Intrinsic uncoupling in proton-pumping cytochrome c oxidase: pH dependence of cytochrome c oxidation in coupled and uncoupled phospholipid vesicles.
The pH dependence of the transient aerobic kinetics of cytochromes c and a has been investigated with cytochrome oxidase reconstituted in phospholipid vesicles in the absence and presence of an uncoupler and an ionophore. The cytochrome a reduction level immediately after the burst phase was 60-80% and was not significantly changed by the addition of uncoupler and/or ionophore. The coupled rate of ferro-cytochrome c oxidation increases linearly with decreasing pH in the range 8.4-5.4. The increase in rate on uncoupling becomes less with decreasing pH and low cytochrome c concentration, being almost zero at pH 5.4. The coupled rate is increased by a lowering of the outside pH when the inside pH is constant. Varying the inside pH with a constant outside pH of 7.4 has little effect on the rate. It is suggested that the electrochemical potential has two separate effects on the coupled rate: the pH gradient mainly slows down the intramolecular electron transfer, but the membrane potential also lowers the second-order rate constant for the reaction with cytochrome c. The results are interpreted in terms of a model in which protonation of an acid-base group with a pKa of 6.4 from the inside increases the catalytic constant. Protonation from the outside, on the other hand, leads to an intrinsic uncoupling, because the protonated enzyme in the output state can return to the input state. This has no adverse physiological effect, since it becomes significant only at pH values well below 7.
Maison-Peteri
B
B
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Malmström
B G
BG
D000818:Animals; D017936:Cytochrome a Group; D003574:Cytochrome c Group / Q000378:metabolism; D003580:Cytochromes / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008081:Liposomes; D010084:Oxidation-Reduction; D011522:Protons; D014475:Uncoupling Agents / Q000494:pharmacology
D017936:Cytochrome a Group; D003574:Cytochrome c Group; D003580:Cytochromes; D008081:Liposomes; D011522:Protons; D014475:Uncoupling Agents; D003576:Electron Transport Complex IV