Rapid Reactions of Peroxynitrite with Heme–Thiolate Proteins as the Basis for Protection of Prostacyclin Synthase from Inactivation by Nitration

Prostacyclin (PGI2) synthase is a heme–thiolate (P450) protein which reacts with low levels of peroxynitrite (PN) under tyrosine nitration and inactivation. Studying heme proteins as models, we have found the heme–thiolate protein NADH–NO reductase (P450NOR) to be highly efficient in decomposing PN under concomitant nitration of phenol. The present study investigates two other P450 proteins, P450BM-3 and chloroperoxidase, in order to test for the specific role of the thiolate ligand in the reaction with PN. A comparison with horseradish peroxidase and microperoxidase gives evidence of kinetic differences that classify heme–thiolate proteins, but not other heme proteins, as effective inhibitors of PGI2 synthase nitration and inactivation. P450BM-3 with PN catalyzes phenol nitration and nitration of its own tyrosine below 10 μM PN, whereas chloroperoxidase and P450NOR at such concentrations also nitrate phenol but not enzyme-bound tyrosine residues. We conclude that heme–thiolate proteins in general exhibit high reactivity with PN and turnover, probably due to the special electronic structure of the presumed thiolate–ferryl intermediate.