title: 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 tau of 40-50 microseconds (reduction of heme a by CuA) 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 approximately 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 tau 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 CuB redox state in controlling the rate of P --> F conversion of heme a3 is discussed.
authors: A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
authors: S
authors: S
authors: Siletsky
authors: A D
authors: AD
authors: Kaulen
authors: A A
authors: AA
authors: Konstantinov
mesh_terms: D000818:Animals; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry*; D005296:Ferrous Compounds / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007700:Kinetics; D008564:Membrane Potentials; D008929:Mitochondria, Heart / Q000201:enzymology; D010100:Oxygen / Q000737:chemistry*; D011190:Potassium Cyanide / Q000737:chemistry
chemical_list: D005296:Ferrous Compounds; D006861:Hydrogen Peroxide; D003576:Electron Transport Complex IV; D011190:Potassium Cyanide; D010100:Oxygen