FLASH-INDUCED MEMBRANE-POTENTIAL GENERATION BY CYTOCHROME-C-OXIDASE

Flash-induced single-electron reduction of cytochrome c oxidase. Compo und F (oxoferryl state) by Ru-Pi(2,2'-bipyridyl)(3)(2+) [Nilsson (1992 ) Proc. Natl. Acad. Sci. USA 89, 6497-6501] gives rise to three phases of membrane potential generation in proteoliposomes with tau values a nd contributions of ca. 45 mu s (20%), 1 ms (20%) and 5 ms (60%). The rapid phase is not sensitive to the binuclear centre ligands, such as cyanide or peroxide, and is assigned to vectorial electron transfer fr om Cu-A to heme a. The two slow phases kinetically match reoxidation o f heme a, require added H2O2 or methyl peroxide for full development, and are completely inhibited by cyanide; evidently, they are associate d with the reduction of Compound F to the Ox state by heme a. The char ge transfer steps associated with the F to Ox conversion are likely to comprise (i) electrogenic uptake of a 'chemical' proton from the N ph ase required for protonation of the reduced oxygen atom and (ii) elect rogenic H+ pumping across the membrane linked to the F to Ox transitio n. Assuming heme a 'electrical location' in the middle of the dielectr ic barrier, the ratio of the rapid to slow electrogenic phase amplitud es indicates that the F to Ox transition is linked to transmembrane tr anslocation of 1.5 charges (protons) in addition to an electrogenic up take of one 'chemical' proton required to form Fe3+-OH- from Fe4+=O2-. The shortfall in the number of pumped protons and the biphasic kineti cs of the millisecond part of the electric response matching biphasic reoxidation of heme a may indicate the presence of 2 forms of Compound F, reduction of only one of which being linked to full proton pumping .