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

Abstract

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 P_m), but it is also formed transiently during the reaction of the fully reduced enzyme with oxygen (called P_r). In recent years, evidence has accumulated to suggest that the O−O bond is cleaved in the P intermediate and that the heme a₃ iron is in the oxo-ferryl state. In this study, we have investigated the kinetic and thermodynamic parameters for formation of P_m and P_r, respectively, in the Rhodobacter sphaeroides enzyme. The rate constants and activation energies for the formation of the P_r and P_m 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 P_m and P_r 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 P_m and P_r intermediates, the formation mechanisms of P_m/P_r, and the involvement of these intermediates in proton pumping.