The kinetics of the oxidation of l-ascorbic acid by peroxynitrite

Peroxynitrite [O=NOO −, oxoperoxonitrate(1-)] is a strong oxidant that may be formed in vivo by the reaction of O 2 ·− and NO ·. Oxoperoxonitrate(1-) reacts with molecules in aqueous acidic solutions via pathways that involve the highly reactive hydrogen oxoperoxonitrate either as an intermediate in a first-order reaction or as a reactive agent in a simple second-order reaction. ESR experiments show that hydrogen oxoperoxonitrate oxidizes monohydrogen l-ascorbate by one electron: when mixed at pH ca. 5 and passed through a flow cell within 0.1 s, the two-line ESR signal of the ascorbyl radical anion ( a H= 0.18 T, g = 2.005) is observed. The overall stoichiometry of the reaction was 1 mol of ascorbate oxidized per mol of oxoperoxonitrate(1-) added. The kinetics of the reaction were studied over the pH range 4.0–7.5 by stopped-flow spectrometry. Hydrogen oxoperoxonitrate, observed between 300 and 350 nm, and the oxoperoxonitrate(1-) anion, at 302 nm, disappear faster than predicted for the first-order isomerization to NO 3 −. The rate increases from pH 4 to 5.8, and then decreases with increasing pH. The rate variation suggests a bimolecular reaction either between the oxoperoxonitrate(1-) anion and ascorbic acid or between hydrogen oxoperoxonitrate and the monohydrogen ascorbate anion. Although the two pathways are kinetically indistinguishable, the pK a values of ascorbic acid and hydrogen oxoperoxonitrate strongly suggest that the reacting species are hydrogen oxoperoxo-nitrate and monohydrogen ascorbate. The second-order rate constant for this reaction is 235 ± 4 M 1s −1 at 25°C. The enthalpy and entropy of activation are ΔH ±= 9.3 ± 0.5 kcal/mol and ΔS ++ = −16 ± 2 cal/(mol. K), respectively.