Role of the carbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite.
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.
Santos
C X
CX
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Cx P. 26077, São Paulo, SP, 05513-970, Brazil.
Bonini
M G
MG
Augusto
O
O
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*
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