Recovery of reduced thiol groups by superoxide-mediated denitrosation of nitrosothiols

Nitrosation of critical thiols has been elaborated as reversible posttranslational modification with regulatory function in multiple disorders. Reversibility of S-nitrosation is generally associated with enzyme-mediated one-electron reductions, catalyzed by the thioredoxin system, or by nitrosoglutathione reductase. In the present study, we confirm previous evidence for a non-enzymatic de-nitrosation of nitrosoglutathione (GSNO) by superoxide. The interaction leads to the release of nitric oxide that subsequently interacts with a second molecule of superoxide (O(2)(•−)) to form peroxynitrite. Despite the formation of peroxynitrite, approximately 40–70% of GSNO yielded reduced glutathione (GSH), depending on the applied analytical assay. The concept of O(2)(•−) dependent denitrosation was then applied to S-nitrosated enzymes. S-nitrosation of isocitrate dehydrogenase (ICDH; NADP(+)-dependent) was accompanied by an inhibition of the enzyme and could be reversed by dithiothreitol. Treatment of nitrosated ICDH with O(2)(•−) indicated ca. 50% recovery of enzyme activity. Remaining inhibition was largely consequence of oxidative modifications evoked either by O(2)(•−) or by peroxynitrite. Recovery of activity in S-nitrosated enzymes by O(2)(•−) appears relevant only for selected examples. In contrast, recovery of reduced glutathione from the interaction of GSNO with O(2)(•−) could represent a mechanism to regain reducing equivalents in situations of excess O(2)(•−) formation, e.g. in the reperfusion phase after ischemia.