Kinetic indication for multiple sites of ubiquinol-1 interaction in ubiquinol-cytochrome c reductase in bovine heart mitochondria

We have assayed the ubiquinol-cytochrome c reductase activity either in situ or in different mitochondrial fractions, including the isolated bc₁ complex, employing ubiquinol-1 and exogenous cytochrome c as substrates. A clear biphasic behavior of both the time courses and the initial rates of cytochrome c reduction have been observed. Two K_m values have been found, one of 1–7 × 10^?6m ubiquinol-1, and another varying from 0.6 to 4.6 × 10^?5m ubiquinol-1, depending on the cytochrome c concentration and the type of mitochondrial fraction used. Either the kinetic phase with the lower K_m or the kinetic phase with the higher K_m exhibits an almost identical antimycin sensitivity. We have also monitored the rapid reduction of endogenous b cytochromes in the presence of antimycin, and the initial rates are again biphasic as a function of ubiquinol-1 concentration. These findings indicate that the steps conferring the biphasic kinetics to the ubiquinol-cytochrome c reductase activity involve the redox equilibria between exogenous ubiquinol-1 and the b cytochromes, and suggest that two redox pathways may be present in the electron transfer from ubiquinol to cytochrome c through the bc₁ segment of the mammalian respiratory chain.