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 alkalinecis-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. 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 alkalinecis-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. In the decade since its discovery, nitric oxide has been shown to have multiple physiological actions and is implicated in the pathology of a wide range of diseases. However, nitric oxide itself is neither highly reactive nor particularly toxic, but rather forms secondary oxidants responsible for tissue injury. A major pathway that enhances the toxicity of nitric oxide is the near diffusion-limited reaction with superoxide to form peroxynitrite (ONOO−). One of the most easily identified products of peroxynitrite attack on proteins is 3-nitrotyrosine (2Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (912) Google Scholar). Nitrotyrosine has been identified in human atherosclerosis, pulmonary and heart disease, acute and chronic kidney rejection, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (3Beckman J.S. Ye Y.Z. Anderson P. Chen J. Accavetti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1069) Google Scholar, 4Kooy N.W. Lewis S.J. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Crit. Care Med. 1997; 25: 812-819Crossref PubMed Scopus (215) Google Scholar, 5Kooy N.W. Royall J.A. Ye Y.Z. Kelly D.R. Beckman J.S. Am. J. Respir. Crit. 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Full Text Full Text PDF PubMed Scopus Google suggested that urate was not a major in the of did not tyrosine However, not urate concentrations to tyrosine nitration under reaction conditions has a and be by by the xanthine oxidase was a nitration in superoxide used in as is to by peroxynitrite N.W. Royall J.A. Ischiropoulos H. Beckman J.S. Biol. Med. 1994; PubMed Scopus Google Scholar). Pfeiffer and Mayer S. Mayer B. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google peroxynitrite in the xanthine NONOate by the of was with the of peroxynitrite. However, peroxynitrite at the of the reaction urate to We that of the xanthine NONOate reaction for as as was to a extent tyrosine nitration by preformed peroxynitrite the of urate was sufficient to account for the lack of T. T. H. J. Biol. Chem. 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