PATHWAYS OF PEROXYNITRITE OXIDATION OF THIOL-GROUPS

Peroxynitrite mediates the oxidation of the thiol group of both cystei ne and glutathione. This process is associated with oxygen consumption . At acidic pH and a cysteine/peroxynitrite molar ratio of less than o r equal to 1.2, there was a single fast phase of oxygen consumption, w hich increased with increasing concentrations of both cysteine and oxy gen. At higher molar ratios the profile of oxygen consumption became b iphasic, with a fast phase (phase I) that decreased with increasing cy steine concentration, followed by a slow phase (phase II) whose rate o f oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5, 5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismut ase also inhibited oxygen consumption in phase a, while catalase added during phase II decreased the rate of oxygen consumption. For both cy steine and glutathione, oxygen consumption in phase I was maximal at n eutral to acidic pH; in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-pheny lnitrone indicated that the yield of thiyl radical adducts had a pH pr ofile comparable with that found for oxygen consumption. The apparent Second-order rate constants for the reactions of peroxynitrite with cy steine and glutathione were 1290 +/- 30 M(-1) . s(-1) and 281 +/- 6 M( -1) . s(-1) respectively at pH 5.75 and 37 degrees C. These results ar e consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction bf the peroxynitrite anion with the protonated thiol group, ina second-or der process likely to involve a two-electron oxidation, and (b) the re action of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reac tion.