Simultaneous reduction of iron-sulfur protein and cytochrome b_L during ubiquinol oxidation in cytochrome bc₁ complex

The key step of the protonmotive Q-cycle mechanism of the cytochrome bc₁ 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 μs with a t_1/2 of 250 μs. 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 Q _p 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.