Kinetic and Mechanistic Studies of the Peroxynitrite-Mediated Oxidation of Oxymyoglobin and Oxyhemoglobin

Kinetic studies of the peroxynitrite-mediated oxidations of oxymyoglobin (MbFeO₂) and oxyhemoglobin (HbFeO₂) showed that the mechanisms of these reactions are more complex than what had previously been reported; both reactions proceed in two steps. For myoglobin, we found that the small amount of deoxymyoglobin (MbFe^II) which is in equilibrium with MbFeO₂ is first oxidized by peroxynitrous acid to ferryl myoglobin (MbFe^IVO). Then, in the 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 °C for the two steps are (5.4 ± 0.2) × 10⁴ and (2.2 ± 0.1) × 10⁴ 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 °C for the two steps are (8.8 ± 0.4) × 10⁴ and (9.4 ± 0.7) × 10⁴ M^-1 s^-1, respectively. A possible mechanism in the absence as well as in the presence of CO₂ and the relevance of these reactions in vivo are discussed.