Respiratory control in cytochrome oxidase vesicles is correlated with the rate of internal electron transfer.
Cytochrome c oxidase, after reconstitution into phospholipid vesicles, displays respiratory control. This appears as an inhibition of substrate oxidation (cytochrome c) or reduction (O2) rates which, in the first few turnovers, can be largely removed upon addition of valinomycin, a specific K+ carrier. We report experiments designed to measure directly the internal electron transfer leading to the reduction of cytochrome a3/CuB, in the presence and the absence of a membrane potential. The results suggest that, after the complete oxidation and partial re-reduction of the protein, electron transfer to the binuclear site is valinomycin-sensitive, i.e. is inhibited by the membrane potential. The first-order rate constants calculated in the absence and presence of valinomycin were 0.5-0.6 and 5-6 s-1 respectively. Kinetic analysis of the reduction process is consistent with the conclusion that the membrane potential is below the critical threshold until the first electron is transferred to the cytochrome a3/CuB site. Furthermore, the respiratory control ratio obtained from the dependence of the internal electron transfer rate constant on valinomycin is always higher (by factor of 2) than that measured under turnover conditions either polarographically or spectrophotometrically. Two possible interpretations of this discrepancy are discussed.
Sarti
P
P
Institute of Biological Chemistry University of Cagliari, Sardinia.
Antonini
G
G
Malatesta
F
F
Brunori
M
M
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology; D003576:Electron Transport Complex IV / Q000378:metabolism / Q000502:physiology*; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D009550:Nigericin / Q000494:pharmacology; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000502:physiology*; D010743:Phospholipids / Q000378:metabolism; D013053:Spectrophotometry
D003574:Cytochrome c Group; D007476:Ionophores; D010743:Phospholipids; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV; D009550:Nigericin