Formation of the “Peroxy” Intermediate in Cytochrome c Oxidase Is Associated with Internal Proton/Hydrogen Transfer

When dioxygen is reduced to water by cytochrome c oxidase a sequence of oxygen intermediates are formed at the reaction site. One of these intermediates is called the “peroxy” (P) intermediate. It can be formed by reacting the two-electron reduced (mixed-valence) cytochrome c oxidase with dioxygen (called Pm), but it is also formed transiently during the reaction of the fully reduced enzyme with oxygen (called Pr). In recent years, evidence has accumulated to suggest that the O−O bond is cleaved in the P intermediate and that the heme a 3 iron is in the oxo-ferryl state. In this study, we have investigated the kinetic and thermodynamic parameters for formation of Pm and Pr, respectively, in the Rhodobacter sphaeroides enzyme. The rate constants and activation energies for the formation of the Pr and Pm intermediates were 1.4 × 104 s-1 (∼20 kJ/mol) and 3 × 103 s-1 (∼24 kJ/mol), respectively. The formation rates of both P intermediates were independent of pH in the range 6.5−9, and there was no proton uptake from solution during P formation. Nevertheless, formation of both Pm and Pr were slowed by a factor of 1.4−1.9 in D2O, which suggests that transfer of an internal proton or hydrogen atom is involved in the rate-limiting step of P formation. We discuss the origin of the difference in the formation rates of the Pm and Pr intermediates, the formation mechanisms of Pm/Pr, and the involvement of these intermediates in proton pumping.