Inhibition of peroxynitrite-induced nitration of tyrosine by glutathione in the presence of carbon dioxide through both radical repair and peroxynitrate formation

Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2.) and trioxocarbonate(1-) (CO3.) radicals. We have studied the mechanism by which glutathione (GSH) i nhibits the NO2./CO3.--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent ty rosine consumption (IC50 = 660 muM) as well as 3-nitrotyrosine formation (I C50 = 265 muM). From the determination of the level of oxygen produced or c onsumed under various initial conditions.. it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS(.)) through reduction of dioxygen to superox ide (O-2(.-)) and reaction with NO2. to form peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effectiv e in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2 . radicals is highly unlikely at physiological levels of GSH.