S-nitrosation of glutathione by nitric oxide, peroxynitrite, and •NO/O2•-

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((NO)-N-.), peroxynitrite, and (NO)-N-./O-2(.-). In the presence of O-2, (NO)-N-. yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (NO)-N-./O-2 is predominantly mediated by (NO2)-N-.. Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (NO)-N-. and O-2(.-) at equal fluxes yielded less GSNO than (NO)-N-. alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (NO)-N-./O-2(.) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O-2(.-) to initiate a chain reaction that forms radical intermediates which combine with (NO)-N-. to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (NO)-N-./O-2 and (NO)-N-./O-2(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (NO)-N-. and O-2(.-) may essentially contribute to nitrosative stress in inflammatory diseases. (C) 2003 Elsevier Inc.