Reactions of Oxymyoglobin with NO, NO2, and NO2- under Argon and in Air

Abstract

Oxymyoglobin under argon reacts with NO₂^- and NO₂ (N₂O₄) to produce metmyoglobin in a spectrally clean process with clear isosbestic points. In both cases, the reactant is NO₂^-. The second-order rate constant for NO₂^- or N₂O₄ is the same: d(Mb⁺)/dt = k(MbO₂)(NO₂^-) where k = 0.21 ± 0.02 L mol^-1 s^-1. The reaction of MbO₂ with NO under argon is a complex process and entails the generation of Mb⁺ and OONO^- (peroxynitrite) in the first step. The latter (λ_max, 302 nm) was poorly resolved from more intense protein absorbtion in the 300-nm region. However, at pH 9, the change in absorbance corresponded exactly to a quantitative production of the OONO^- ion. Hydroxyl radicals from it were trapped with ethylene-1,2-¹³C. The initial step is followed in sequence by the rapid formation of MbNO⁺. The iron(III)−nitrosyl adduct hydrolyzes slowly to Mb^II and NO₂^- (k = 8.0 ± 0.8 × 10^-5 s^-1). Mb^II then rapidly associates with NO, and MbNO is the final product of this reaction. Oxymyoglobin is inert to NO₃^-. In contrast to the results under argon, in air the reactions of MbO₂ with NO₂^-, NO, and NO₂ (N₂O₄) all proceed in the same autocatalytic fashion with k_ave (for the autocatalytic rates) ≅ 9 ± 5 L mol^-1 s^-1. Nitrite is the initial reactant in all cases. Isosbestic points are not observed in the visible spectrum, and additional porphyrin iron-ligated species are intermediates. Based upon work with iron porphyrins [J. Org. Chem. (1996) 61, 6388−6395 and J. Am. Chem. Soc. (1996) 118, 3894−3895], it is proposed that ozone may be an intermediate in the autocatalysis.