Kinetic and mechanistic studies of the peroxynitrite-mediated oxidation ofoxymyoglobin and oxyhemoglobin

Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin (M bFeO(2)) and oxyhemoglobin (HbFeO(2)) showed that the mechanisms of these r eactions are more complex than what had previously been reported; both reac tions proceed in two steps. For myoglobin, we found that the small amount o f deoxymyoglobin (MbFe(II)) which is in equilibrium with MbFeO(2) is first oxidized by peroxynitrous acid to ferryl myoglobin (MbFe(IV)=O). Then, in t he second step, MbFe(IV)=O is reduced by peroxynitrous acid to metmyoglobin (metMb). The second-order rate constant values obtained at pH 7.3 and 20 d egrees C for the two steps are (5.4 +/- 0.2) x 10(4) and (2.2 +/- 0.1) x 10 (4) M-1 s(-1), respectively. Analogous studies with hemoglobin suggest that its reaction with peroxynitrite follows the same mechanism. In this case, the second-order rate constant values measured at pH 7.0 and 20 degrees C f or the two steps are (8.8 +/- 0.4) x 10(4) and (9.4 +/- 0.7) x 10(4) M-1 s( -1), respectively. A possible mechanism in the absence as well as in the pr esence of CO2 and the relevance of these reactions in vivo are discussed.