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* (k*2) and the decay of HOONO to nitrate (kN), giving the value of k*2/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.