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 (kobs) 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* (k2*) and the decay of HOONO to nitrate (kN), giving the value of k2*/kN = 3158 +/- 505 M-1; and of the rate constant (k2) for the reaction between AH- and HOONO, k2 = 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.