title: Nitric oxide trapping of the tyrosyl radical of prostaglandin H synthase-2 leads to tyrosine iminoxyl radical and nitrotyrosine formation.
abstract: The determination of protein nitrotyrosine content has become a frequently used technique for the detection of oxidative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e. g. peroxynitrite) formed in reactions between nitric oxide (NO.) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of prostaglandin formation. In the presence of the NO.-generator diethylamine nonoate, the electron spin resonance spectrum of the PHS-2-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO. to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicating that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO.-generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.
authors.affiliation: Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. gunther@niehs.nih.gov
authors.forename: M R
authors.initials: MR
authors.lastname: Gunther
authors.forename: L C
authors.initials: LC
authors.lastname: Hsi
authors.forename: J F
authors.initials: JF
authors.lastname: Curtis
authors.forename: J K
authors.initials: JK
authors.lastname: Gierse
authors.forename: L J
authors.initials: LJ
authors.lastname: Marnett
authors.forename: T E
authors.initials: TE
authors.lastname: Eling
authors.forename: R P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D009153:Mutagens / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009589:Nitrogen Oxides; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D006834:Hydrazines; D009153:Mutagens; D009566:Nitrates; D009589:Nitrogen Oxides; C039642:peroxynitric acid; D016718:Arachidonic Acid; D009569:Nitric Oxide; D014443:Tyrosine; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D011451:Prostaglandin-Endoperoxide Synthases; D013482:Superoxide Dismutase