Asymmetric and redox-specific binding of quinone and quinol at center N of the dimeric yeast cytochrome bc1 complex -: Consequences for semiquinone stabilization

The cytochrome bc(1) complex recycles one of the two electrons from quinol (QH(2)) oxidation at center P by reducing quinone ( Q) at center N to semiquinone ( SQ), which is bound tightly. We have analyzed the properties of SQ bound at center N of the yeast bc1 complex. The EPR- detectable signal, which reports SQ bound in the vicinity of reduced b(H) heme, was abolished by the center N inhibitors antimycin, funiculosin, and ilicicolin H, but was unchanged by the center P inhibitors myxothiazol and stigmatellin. After correcting for the EPR- silent SQ bound close to oxidized bH, we calculated a midpoint redox potential ( Em) of similar to 90 mV for all bound SQ. Considering the Em values for bH and free Q, this result indicates that center N preferentially stabilizes SQ center dot bH 3(+) complexes. This favors recycling of the electron coming from center P and also implies a > 2.5- fold higher affinity for QH2 than for Q at center N, which would potentially inhibit bH oxidation by Q. Using pre- steady- state kinetics, we show that Q does not inhibit the initial rate of bH reduction by QH2 through center N, but does decrease the extent of reduction, indicating that Q binds only when bH is reduced, whereas QH2 binds when bH is oxidized. Kinetic modeling of these results suggests that formation of SQ at one center N in the dimer allows stabilization of SQ in the other monomer by Q reduction after intradimer electron transfer. This model allows maximum SQ (center dot)bH 3(+) formation without inhibition of Q binding by QH2.