title: Role of the carbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite.
abstract: Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate-carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3, 3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate-carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate-carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Cx P. 26077, São Paulo, SP, 05513-970, Brazil.
authors.forename: C X
authors.initials: CX
authors.lastname: Santos
authors.forename: M G
authors.initials: MG
authors.lastname: Bonini
authors.forename: O
authors.initials: O
authors.lastname: Augusto
mesh_terms: D000838:Anions / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002254:Carbonates / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*
chemical_list: D000838:Anions; D001639:Bicarbonates; D002254:Carbonates; D005609:Free Radicals; D009566:Nitrates; C408694:tyrosine radical; D002245:Carbon Dioxide; C039642:peroxynitric acid; D009569:Nitric Oxide; D017665:Hydroxyl Radical; C002744:3-nitrotyrosine; D014443:Tyrosine; C020912:N-hydroxytyrosine; C007543:dityrosine