Resolution of Electrogenic Steps Coupled to Conversion of Cytochrome c Oxidase from the Peroxy to the Ferryl−Oxo State

Abstract

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl−oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl−oxo-to-oxidized (F → O) transition of the enzyme [Zaslavsky, D., et al. (1993) FEBS Lett. 336, 389−393]. As in the case of the F → O transition, the electric response associated with photoreduction of P to F includes a rapid KCN-insensitive electrogenic phase with a τ of 40−50 μs (reduction of heme a by Cu_A) and a multiphasic slower part; this part is cyanide-sensitive and is assigned to vectorial transfer of protons coupled to reduction of oxygen intermediate in the binuclear center. The net KCN-sensitive phase of the response is ∼4-fold more electrogenic than the rapid phase, which is similar to the characteristics of the F → O electrogenic transition and is consistent with net transmembrane translocation of two protons per electron, including vectorial movement of both “chemical” and “pumped” protons. The protonic part of the P → F electric response is faster than in the F → O transition and can be deconvoluted into three exponential phases with τ values varying for different samples in the range of 0.25−0.33, 1−1.5, and 6−7.5 ms at pH 8. Of these three phases, the 1−1.5 ms component is the major one contributing 50−60%. The P → F conversion induced by single electron photoreduction of the peroxy state as studied in this work is several times slower than the P → F transition resolved during oxidation of the fully reduced oxidase by molecular oxygen. The role of the Cu_B redox state in controlling the rate of P → F conversion of heme a₃ is discussed.