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Lack of Tyrosine Nitration by Peroxynitrite Generated at Physiological pH

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27280

ISSN

1083-351X

Autores

Silvia Pfeiffer, Bernd Mayer,

Tópico(s)

Electron Spin Resonance Studies

Resumo

Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of ⋅NO and O⨪2 at physiological pH. In the present study, we measured the nitration of free tyrosine by ⋅NO and O⨪2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]-diazen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mm) to tyrosine (1 mm) at pH 7.4 yielded 36.77 ± 1.67 μm 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from ⋅NO and O⨪2, which were generated at equal rates (∼5 μm × min−1) from 1 mmspermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mm hypoxanthine was much less efficient (0.67 ± 0.01 μm; ∼0.07% of total product flow). At O⨪2fluxes exceeding the ⋅NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 μm). Nitration was most efficient (∼0.3%) with the ⋅NO donor alone, i.e.without concomitant generation of O⨪2. Nitration by ⋅NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1–2 mm), and was not enhanced by NaHCO3 (up to 20 mm), indicating that it was mediated by ⋅NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from ⋅NO and O⨪2 as a mediator of tyrosine nitration in vivo. Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of ⋅NO and O⨪2 at physiological pH. In the present study, we measured the nitration of free tyrosine by ⋅NO and O⨪2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]-diazen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mm) to tyrosine (1 mm) at pH 7.4 yielded 36.77 ± 1.67 μm 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from ⋅NO and O⨪2, which were generated at equal rates (∼5 μm × min−1) from 1 mmspermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mm hypoxanthine was much less efficient (0.67 ± 0.01 μm; ∼0.07% of total product flow). At O⨪2fluxes exceeding the ⋅NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 μm). Nitration was most efficient (∼0.3%) with the ⋅NO donor alone, i.e.without concomitant generation of O⨪2. Nitration by ⋅NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1–2 mm), and was not enhanced by NaHCO3 (up to 20 mm), indicating that it was mediated by ⋅NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from ⋅NO and O⨪2 as a mediator of tyrosine nitration in vivo. nitric oxide dihydrorhodamine 123 high performance liquid chromatography nitrosoperoxycarbonate anion superoxide anion oxyhemoglobin 3-morpholinosydnonimine (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]-diazen-1-ium-1,2-diolate] xanthine oxidase. Nitric oxide (⋅NO)1 is a cellular messenger regulating numerous biological processes, including relaxation of blood vessels and neurotransmitter release in the brain, but overproduction of ⋅NO appears to contribute essentially to tissue injury in inflammatory and ischemic conditions (1Mayer B. Hemmens B. Trends Biochem. Sci. 1997; 22: 453-498Abstract Full Text PDF PubMed Scopus (511) Google Scholar). The molecular mechanisms underlying the cytotoxicity of ⋅NO are not well understood. The potent oxidant peroxynitrite, which is formed in a rapid reaction from ⋅NO and O⨪2, is thought to be a key mediator of ⋅NO toxicity in atherosclerosis, congestive heart failure, glutamate excitotoxicity, and other disease states involving inflammatory oxidative stress (2Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 40: C1424-C1437Crossref Google Scholar). Formation of peroxynitrite from ⋅NO and O⨪2 occurs at nearly diffusion-controlled rates (4.3–6.7 × 109m−1 s−1) (3Huie R.E. Padmaja S. Free Radic. Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2015) Google Scholar, 4Goldstein S. Czapski G. Free Radical Biol. Med. 1995; 117: 12078-12084Google Scholar). Therefore,⋅NO out-competes the reaction of O⨪2 with superoxide dismutase at steady-state concentrations that are likely to occurin vivo (5Malinski T. Bailey F. Zhang Z.G. Chopp M. J. Cereb. Blood Flow Metab. 1993; 13: 355-358Crossref PubMed Scopus (571) Google Scholar). Peroxynitrite is stable at alkaline pH but has a half-life of less than 1 s at pH 7.4 (pK a = 6.8) (6Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1277) Google Scholar). Depending on the pH, the corresponding peroxynitrous acid either rearranges to NO3− or decomposes to NO2− and O2 (7Pfeiffer S. Gorren A.C.F. Schmidt K. Werner E.R. Hansert B. Bohle D.S. Mayer B. J. Biol. Chem. 1997; 272: 3465-3470Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Peroxynitrite has been shown to react with virtually all classes of biomolecules (8Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 12: L699-L722Google Scholar). The reaction with phenolic compounds, including free and protein-bound tyrosine, results in the formation of nitrated, hydroxylated, and dimeric products (9Halfpenny, E., and Robinson, P. L. (1952) J. Chem. Soc. 939–946Google Scholar, 10Beckman J.S. Ischiropoulos H. Zhu L. van der Woerd M. Smith C. Chen J. Harrison J. Martin J.C. Tsai M. Arch. Biochem. Biophys. 1992; 298: 438-445Crossref PubMed Scopus (733) Google Scholar, 11van der Vliet A. O'Neill C.A. Halliwell B. Cross C.E. Kaur H. FEBS Lett. 1994; 339: 89-92Crossref PubMed Scopus (358) Google Scholar, 12van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Crossref PubMed Scopus (375) Google Scholar). The nitration of tyrosine, yielding mainly 3-nitrotyrosine, is markedly enhanced by CO2, which reacts with peroxynitrite anion at physiological pH to form the potent nitrating species ONO2CO2− (13Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (494) Google Scholar, 14Lymar S.V. Jiang Q. Hurst J.K. Biochemistry. 1996; 35: 7855-7861Crossref PubMed Scopus (315) Google Scholar, 15Gow A. Duran D. Thom S.R. Ischiropoulos H. Arch. Biochem. Biophys. 1996; 333: 42-48Crossref PubMed Scopus (279) Google Scholar). Tyrosine nitration appears to represent a prominent pathway of pathophysiological protein modification under inflammatory conditions associated with increased expression and/or activity of ⋅NO synthases (16Xie Q.W. Nathan C. J. Leukocyte Biol. 1994; 56: 576-582Crossref PubMed Scopus (470) Google Scholar). Based on the detection of nitrotyrosine, peroxynitrite has been suggested to be involved in the pathology of a wide range of diseases, including neurodegenerative diseases (17Coyle J.T. Puttfarcken P. Science. 1993; 262: 689-695Crossref PubMed Scopus (3525) Google Scholar, 18Bagasra O. Michaels F.H. Zheng Y.M. Bobroski L.E. Spitsin S.V. Fu Z.F. Tawadros R. Koprowski H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12041-12045Crossref PubMed Scopus (434) Google Scholar), acute lung injury (19Haddad I.Y. Ischiropoulos H. Holm B.A. Beckman J.S. Baker J.R. Matalon S. Am. J. Physiol. 1993; 265: L555-L564PubMed Google Scholar), atherosclerosis (20Beckman J.S. Carson M. Smith C.D. Koppenol W.H. Nature. 1993; 364: 584Crossref PubMed Scopus (787) Google Scholar, 21Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1069) Google Scholar), bacterial and viral infections (22Mannick E.E. Bravo L.E. Zarama G. Realpe J.L. Zhang X.J. Ruiz B. Fontham E.T. Mera R. Miller M.J. Correa P. Cancer Res. 1995; 56: 3238-3243Google Scholar, 23Akaike T. Noguchi Y. Ijiri S. Setoguchi K. Suga M. Zheng Y. Dietzschold B. Maeda H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2448-2453Crossref PubMed Scopus (440) Google Scholar), and chronic inflammation (24Kaur H. Halliwell B. FEBS Lett. 1994; 350: 9-12Crossref PubMed Scopus (657) Google Scholar). Peroxynitrite-triggered tyrosine nitration has been extensively studiedin vitro by bolus addition of alkaline solutions of peroxynitrite to tyrosine-containing samples. However, this experimental approach does not reflect the in vivo situation in which peroxynitrite is thought to be formed by the rapid reaction of⋅NO with O⨪2 at physiological pH. Intriguingly, we 2S. Pfeiffer and B. Mayer, unpublished observation.2S. Pfeiffer and B. Mayer, unpublished observation. and others (12van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Crossref PubMed Scopus (375) Google Scholar) found that the nitrating potential of the sydnonimine-based nitrovasodilator SIN-1, which releases ⋅NO and O⨪2 at the same time (25Feelisch M. Ostrowski J. Noack E. J. Cardiovasc. Pharmacol. 1989; 14: 13-22Crossref PubMed Scopus (396) Google Scholar), is much lower than that of authentic peroxynitrite. However, the decomposition of SIN-1 is highly pH-dependent, its decomposition pathways may be more complicated than hitherto assumed, and co-products of SIN-1 metabolism may interfere with tyrosine nitration, making this drug an inappropriate tool to investigate the nitrating potential of ⋅NO/O⨪2. In the present study, we have addressed this issue with ⋅NO and O⨪2 generated simultaneously by a system that has been used previously to study hydroxylation and S-nitrosation reactions by⋅NO/O⨪2 (26Miles A.M. Bohle D.S. Glassbrenner P.A. Hansert B. Wink D.A. Grisham M.B. J. Biol. Chem. 1996; 271: 40-47Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 27Wink D.A. Cook J.A. Kim S.Y. Vodovotz Y. Pacelli R. Krishna M.C. Russo A. Mitchell J.B. Jourd'heuil D. Miles A.M. Grisham M.B. J. Biol. Chem. 1997; 272: 11147-11151Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Spermine NONOate was from Alexis (Läufelfingen, Switzerland). Hypoxanthine and DHR were from Fluka (Vienna, Austria). ⋅NO gas (99% pure) was from Linde (Munich, Germany). SIN-1 was a generous gift from Höchst Marion Roussel Inc. (Frankfurt, Germany). XO (from buttermilk, 1.4 units/mg) and all other chemicals were from Sigma. All solutions were prepared fresh each day. Water was ultrafiltered type I (resistance, ≥18 mΩ × cm−1) from a Barnstead NanoPure apparatus. Spermine NONOate was prepared as a 10-fold stock solution in 10 mmNaOH. SIN-1 was dissolved to 10 mm at pH 5.0. DHR was dissolved to 10 mm in acetonitrile and kept in the dark until use. ⋅NO solutions were prepared as described (28Kukovetz W.R. Holzmann S. J. Cardiovasc. Pharmacol. 1989; 14: S40-S46Crossref PubMed Google Scholar). Alkaline solutions of peroxynitrite were prepared from acidified NO2− and H2O2as described (7Pfeiffer S. Gorren A.C.F. Schmidt K. Werner E.R. Hansert B. Bohle D.S. Mayer B. J. Biol. Chem. 1997; 272: 3465-3470Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Stock solutions were diluted with H2O to 10 mm (pH ∼12.8) immediately before the experiments and added to 50 mmK2HPO4/KH2PO4 buffer, pH 7.4, to obtain final peroxynitrite concentrations of 1 mm. Changes of buffer pH were <0.1 unit. oxyHb was prepared as described (29Mayer B. Klatt P. Böhme E. Schmidt K. J. Neurochem. 1992; 59: 2024-2029Crossref PubMed Scopus (99) Google Scholar). Fluxes of ⋅NO and O⨪2 were determined photometrically using a previously described method that is based on the simultaneous scavenging of⋅NO and O⨪2 by oxyHb and cytochrome c, respectively (30Kelm M. Dahmann R. Wink D. Feelisch M. J. Biol. Chem. 1997; 272: 9922-9932Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The absorbance changes at 542 nm (oxidation of oxyHb), 465 nm (reduction of cytochrome c), and 525 nm (isosbestic point for both reactions) were monitored with a Hewlett Packard 8452A diode array spectrophotometer. Flux rates of ⋅NO and O⨪2 were calculated using extinction coefficients of 6.6 mm−1 cm−1 (542–525 nm) and 7.3 mm−1 cm−1 (465–525 nm), respectively. The signal averaging time was 0.5 s; spectra were recorded every second for 1–3 min. All measurements were performed at ambient temperature in a total volume of 0.2 ml of a 50 mmK2HPO4/KH2PO4 buffer, pH 7.4, containing 1 mm hypoxanthine, 5 μmferricytochrome c, 20 μm oxyHb in the presence of XO and spermine NONOate at the concentrations indicated. The kinetics of spermine NONOate decomposition was simulated with the software package Mathematica® (Version 2.2.2., Wolfram Research Inc., Champaign, IL) based on a half-life of 230 min at 22 °C and pH 7.4 as described (31Schmidt K. Desch W. Klatt P. Kukovetz W.R. Mayer B. Naunyn-Schmiedeberg′s Arch. Pharmacol. 1997; 355: 457-462Crossref PubMed Scopus (75) Google Scholar). Unless indicated otherwise, hypoxanthine (1 mm) and tyrosine (1 mm) were incubated at ambient temperature for 12 h in 50 mmK2HPO4/KH2PO4 buffer, pH 7.4, in the presence of various concentrations of XO and spermine NONOate or SIN-1 (final concentration, 1 mm). Authentic 3-nitrotyrosine was stable under these conditions (recovery, 97.2 ± 3.44% (n = 4) after 12 h of incubation). In some experiments, reaction mixtures were incubated for 1 h with a solution of authentic ⋅NO (final concentration, ∼1 mm) instead of the ⋅NO donor. To study tyrosine nitration by authentic peroxynitrite, alkaline stock solutions of peroxynitrite (final concentration, 1 mm) were added dropwise under vigorous vortexing to tyrosine (1 mm) in 50 mmK2HPO4/KH2PO4 buffer, pH 7.4, followed by incubation for 1 h. The combined presence of hypoxanthine (1 mm), XO (28 milliunits/ml), and spermine NONOate (1 mm) had no effect on tyrosine nitration triggered by authentic peroxynitrite. The following buffers were used to study the pH dependence of tyrosine nitration: pH 6.0–8.0, 0.2mK2HPO4/KH2PO4; pH 8.2–9.0, 0.2 m Tris/HCl; pH ≥ 10, solutions of NaOH. The effect of CO2 was studied by incubation in the presence of up to 20 mm NaHCO3. HPLC analysis of 3-nitrotyrosine was performed on a C18 reversed phase column with 50 mmKH2PO4/H3PO4 buffer (pH 3) containing 10% (v/v) methanol at 1 ml/min and detection at 274 nm as described (32Pfeiffer S. Leopold E. Hemmens B. Schmidt K. Werner E.R. Mayer B. Free Radical Biol. Med. 1997; 22: 787-794Crossref PubMed Scopus (79) Google Scholar). Calibration curves were recorded daily with authentic 3-nitrotyrosine (0.06–5 μm for experiments with ⋅NO/O⨪2 and ⋅NO, and 10–100 μm for experiments with authentic peroxynitrite). DHR oxidation was measured at 501 nm with a Shimadzu 160A spectrophotometer at ambient temperature in a total volume of 0.2 ml of a 50 mmK2HPO4/KH2PO4 buffer, pH 7.4, as described (33Kooy N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Free Radical Biol. Med. 1994; 16: 149-156Crossref PubMed Scopus (670) Google Scholar, 34Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (553) Google Scholar). The amount of oxidized DHR was calculated using an extinction coefficient of 78.78 mm−1 cm−1. With authentic peroxynitrite, the recovery of DHR oxidation was 37.4 ± 1.43% (n = 4). Spermine NONOate (1 mm) or hypoxanthine/XO (28 milliunits/ml) alone led to DHR oxidation rates of <0.06 and 109m−1s−1) (38Eiserich J.P. Butler J. Van der Vliet A. Cross C.E. Halliwell B. Biochem. J. 1995; 310: 745-749Crossref PubMed Scopus (137) Google Scholar) leads to formation of C-nitroso and/or O-nitrosotyrosine products that can be converted to 3-nitrotyrosine in a two-electron oxidation reaction (39Gunther M.R. Hsi L.C. Curtis J.F. Gierse J.K. Marnett L.J. Eling T.E. Mason R.P. J. Biol. Chem. 1997; 272: 17086-17090Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 40Goodwin D.C. Gunther M.R. Hsi L.C. Crews B.C. Eling T.E. Mason R.P. Marnett L.J. J. Biol. Chem. 1998; 273: 8903-8909Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). It is unlikely that this mechanism accounts for tyrosine nitration under our experimental conditions, i.e. in the absence of additional oxidants required for tyrosyl radical formation and oxidative rearrangement of the intermediate(s). The recovery of 3-nitrotyrosine was considerably higher when a high initial concentration of ⋅NO was applied as a bolus as compared with the continuous release of⋅NO from spermine NONOate giving low steady state concentrations of free ⋅NO. This observation hints at an essential involvement of ⋅NO autoxidation, a reaction that follows second order kinetics with respect to ⋅NO and, therefore, occurs at significant rates only at relatively high ⋅NO concentrations (41Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1994; 269: 5881-5883Abstract Full Text PDF PubMed Google Scholar). The initial product of ⋅NO autoxidation,⋅NO2, was shown to nitrate free tyrosine and tyrosine residues of proteins in aqueous solution (12van der Vliet A. Eiserich J.P. O'Neill C.A. Halliwell B. Cross C.E. Arch. Biochem. Biophys. 1995; 319: 341-349Crossref PubMed Scopus (375) Google Scholar, 42Prütz W.A. Mönig H. Butler J. Land E.J. Arch. Biochem. Biophys. 1985; 243: 125-134Crossref PubMed Scopus (327) Google Scholar, 43Kigugawa K. Kato T. Okamoto Y. Free Radical Biol. Med. 1994; 16: 373-382Crossref PubMed Scopus (78) Google Scholar). The pH dependence of tyrosine nitration caused by ⋅NO (Fig. 4) resembles that obtained by others with authentic⋅NO2 (42Prütz W.A. Mönig H. Butler J. Land E.J. Arch. Biochem. Biophys. 1985; 243: 125-134Crossref PubMed Scopus (327) Google Scholar), suggesting that the nitration of tyrosine observed under our experimental conditions was mediated by⋅NO2 formed in the course of ⋅NO autoxidation. The most interesting finding of this study was the apparent lack of significant tyrosine nitration by peroxynitrite generated from⋅NO and O⨪2 at physiological pH, even though alkaline solutions of peroxynitrite efficiently nitrated tyrosine under identical conditions. These results suggest that the peroxynitrite formed from ⋅NO and O⨪2 at physiological pH differs from the species present in alkaline solutions. The efficiency of tyrosine nitration triggered by alkaline solutions of peroxynitrite is markedly enhanced by CO2, which reacts with peroxynitrite anion to give the potent nitrating species ONO2CO2− (13Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (494) Google Scholar, 15Gow A. Duran D. Thom S.R. Ischiropoulos H. Arch. Biochem. Biophys. 1996; 333: 42-48Crossref PubMed Scopus (279) Google Scholar, 35Lemercier J.N. Padmaja S. Cueto R. Squadrito G.L. Uppu R.M. Pryor W.A. Arch. Biochem. Biophys. 1997; 345: 160-170Crossref PubMed Scopus (110) Google Scholar,44Lymar S.V. Hurst J.K. Chem. Res. Toxicol. 1996; 9: 845-850Crossref PubMed Scopus (184) Google Scholar, 45Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (505) Google Scholar, 46Uppu R.M. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1996; 327: 335-343Crossref PubMed Scopus (284) Google Scholar). We observed that NaHCO3 led to an about 2-fold increase in tyrosine nitration by alkaline peroxynitrite, a result that agrees well with previous data from other laboratories (14Lymar S.V. Jiang Q. Hurst J.K. Biochemistry. 1996; 35: 7855-7861Crossref PubMed Scopus (315) Google Scholar, 35Lemercier J.N. Padmaja S. Cueto R. Squadrito G.L. Uppu R.M. Pryor W.A. Arch. Biochem. Biophys. 1997; 345: 160-170Crossref PubMed Scopus (110) Google Scholar, 45Denicola A. Freeman B.A. Trujillo M. Radi R. Arch. Biochem. Biophys. 1996; 333: 49-58Crossref PubMed Scopus (505) Google Scholar,46Uppu R.M. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1996; 327: 335-343Crossref PubMed Scopus (284) Google Scholar). However, Berlett et al. (47Berlett B.S. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2784-2789Crossref PubMed Scopus (137) Google Scholar) have recently suggested that formation of ONO2CO2−is obligatory for peroxynitrite-mediated nitration (47Berlett B.S. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2784-2789Crossref PubMed Scopus (137) Google Scholar). The apparently CO2-independent reaction observed in the absence of added bicarbonate would accordingly be due to contaminating bicarbonate present in appreciable concentrations (0.1–0.2 mm) in buffer solutions (35Lemercier J.N. Padmaja S. Cueto R. Squadrito G.L. Uppu R.M. Pryor W.A. Arch. Biochem. Biophys. 1997; 345: 160-170Crossref PubMed Scopus (110) Google Scholar). Assuming that formation of ONO2CO2− is indeed essential for peroxynitrite-triggered tyrosine nitration as suggested by Berlett et al. (47Berlett B.S. Levine R.L. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2784-2789Crossref PubMed Scopus (137) Google Scholar), our findings suggest that the peroxynitrite species that is generated from ⋅NO and O⨪2at physiological pH does not react with CO2. This would explain both the inability of ⋅NO/O⨪2 to nitrate tyrosine and the lack of effect of bicarbonate on the observed residual nitration reaction. We can only speculate about the nature of the two postulated peroxynitrite species. The most obvious assumption is the involvement of the cis- and trans-rotamers. Interestingly, the cis- and trans-isomers exhibit clearly different pK a values of 6.8 and 8.0 (48Tsai J.H.M. Harrison J.G. Martin J.C. Hamilton T.P. Woerd van der M. Jablonsky M.J. Beckman J.S. J. Am. Chem. Soc. 1994; 116: 4115-4116Crossref Scopus (119) Google Scholar,49Crow J.P. Spruell C. Chen J. Gunn C. Ischiropoulos H. Tsai M. Smith C.D. Radi R. Koppenol W.H. Beckman J.S. Free Radical Biol. Med. 1994; 16: 331-338Crossref PubMed Scopus (200) Google Scholar), respectively, and data obtained by Raman spectroscopy indicate that peroxynitrite is present exclusively in thecis-conformation in alkaline solution (48Tsai J.H.M. Harrison J.G. Martin J.C. Hamilton T.P. Woerd van der M. Jablonsky M.J. Beckman J.S. J. Am. Chem. Soc. 1994; 116: 4115-4116Crossref Scopus (119) Google Scholar). Based on this information, we propose the scheme shown in Fig. 6 as a hypothetical explanation of our data. It is postulated that trans-peroxynitrite is the initial product of the reaction between ⋅NO and O⨪2 at pH 7.4. Because CO2 reacts with peroxynitrite anion but not with peroxynitrous acid (13Lymar S.V. Hurst J.K. J. Am. Chem. Soc. 1995; 117: 8867-8868Crossref Scopus (494) Google Scholar), protonation of trans-peroxynitrite at pH 7.4 (pK a = 8.0) is expected to prevent formation of the nitrating ONO2CO2− adduct. In contrast, ∼80% of the cis-isomer (pK a= 6.8) exist as anion at physiological pH allowing for the reaction with CO2 and consequent tyrosine nitration. What are the alternative possibilities to explain our observations? Peroxynitrite in statu nascendi might represent a novel as yet unrecognized form of this molecule, but there are no indications that such a species exists. Alternatively, peroxynitrite generated from⋅NO and O⨪2 at physiological pH could react with CO2 to yield an unreactive form of ONO2CO2−. Formation of unreactive ONO2CO2−, kinetically distinguishable from the reactive form, has been reported (50Lymar S.V. Hurst J.K. Inorg. Chem. 1998; 37: 294-301Crossref Scopus (165) Google Scholar), but we cannot prove or disprove formation of such a species under our experimental conditions. Finally, the obligatory role of ONO2CO2− in peroxynitrite-triggered tyrosine nitration is not generally accepted. 3Dr. Willem H. Koppenol, personal communication. Thus, it cannot be excluded that peroxynitrous acid acts as nitrating species even in the complete absence of CO2. In this case, the explanation of our data would require the additional assumption that thecis- but not the trans-form of peroxynitrous acid nitrates tyrosine. Our data demonstrate that the simultaneous generation of ⋅NO and O⨪2 does not cause tyrosine nitration under physiological conditions. Together with the unequivocal demonstration of highly elevated nitrotyrosine levels in several human disease states (21Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1069) Google Scholar, 24Kaur H. Halliwell B. FEBS Lett. 1994; 350: 9-12Crossref PubMed Scopus (657) Google Scholar,51Kooy N.W. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Am. J. Respir. Crit. Care Med. 1995; 151: 1250-1254PubMed Google Scholar, 52Saleh D. Barnes P.J. Giaid A. Am. J. Respir. Crit. 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Chem. 1993; 268: 4069-4077Abstract Full Text PDF PubMed Google Scholar) that can be trapped by⋅NO, leading to formation of 3-nitrotyrosine in the presence of additional oxidants, e.g. H2O2 (40Goodwin D.C. Gunther M.R. Hsi L.C. Crews B.C. Eling T.E. Mason R.P. Marnett L.J. J. Biol. Chem. 1998; 273: 8903-8909Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In the presence of H2O2, myeloperoxidase may also cause nitration by oxidation of NO2− to ⋅NO2 (56van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar). Finally, myeloperoxidase catalyzes formation of hypochlorous acid (HOCl), which reacts nonenzymatically with NO2− to form the potent nitrating agent nitryl chloride (NO2Cl) (57Eiserich J.P. Cross C.E. Jones A.D. Halliwell B. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 58Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1358) Google Scholar). Thus, tyrosine nitration may become significant at sites where ⋅NO synthase is activated together with oxidative pathways generating tyrosyl radicals and H2O2 or other oxidants. The presence of myeloperoxidase appears to be important but would not be obligatory if other enzymatic pathways acting in a similar manner were activated together with ⋅NO synthase in tissue inflammation. In conclusion, our results show that peroxynitrite generated from⋅NO and O⨪2 at physiological pH does not nitrate tyrosine. This forces us to question the relevance of previous reports on protein nitration by bolus addition of alkaline peroxynitrite. It should also strongly stimulate new efforts to discover the true mechanism responsible for tyrosine nitration in inflammatory and infectious disease. We thank Eva Pitters and Margit Rehn for excellent technical assistance and Dr. Benjamin Hemmens for careful reading of the manuscript.

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