Thiols Protect the Inhibition of Myocardial Aconitase by Peroxynitrite

Peroxynitrite (ONOO−) is a potent inhibitor of myocardial aconitase. Because ONOO−reacts with sulfhydryl moieties, we investigated whether thiols protect against ONOO−-mediated inhibition of aconitase. Aconitase activity was examined in ventricular homogenates prepared from freshly isolated rat hearts. Peroxynitrite, but not the nitric oxide donorS-nitroso-N-acetyl-d,l-penicillamine (0.03–300 μM), inhibited aconitase activity (IC50= 47 ± 6 μM).l-Cysteine (0.03–3 mM), glutathione (0.03–3 mM), andN-(2-mercaptoproprionyl)-glycine (MPG, 0.1–3 mM) protected against the inhibitory effect of ONOO−(100 μM) with the rank order of potency of MPG > glutathione >l-cysteine.d-Cysteine (3 mM) had a protective effect similar tol-cysteine, butl-cystine, the oxidized form ofl-cysteine, offered no protection. Ferrous ammonium sulfate (1 mM) markedly enhanced the protection provided byl-cysteine, but not by glutathione or MPG. Thiols protect myocardial aconitase against inhibition by ONOO−in a manner which is sulfhydryl group dependent and not stereospecific. The protection is related to the maintenance of the redox state of the iron–sulfur cubane cluster and cysteine residues at the active site of the enzyme. Both naturally occurring thiols and thiol-based drugs may be useful to protect the heart during ischemia–reperfusion injury where there is an excessive production of ONOO−.