Resolution of electrogenic steps coupled to conversion of cytochrome c oxidase from the peroxy to the ferryl-oxo state

Charge translocation across the membrane coupled to transfer of the third e lectron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the fer ryl-oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstit uted 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) FEES 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 phas e with a tau of 40-50 mu s (reduction of heme a by CUA) and a multiphasic s lower part; this part is cyanide-sensitive and is assigned to vectorial tra nsfer of protons coupled to reduction of oxygen intermediate in the binucle ar center. The net KCN-sensitive phase of the response is similar to 4-fold more electrogenic than the rapid phase, which is similar to the characteri stics of the F --> O electrogenic transition and is consistent with net tra nsmembrane translocation of two protons per electron, including vectorial m ovement 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 b e deconvoluted into three exponential phases with tau values varying for di fferent 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 t he 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 a(3) is discussed.