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

Peroxynitrite [O=NOO−, oxoperoxonitrate(1-)] is a strong oxidant that may be formed in vivo by the reaction of O2·− 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 image-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 (aH= 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 NO3−. 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 pKa 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.