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.
Reiter
C D
CD
Departments of Anesthesiology, Biochemistry and Molecular Genetics, and Pediatrics, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233, USA.
Teng
R J
RJ
Beckman
J S
JS
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009566:Nitrates / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry*; D020030:Nitric Oxide Donors / Q000737:chemistry; D009589:Nitrogen Oxides; D016877:Oxidants / Q000737:chemistry; D010777:Photochemistry; D013053:Spectrophotometry / Q000379:methods; D013096:Spermine / Q000031:analogs & derivatives / Q000737:chemistry; D013481:Superoxides / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*; D014969:Xanthine Oxidase / Q000378:metabolism
D009566:Nitrates; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D016877:Oxidants; D013481:Superoxides; C091861:spermine nitric oxide complex; C039642:peroxynitric acid; D013096:Spermine; D009569:Nitric Oxide; D014443:Tyrosine; D014969:Xanthine Oxidase