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

A new mechanism is presented for the oxidation of ascorbate by peroxyn itrite. Our mechanism involves the reaction of ascorbate both with gro und-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 mon oanion (AH(-)) is the predominant ascorbate species. The plot of the o bserved rate constant for peroxynitrite decay (k(obs)) vs AH(-) for th e reaction of peroxynitrite with AH shows two regions, one linear and one curved. In the linear region, which involves high AH(-) concentrat ions, the reaction is dominated by the bimolecular reaction between HO ONO and AH(-). At lower AH(-) concentrations, this bimolecular reactio n slows and reactions with both HOONO and HOONO produce the observed curvature, Analysis of the data leads to the estimation of the ratio o f 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) = 31 58 +/- 505 M(-1); and of the rate constant (k(2)) for the reaction bet ween AH- and HOONO, k(2) = 236 +/- 14 M(-1) s(-1). Ascorbate displays higher selectivity for HOONO than does methionine or 2-keto-4-thiomet hylbutanoic acid, two substrates whose reactivity toward HOONO and HOO NO has previously been reported. The biological relevance of the reac tion of ascorbate with peroxynitrite is discussed in terms of the rate constants and the concentrations of AH(-) typically found in biologic al systems; ascorbate may react with HOONO, although the reaction wit h ground-state HOONO probably is too slow to occur in vivo. (C) 1995 A cademic Press, Inc.