Cytochrome c oxidase as an electron-transport-driven proton pump: pH dependence of the reduction levels of the redox centers during turnover.
The pH dependence of the steady-state kinetic parameters of cytochrome oxidase has been determined in the pH range 5.4-8.4 with the enzyme in detergent solution at high ionic strength. The catalytic constant increases continuously with decreasing pH, whereas the specificity constant for reduced cytochrome c is essentially unchanged. The effect of pH on the aerobic transient kinetics has also been investigated in two types of experiments in a stopped-flow apparatus. In one series, a 20-fold molar excess of reduced cytochrome c was the only reducing substrate, whereas in the other an excess of ascorbate was used together with a mediator and varying concentrations of cytochrome c. In both sets the time course of the reduction levels of cytochrome c, cytochrome a, and CuA was monitored at specific wavelengths. In the first type of experiment, the reoxidation of cytochrome a was slower than cytochrome c oxidation. In the second type, four kinetic phases were observed, including a long steady state. The time courses, including these features, were simulated on the basis of a mechanistic model for cytochrome oxidase as a proton pump. In this model the enzyme exists in two conformations, E1 and E2. The intramolecular electron transfer from the primary electron acceptors to the dioxygen-reducing site is rapid in E2 only. The transition from E1 to E2 is triggered by the reduction of cytochrome a and CuA. For the conversion to be rapid, the enzyme must also be doubly protonated.(ABSTRACT TRUNCATED AT 250 WORDS)
Thörnström
P E
PE
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Brzezinski
P
P
Fredriksson
P O
PO
Malmström
B G
BG
D000332:Aerobiosis; D000818:Animals; D001693:Biological Transport, Active; D002417:Cattle; D017936:Cytochrome a Group; D003580:Cytochromes / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction
D017936:Cytochrome a Group; D003580:Cytochromes; D003576:Electron Transport Complex IV