NITROSATION BY PEROXYNITRITE - USE OF PHENOL AS A PROBE

Nitrosation is an important pathway in the metabolism of nitric oxide, producing S-nitrosothiols that may be critical signal transduction sp ecies. The reaction of peroxynitrite with aromatic compounds ire the p H range of 5 to 8 has long been known to produce hydroxylated and nitr ated products. However, we here present evidence that peroxynitrite al so can promote the nitrosation of nucleophiles. We chose phenol as a s ubstrate because the nitrosation reaction was first recognized during a study of the CO2-modulation of the patterns of hydroxylation and nit ration of phenol by peroxynitrite (Lemercier et al, Arch. Biochem. Bio phys. 345, 160-170, 1997), 4-Nitrosophenol, the principal nitrosation product, is detected at pH 7.0, along with 2- and 4-nitrophenols; 4-ni trosophenol becomes the dominant product at pH greater than or equal t o 8.0. The yield of Li-nitrosophenol continues to increase even after pH 11.1, 1.2 units above the pK(a) of phenol, suggesting that the phen olate ion, and not phenol, is involved in the reaction. Hydrogen perox ide is not formed as a by-product. The nitrosation reaction is zero-or der in phenol and first-order in peroxynitrite, suggesting the phenola te ion reacts with an activated nitrosating species derived from perox ynitrite, and not with peroxynitrite itself. Under optimal conditions, the yields of 4-nitrosophenol are comparable to those of 2- and 4-nit rophenols, indicating that the nitrosation reaction is as significant as the nitration of phenolic compounds by peroxynitrite, Low concentra tions of CO2 facilitate the nitrosation reaction, but excess CO2 drama tically reduces the yield of 4-nitrosophenol, The dual effects of CO2 can be rationalized if O=N-OO- reacts with the peroxynitrite anion-CO2 adduct (O=N-OOCO2-) or secondary intermediates derived from it, inclu ding the nitrocarbonate anion (O2N-OCO2-), the carbonate radical (CO3. -), and (NO2)-N-.. The product resulting from these reactions can be e nvisioned as an activated intermediate X-N=O (where X is -OONO2, -NO2, or -CO3-) that could transfer a nitrosyl cation (NO+) to the phenolat e ion. An alternative mechanism for the nitrosation of phenol involves the one-electron oxidation of the phenolate ion by CO3.- to give the phenoxyl radical and the oxidation of O=N-OO- by CO3.- to give a nitro syldioxyl radical (O=N-OO.), which decomposes to give (NO)-N-. and O-2 ; the (NO)-N-. then reacts with the phenoxyl radical giving nitrosophe nol. Both mechanisms are consistent with the high yields of NO2- and O -2 during the alkaline decomposition of peroxynitrite and the potent i nhibitory effect of N-3(-) fin the nitrosation of phenol by peroxynitr ite and peroxynitrite/CO2 adducts. The biological significance of the peroxynitrite-mediated nitrosations is discussed. (C) 1998 Academic Pr ess.