Effect of membrane potential and pH gradient on electron transfer in cytochrome oxidase.
Steady-state spectra of cytochrome oxidase in phospholipid vesicles were obtained by using hexaammineruthenium(II) and ascorbate as reductants. Cytochrome a was up to 80% reduced in the steady state in coupled vesicles. Upon addition of nigericin or acetate, which decrease delta pH, resulting in an increase in delta psi, cytochrome a became more oxidized in the steady state with no change in the rate of respiration. On the other hand, uncouplers or valinomycin plus nigericin, which lower both delta psi and delta pH, stimulated respiration 2-8-fold and also lowered the steady-state level of reduction of cytochrome a. These experiments indicate that electron transfer between cytochromes a and a 3 is sensitive primarily to the pH gradient. Studies with the reconstituted and the soluble enzyme at various pH values indicated that the pH on the matrix side of the membrane, rather than delta pH, controlled the steady-state level of reduced cytochrome a. Hexaammineruthenium(II) substituted for cytochrome c in measurements of proton pumping by cytochrome oxidase. Dicyclohexylcarbodiimide, which eliminated proton pumping by cytochrome oxidase, decreased the effect of ionophores on the steady-state level of reduced cytochrome a.
D017936:Cytochrome a Group; D003580:Cytochromes / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D012428:Ruthenium; D017975:Ruthenium Compounds; D013053:Spectrophotometry
D017936:Cytochrome a Group; D003580:Cytochromes; D008081:Liposomes; D017975:Ruthenium Compounds; C031584:hexammineruthenium; D004024:Dicyclohexylcarbodiimide; D012428:Ruthenium; D003576:Electron Transport Complex IV