STOPPED-FLOW KINETIC-STUDY OF THE REACTION OF ASCORBIC-ACID WITH PEROXYNITRITE

A new mechanism is presented for the oxidation of ascorbate by peroxynitrite. Our mechanism involves the reaction of ascorbate both with ground-state peroxynitrous acid (HOONO) and with a reactive intermediate (HOONO*); the reactive intermediate is postulated to be formed in the decay of HOONO to form nitrate. At physiological pH, the ascorbate monoanion (AH(-)) is the predominant ascorbate species. The plot of the observed rate constant for peroxynitrite decay (k(obs)) vs AH(-) for the reaction of peroxynitrite with AH shows two regions, one linear and one curved. In the linear region, which involves high AH(-) concentrations, the reaction is dominated by the bimolecular reaction between HOONO and AH(-). At lower AH(-) concentrations, this bimolecular reaction slows and reactions with both HOONO and HOONO* produce the observed curvature, Analysis of the data leads to the estimation of the ratio of rate constants for the reaction of AH(-) with HOONO* (k(2)*) and the decay of HOONO to nitrate (k(N)), giving the value of k(2)*/k(N) = 3158 +/- 505 M(-1); and of the rate constant (k(2)) for the reaction between AH- and HOONO, k(2) = 236 +/- 14 M(-1) s(-1). Ascorbate displays higher selectivity for HOONO* than does methionine or 2-keto-4-thiomethylbutanoic acid, two substrates whose reactivity toward HOONO and HOONO* has previously been reported. The biological relevance of the reaction of ascorbate with peroxynitrite is discussed in terms of the rate constants and the concentrations of AH(-) typically found in biological systems; ascorbate may react with HOONO*, although the reaction with ground-state HOONO probably is too slow to occur in vivo. (C) 1995 Academic Press, Inc.