Tyrosine Modification by Reactive Nitrogen Species: A Closer Look

Peroxynitrite (ONOO−) is a powerful oxidant and cytotoxic species formed by the rapid reaction between nitrogen monoxide (nitric oxide, . NO) and superoxide (O . 2 −). At neutral pH ONOO− is partly protonated and this protonated form, peroxynitrous acid (ONOOH), decomposes rapidly to nitrate, forming (an) intermediate(s) with reactivity similar to . OH and . NO2. Peroxynitrite can hydroxylate and nitrate aromatic rings, and aromatic nitration of phenols such as tyrosine by ONOOH is proposed to proceed via a radical mechanism, with intermediate formation of . NO2. Modification of tyrosine by . NO2 also involves nitration via a radical mechanism. Aromatic nitration of phenols by ONOO− has been shown to be enhanced by superoxide dismutase or Fe3+-EDTA, which were proposed to catalyze heterolytic cleavage of ONOOH to form a nitrating species similar to the nitronium ion NO+ 2. We investigated possible mechanisms of tyrosine modification by various reactive nitrogen species, including ONOO−, 3-morpholinosydnonimine (SIN-1), and . NO2. Reaction of tyrosine with ONOO− leads to formation of 3-nitrotyrosine and dityrosine, indicating intermediate formation of tyrosyl radicals. The pH dependence of formation of both 3-nitrotyrosine and dityrosine by ONOO− suggests that intermediate formation of ONOOH is required. Qualitatively similar results were obtained when ONOOH was generated continuously by H2O2 and NaNO2 at mildy acidic pH or with SIN-1, a compound which at neutral pH releases both . NO and O . 2 −, presumably producing ONOO−. However, relatively low yields of nitrotyrosine were obtained with SIN-1, possibly because of competing reactions of tyrosyl radicals with . NO or O . 2 −. Possible involvement of . NO2 in tyrosine modification by ONOO− was studied using hydroxyl radical scavengers, which can increase the radical yield during decomposition of ONOOH and thereby enhance generation of . NO2. Hydroxyl radical scavengers did not affect tyrosine modification by . NO2 directly and slightly inhibited tyrosine modification by authentic ONOO−. However, when ONOO− was produced at a slower rate, either by SIN-I or by H2O2/NaNO2 at acidic pH, hydroxyl radical scavengers were found to significantly enhance tyrosine nitration. Our results suggest that ONOO− or ONOO−-generating systems induce nitration of tyrosine (or tyrosine residues in proteins) via intermediate formation of tyrosyl radicals and . NO2.