文摘
We examined the possible role of methionines as oxidant scavengers that prevent theperoxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. Thepossible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-typeCaM and following substitution of all nine methionines in CaM with leucines. We find thatperoxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite perCaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation ofbetween 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent ofnitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitratedby peroxynitrite and that methionine side chains do not function as an antioxidant in scavengingperoxynitrite. Thus, separate reactive species are involved in the oxidation of methionine andnitration of Tyr99 whose relative concentrations are determined by solution conditions. Thesensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways aresensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker ofperoxynitrite-induced changes in cellular function.