IONIC-STRENGTH DEPENDENCE OF THE KINETICS OF ELECTRON-TRANSFER FROM BOVINE MITOCHONDRIAL CYTOCHROME-C TO BOVINE CYTOCHROME-C-OXIDASE

The effect of ionic strength on the one-electron reduction of oxidized bovine cytochrome c oxidase by reduced bovine cytochrome c has been studied by using flavin semiquinone reductants generated in situ by laser flash photolysis. In the absence of cytochrome c, direct reduction of the heme alpha prosthetic group of the oxidase by the one-electron reductant 5-deazariboflavin semiquinone occurred slowly, despite a driving force of approximately +1 V. This is consistent with a sterically inaccessible heme alpha center. This reduction process was independent of ionic strength from 10 to 100 mM. Addition of cytochrome c resulted in a marked increase in the amount of reduced oxidase generated per laser flash. Reduction of the oxidase at the heme alpha site was monophasic, whereas oxidation of cytochrome c was multiphasic, the fastest phase corresponding in rate constant to the reduction of the heme alpha. During the fast kinetic phase, 2 equiv of cytochrome c was oxidized per heme alpha reduced. We presume that the second equivalent was used to reduce the Cu(a) center, although this was not directly measured. The first-order rate-limiting process which controls electron transfer to the heme alpha showed a marked ionic strength effect, with a maximum rate constant occurring at mu = 110 mM (1470 s-1), whereas the rate constant obtained at mu = 10 mM was 630 s-1 and at mu = 510 mM was 45 s-1. There was no effect of "pulsing" the enzyme on this rate-limiting one-electron transfer process. These results suggest that there are structural differences in the complex(es) formed between mitochondrial cytochrome c and cytochrome c oxidase at very low and more physiologically relevant ionic strengths, which lead to differences in electron-transfer rate constants.