Proton translocation stoichiometry of cytochrome oxidase: use of a fast-responding oxygen electrode.
The mechanistic stoichiometry of vectorial H+ ejection coupled to electron transport from added ferrocytochrome c to oxygen by the cytochrome oxidase (EC 1.9.3.1) of rat liver mitoplasts was determined from measurements of the initial rates of electron flow and H+ ejection in the presence of K+ (with valinomycin). Three different methods of measuring electron flow were used: (a) dual-wavelength spectrophotometry of ferrocytochrome c oxidation, (b) uptake of scalar H+ for the reduction of O2 in the presence of a protonophore, and (c) a fast-responding membraneless oxygen electrode. The reliability of the rate measurements was first established against the known stoichiometry of the scalar reaction of cytochrome oxidase (2ferrocytochrome c + 2H+ + 1/2O2 leads to 2ferricytochrome c + H2O) in the presence of excess protonophore. With all three methods the directly observed vectorial H+/O ejection ratios in the presence of K+ + valinomycin significantly exceeded 3.0. However, because the rate of backflow of the ejected H+ into the mitoplasts is very high and increases with the increasing delta pH generated across the membrane, there is a very rapid decline in the observed H+/O ratio from the beginning of the reaction. Kinetic analysis of ferrocytochrome c oxidation by the mitoplasts, carried out with a fast-responding membraneless oxygen electrode, showed the reaction to be first order in O2 and allowed accurate extrapolation of the rates of O2 uptake and H+ ejection to zero time. At this point, at which there is zero delta pH across the membrane, the H+/O ejection ratio of the cytochrome oxidase reaction, obtained from the rates at zero time, is close to 4.0.
D055598:Chemical Phenomena; D002621:Chemistry; D003574:Cytochrome c Group; D004566:Electrodes; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010100:Oxygen; D011188:Potassium; D013053:Spectrophotometry; D014634:Valinomycin
D003574:Cytochrome c Group; D014634:Valinomycin; D006859:Hydrogen; D003576:Electron Transport Complex IV; D011188:Potassium; D010100:Oxygen