Simultaneous reduction of iron-sulfur protein and cytochrome b(L) during ubiquinol oxidation in cytochrome bc(1) complex.

The key step of the protonmotive Q-cycle mechanism of the cytochrome bc(1) complex is the bifurcated oxidation of ubiquinol at the Qp site. It was postulated that the iron-sulfur protein (ISP) accepts the first electron from ubiquinol to generate ubisemiquinone anion to reduce b(L). Because of the difficulty of following the reduction of ISP optically, direct evidence for the early involvement of ISP in ubiquinol oxidation is not available. Using the ultra-fast microfluidic mixer and the freeze-quenching device, coupled with EPR, we have been able to determine the presteady-state kinetics of ISP and cytochrome b(L) reduction by ubiquinol. The first-phase reduction of ISP starts as early as 100 micros with a t(1/2) of 250 micros. A similar reduction kinetic is also observed for cytochrome b(L), indicating a simultaneous reduction of both ISP and b(L). These results are consistent with the fact that no ubisemiquinone was detected at the Qp site during oxidation of ubiquinol. Under the same conditions, by using stopped flow, the reduction rates of cytochromes b(H) and c(1) were 403 s(-1) (t(1/2) 1.7 ms) and 164 s(-1) (t(1/2) 4.2 ms), respectively.