Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.     

Peroxynitrite oxidation of tubulin sulfhydryls inhibits microtubule polymerization

Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol and beta-mercaptoethanol restored a significant portion of the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-induced disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization.     

Dopamine prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide: is nitrotyrosine formation an early step in dopamine neuronal damage?

Peroxynitrite and nitrogen dioxide (NO2) are reactive nitrogen species that have been implicated as causal factors in neurodegenerative conditions. Peroxynitrite-induced nitration of tyrosine residues in tyrosine hydroxylase (TH) may even be one of the earliest biochemical events associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced damage to dopamine neurons. Exposure of TH to peroxynitrite or NO2 results in nitration of tyrosine residues and modification of cysteines in the enzyme as well as inactivation of catalytic activity. Dopamine (DA), its precursor 3,4-dihydroxyphenylalanine, and metabolite 3,4-dihydroxyphenylacetic acid completely block the nitrating effects of peroxynitrite and NO2 on TH but do not relieve the enzyme from inhibition. o-Quinones formed in the reaction of catechols with either peroxynitrite or NO2 react with cysteine residues in TH and inhibit catalytic function. Using direct, real-time evaluation of tyrosine nitration with a green fluorescent protein-TH fusion protein stably expressed in intact cells (also stably expressing the human DA transporter), DA was also found to prevent NO2-induced nitration while leaving TH activity inhibited. These results show that peroxynitrite and NO2 react with DA to form quinones at the expense of tyrosine nitration. Endogenous DA may therefore play an important role in determining how DA neurons are affected by reactive nitrogen species by shifting the balance of their effects away from tyrosine nitration and toward o-quinone formation.     

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.     

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.     

Nitration of a critical tyrosine residue in the allosteric inhibitor site of muscle glycogen phosphorylase impairs its catalytic activity

Muscle glycogen phosphorylase (GP) is a key enzyme in glucose metabolism, and its impairment can lead to muscle dysfunction. Tyrosine nitration of glycogen phosphorylase occurs during aging and has been suggested to be involved in progressive loss of muscle performance. Here, we show that GP (in its T and R form) is irreversibly impaired by exposure to peroxynitrite, a biological nitrogen species known to nitrate reactive tyrosine residues, and to be involved in physiological and pathological processes. Kinetic and biochemical analysis indicated that irreversible inactivation of GP by peroxynitrite is due to the fast (k(inact)=3 x 10(4) M(-1) s(-1)) nitration of a unique tyrosine residue of the enzyme. Endogenous GP was tyrosine nitrated and irreversibly inactivated in skeletal muscle cells upon exposure to peroxynitrite, with concomitant impairment of glycogen mobilization. Ligand protection assays and mass spectrometry analysis using purified GP suggested that the peroxynitrite-dependent inactivation of the enzyme could be due to the nitration of Tyr613, a key amino acid of the allosteric inhibitor site of the enzyme. Our findings suggest that GP functions may be regulated by tyrosine nitration.     

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.     

Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite.

Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.     

Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences

Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.     

Nitration Transforms a Sensitive Peroxiredoxin 2 into a More Active and Robust Peroxidase

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H₂O₂-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H₂O₂ accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.     

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: Does the nitration of Tyr in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function [Sharov et al., Exp. Gerontol. 41 (2006) 407-416]; in another report, the nitration of one specific residue, Tyr⁶¹³, located in the allosteric inhibition site was hypothesized as a rationale for peroxynitrite inactivation [Dairou et al., J. Mol. Biol. 372 (2007) 1009-1021]. In this study, we have optimized the analysis of in-gel Ph-b digests by high performance liquid chromatography-electro spray ionization-tandem mass spectrometry, in order to achieve a quantitative analysis of nitration of individual Tyr residues at a high coverage of Tyr-containing sequences (92%). Our data do not confirm the role of Tyr⁶¹³ nitration in the control of enzymatic function. Furthermore, we show here that the enzymatic activity of Ph-b does not directly correlate with the protein nitration levels, and that the modification of Cys and, potentially, other amino acid residues can better rationalize Ph-b inactivation by peroxynitrite.     

Hemin-H2O2-NO2(-) induced protein oxidation and tyrosine nitration are different from those of SIN-1: a study on glutamate dehydrogenase nitrative/oxidative modification

Protein oxidation and tyrosine nitration are two major post-translational modifications of protein by reactive nitrogen oxide species, which are mainly produced by peroxynitrite and heme peroxidases (hemin)-H(2)O(2)-NO(2)(-) system. We report herein some novel phenomena between hemin-H(2)O(2)-NO(2)(-) and 3-morpholinosydnonimine hydrochloride (SIN-1)-mediated oxidation and nitration reactions of glutamate dehydrogenase (GDH). Hemin-H(2)O(2) could effectively induce GDH protein oxidation and reduce its activity. Although the addition of low concentration of nitrite promoted protein oxidation, protein oxidation was weakened with the increase of nitrite concentration, meanwhile, tyrosine nitration was increased and the enzyme activity was partially restored. However, with the increase of SIN-1 concentration, protein oxidation and tyrosine nitration were increased, enzyme activity was decreased. The presence of desferrioxamine and/or catechin inhibit tyrosine nitration both in hemin-H(2)O(2)-NO(2)(-) and in SIN-1, but they promoted protein oxidation and reduced the enzyme activity in hemin-H(2)O(2)-NO(2)(-) system, while inhibited protein oxidation and recover the enzyme activity in SIN-1 system. These results reveal both hemin-H(2)O(2)-NO(2)(-) and SIN-1 can cause inactivation of GDH through protein oxidation and tyrosine nitration, but the impact of the effect of protein oxidation (not thiol oxidation) on enzyme activity is stronger than that of protein tyrosine nitration. Moreover, mass spectrometric analysis indicated that nitrated tyrosine residues by hemin-H(2)O(2)-NO(2)(-) were Tyr262 and Tyr471 while by SIN-1 were Tyr401 and Tyr493. It meant that protein oxidation and tyrosine nitration of GDH induced by hemin-H(2)O(2)-NO(2)(-) were different from those induced by SIN-1.     

Inactivation of glutathione S-transferases by nitric oxide-derived oxidants: exploring a role for tyrosine nitration

Reactive intermediates derived from nitric oxide ((*)NO) are thought to play a contributing role in disease states associated with inflammation and infection. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxification of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen species (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloperoxidase/hydrogen peroxide/nitrite system, or tetranitromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-associated 3-nitrotyrosine (NO(2)Tyr). This inactivation was only partially (<25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide or S-nitrosoglutathione was only partially inhibitory (<25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have contributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site tyrosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly investigate this possibility, we analyzed purified mouse liver GST-mu, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-assisted laser desorption/ionization-time of flight analysis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contribute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollutants in association with inflammation.     

The effect of neighboring methionine residue on tyrosine nitration and oxidation in peptides treated with MPO, H2O2, and NO2 or peroxynitrite and bicarbonate: Role of intramolecular electron transfer mechanism?

Recent reports suggest that intramolecular electron transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H₂O₂ and NO₂⁻ and peroxynitrite (ONOO⁻) or ONOO⁻ and bicarbonate (HCO₃⁻) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analyzed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. 280 (2005) 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO⁻ (or ONOOCO₂⁻) or MPO/H₂O₂/NO₂⁻ system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO⁻ (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer in these peptides. Further support for the interpretation was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed.     

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.     

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of 相似文献