosine in aqueous solution. Evidence for hydroxyl radical formation from decomposition of peroxynitrite in a dimethylformamide/water mixture was obtained by electron spin resonance spin trapping. Mechanisms for nitration of the tyrosyl probe in the membrane are discussed. We conclude that nitration but not oxidation of a tyrosyl probe by peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing its propensity to undergo nitration in the presence of peroxynitrite. This work provides a new perspective on selective nitration of membrane-incorporated tyrosine analogues.     

Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite. Implications for tyrosine modification by nitric oxide/superoxide in vivo

Formation of peroxynitrite from NO and O-(*2) is considered an important trigger for cellular tyrosine nitration under pathophysiological conditions. However, this view has been questioned by a recent report indicating that NO and O-(*2) generated simultaneously from (Z)-1-(N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino) diazen-1-ium-1,2-diolate] (SPER/NO) and hypoxanthine/xanthine oxidase, respectively, exhibit much lower nitrating efficiency than authentic peroxynitrite (Pfeiffer, S. and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285). The present study extends those earlier findings to several alternative NO/O-(*2)-generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/O-(*2) is due to a pronounced decrease of nitration efficiency at low steady-state concentrations of authentic peroxynitrite. The decrease in the yields of 3-nitrotyrosine was accompanied by an increase in the recovery of dityrosine, showing that dimerization of tyrosine radicals outcompetes the nitration reaction at low peroxynitrite concentrations. The observed inverse dependence on peroxynitrite concentration of dityrosine formation and tyrosine nitration is predicted by a kinetic model assuming that radical formation by peroxynitrous acid homolysis results in the generation of tyrosyl radicals that either dimerize to yield dityrosine or combine with (*)NO(2) radical to form 3-nitrotyrosine. The present results demonstrate that very high fluxes (>2 microM/s) of NO/O-(*2) are required to render peroxynitrite an efficient trigger of tyrosine nitration and that dityrosine is a major product of tyrosine modification caused by low steady-state concentrations of peroxynitrite.     

Tyrosine nitration by simultaneous generation of (.)NO and O-(2) under physiological conditions. How the radicals do the job

Radiation chemical experiments demonstrate that the reaction of tyrosyl radical (TyrO(.)) with (.)NO(2) yields 45 +/- 3% 3-nitrotyrosine and that a major product of the reaction of TyrO(.) with (.)NO is 3,3'-dityrosine. Radiolysis was used to generate (.)NO and O-(2) in the presence of tyrosine and bicarbonate at pH 7.5 +/- 0.1. The nitration yield was found to be dose rate-dependent, and the yield per radical produced by pulse radiolysis was identical to that obtained with authentic peroxynitrite. The proposed mechanism that accounts for the data is as follows: (i) In the presence of CO(2) the reaction of (.)NO with O-(2) yields 33% (.)NO(2) and CO-(3), where the latter reacts rapidly with tyrosine to form TyrO(.); (ii) The formation of 3-nitrotyrosine takes place via the reaction of (.)NO(2) with TyrO(.), which is the main process at high dose rates; and (iii) Under continuous generation of (.)NO and O-(2), the formation of 3-nitrotyrosine is strongly suppressed because of efficient scavenging of (.)NO(2) by tyrosine. The proposed model shows that the highest nitration yield is obtained for similar fluxes of (.)NO and O-(2) and is completely inhibited upon excess production of O-(2) because of efficient scavenging of TyrO(.) by O-(2). The biological implications of these findings are discussed.     

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.     

Formation of nitrotyrosine by methylene blue photosensitized oxidation of tyrosine in the presence of nitrite.

Methylene blue photosensitized oxidation of tyrosine in the presence of nitrite produces 3-nitrotyrosine, with maximum yield at pH 6. The formation of 3-nitrotyrosine requires oxygen and increases using deuterium oxide as solvent, suggesting the involvement of singlet oxygen in the reaction. The detection of dityrosine as an additional reaction product suggests that the first step in the interaction of tyrosine with singlet oxygen generates tyrosyl radicals which can dimerize to form dityrosine or react with a nitrite-derived species to produce 3-nitrotyrosine. Although the chemical identity of the nitrating species has not been established, the possible generation of nitrogen dioxide (*NO(2)) by indirect oxidation of nitrite by intermediately produced tyrosyl radical, via electron transfer, is proposed. One important implication of the results of this study is that the oxidation of tyrosine by singlet oxygen in the presence of nitrite may represent an alternative or additional pathway of 3-nitrotyrosine formation of potential importance in oxidative injures such as during inflammatory processes.     

Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation

Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mM), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low.     

Protein tyrosine nitration in hydrophilic and hydrophobic environments

Summary. In this review we address current concepts on the biological occurrence, levels and consequences of protein tyrosine nitration in biological systems. We focused on mechanistic aspects, emphasizing on the free radical mechanisms of protein 3-nitrotyrosine formation and critically analyzed the restrictions for obtaining large tyrosine nitration yields in vivo, mainly due to the presence of strong reducing systems (e.g. glutathione) that can potently inhibit at different levels the nitration process. Evidence is provided to show that the existence of metal-catalyzed processes, the assistance of nitric oxide-dependent nitration steps and the facilitation by hydrophobic environments, provide individually and/or in combination, feasible scenarios for nitration in complex biological milieux. Recent studies using hydrophobic tyrosine analogs and tyrosine-containing peptides have revealed that factors controlling nitration in hydrophobic environments such as biomembranes and lipoproteins can differ to those in aqueous compartments. In particular, exclusion of key soluble reductants from the lipid phase will more easily allow nitration and lipid-derived radicals are suggested as important mediators of the one-electron oxidation of tyrosine to tyrosyl radical in proteins associated to hydrophobic environments. Development and testing of hydrophilic and hydrophobic probes that can compete with endogenous constituents for the nitrating intermediates provide tools to unravel nitration mechanisms in vitro and in vivo; additionally, they could also serve to play cellular and tissue protective functions against the toxic effects of protein tyrosine nitration.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: modeling the impact of superoxide dismutase and nitric oxide diffusion

Tyrosine nitration is a posttranslational modification observed in many pathologic states that can be associated with peroxynitrite (ONOO(-)) formation. However, in vitro, peroxynitrite-dependent tyrosine nitration is inhibited when its precursors, superoxide (O(2)*(-)) and nitric oxide ((*)NO), are formed at ratios (O(2)*(-)/(*)NO) different from one, severely questioning the use of 3-nitrotyrosine as a biomarker of peroxynitrite-mediated oxidations. We herein hypothesize that in biological systems the presence of superoxide dismutase (SOD) and the facile transmembrane diffusion of (*)NO preclude accumulation of O(2)*(-) and (*)NO radicals under flux ratios different from one, preventing the secondary reactions that result in the inhibition of 3-nitrotyrosine formation. Using an array of reactions and kinetic constants, computer-assisted simulations were performed in order to assess the flux of 3-nitrotyrosine formation (J(NO(2(-))Y)) during exposure to simultaneous fluxes of superoxide (J(O(2)*(-))) and nitric oxide (J((*)NO)), varying the radical flux ratios (J(O(2)*(-))/ J((*)NO)), in the presence of carbon dioxide. With a basic set of reactions, J(NO(2(-))Y) as a function of radical flux ratios rendered a bell-shape profile, in complete agreement with previous reports. However, when superoxide dismutation by SOD and (*)NO decay due to diffusion out of the compartment were incorporated in the model, a quite different profile of J(NO(2(-))Y) as a function of the radical flux ratio was obtained: despite the fact that nitration yields were much lower, the bell-shape profile was lost and the extent of tyrosine nitration was responsive to increases in either O(2)*(-) or (*)NO, in agreement with in vivo observations. Thus, the model presented herein serves to reconcile the in vitro and in vivo evidence on the role of peroxynitrite in promoting tyrosine nitration.     

Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite.

Superoxide dismutase and Fe3+EDTA catalyzed the nitration by peroxynitrite (ONOO-) of a wide range of phenolics including tyrosine in proteins. Nitration was not mediated by a free radical mechanism because hydroxyl radical scavengers did not reduce either superoxide dismutase or Fe3+EDTA-catalyzed nitration and nitrogen dioxide was not a significant product from either catalyst. Rather, metal ions appear to catalyze the heterolytic cleavage of peroxynitrite to form a nitronium-like species (NO2+). The calculated energy for separating peroxynitrous acid into hydroxide ion and nitronium ion is 13 kcal.mol-1 at pH 7.0. Fe3+EDTA catalyzed nitration with an activation energy of 12 kcal.mol-1 at a rate of 5700 M-1.s-1 at 37 degrees C and pH 7.5. The reaction rate of peroxynitrite with bovine Cu,Zn superoxide dismutase was 10(5) M-1.s-1 at low superoxide dismutase concentrations, but the rate of nitration became independent of superoxide dismutase concentration above 10 microM with only 9% of added peroxynitrite yielding nitrophenol. We propose that peroxynitrite anion is more stable in the cis conformation, whereas only a higher energy species in the trans conformation can fit in the active site of Cu,Zn superoxide dismutase. At high superoxide dismutase concentrations, phenolic nitration may be limited by the rate of isomerization from the cis to trans conformations of peroxynitrite as well as by competing pathways for peroxynitrite decomposition. In contrast, Fe3+EDTA appears to react directly with the cis anion, resulting in greater nitration yields.     

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

Scavenging of peroxynitrite by oxyhemoglobin and identification of modified globin residues

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with (*)NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species (*)NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.     

Mechanistic studies of peroxynitrite-mediated tyrosine nitration in membranes using the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester

Most of the mechanistic studies of tyrosine nitration have been performed in aqueous solution. However, many protein tyrosine residues shown to be nitrated in vitro and in vivo are associated to nonpolar compartments. In this work, we have used the stable hydrophobic tyrosine analogue N-t-BOC-L-tyrosine tert-butyl ester (BTBE) incorporated into phosphatidylcholine (PC) liposomes to study physicochemical and biochemical factors that control peroxynitrite-dependent tyrosine nitration in phospholipid bilayers. Peroxynitrite leads to maximum 3-nitro-BTBE yields (3%) at pH 7.4. In addition, small amounts of 3,3'-di-BTBE were formed at pH 7.4 (0.02%) which increased over alkaline pH; at pH 6, a hydroxylated derivative of BTBE was identified by HPLC-MS analysis. BTBE nitration yields were similar in dilauroyl- and dimyristoyl-PC and were also significant in the polyunsaturated fatty acid-containing egg PC. *OH and *NO2 scavengers inhibited BTBE nitration. In contrast to tyrosine in the aqueous phase, the presence of CO2 decreased BTBE nitration, indicating that CO3*- cannot permeate to the compartment where BTBE is located. On the other hand, micromolar concentrations of hemin and Mn-tccp strongly enhanced BTBE nitration. Electron spin resonance (ESR) detection of the BTBE phenoxyl radical and kinetic modeling of the pH profiles of BTBE nitration and dimerization were in full agreement with a free radical mechanism of oxidation initiated by ONOOH homolysis in the immediacy of or even inside the bilayer and with a diffusion coefficient of BTBE phenoxyl radical 100 times less than for the aqueous phase tyrosyl radical. BTBE was successfully applied as a hydrophobic probe to study nitration mechanisms and will serve to study factors controlling protein and lipid nitration in biomembranes and lipoproteins.     

Mass spectrometric quantification of 3-nitrotyrosine, ortho-tyrosine, and o,o'-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson's disease

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o, o'-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o'-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies.     

Myoglobin-catalyzed tyrosine nitration: no need for peroxynitrite.

The nitration of tyrosine residues in protein to yield 3-nitrotyrosine derivatives has been suggested to represent a specific footprint for peroxynitrite formation in vivo. However, recent studies suggest that certain hemoproteins such as peroxidases catalyze the H(2)O(2)-dependent nitration of tyrosine to yield 3-nitrotyrosine in a peroxynitrite-independent reaction. Because 3-nitrotyrosine has been shown to be present in the postischemic myocardium, we wished to assess the ability of myoglobin to catalyze the nitration of tyrosine in vitro. We found that myoglobin catalyzed the oxidation of nitrite and promoted the nitration of tyrosine. Both nitrite oxidation and tyrosine nitration were H(2)O(2)-dependent and required the formation of ferryl (Fe(+4)) myoglobin. In addition, nitrite oxidation and tyrosine nitration were pH-dependent with a pH optimum of approximately 6.0. Taken together, these data suggest that the acidic pH and low oxygen tension produced during myocardial ischemia will facilitate myoglobin-catalyzed, peroxyntrite-independent formation of 3-nitrotyrosine.     

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization,characterization, mechanism of nitration,and biological implications

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.     

Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO(-)) produced from the reaction of nitric oxide with superoxide. Pfeiffer and Mayer (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) reported that superoxide produced from hypoxanthine plus xanthine oxidase in combination with nitric oxide produced from spermine NONOate did not nitrate tyrosine at neutral pH. They suggested that nitric oxide and superoxide at neutral pH form a less reactive intermediate distinct from preformed alkaline peroxynitrite that does not nitrate tyrosine. Using a stopped-flow spectrophotometer to rapidly mix potassium superoxide with nitric oxide at pH 7.4, we report that an intermediate spectrally and kinetically identical to preformed alkaline cis-peroxynitrite was formed in 100% yield. Furthermore, this intermediate nitrated tyrosine in the same yield and at the same rate as preformed peroxynitrite. Equivalent concentrations of nitric oxide under aerobic conditions in the absence of superoxide did not produce detectable concentrations of nitrotyrosine. Carbon dioxide increased the efficiency of nitration by nitric oxide plus superoxide to the same extent as peroxynitrite. In experiments using xanthine oxidase as a source of superoxide, tyrosine nitration was substantially inhibited by urate formed from hypoxanthine oxidation, which was sufficient to account for the lack of tyrosine nitration previously reported. We conclude that peroxynitrite formed from the reaction of nitric oxide with superoxide at physiological pH remains an important species responsible for tyrosine nitration in vivo.