The cytochrome bc1 complex recycles one of the two electrons from quinol oxidation at center P by reducing quinone at center N to semiquinone, which is bound tightly. We have analyzed the properties of semiquinone bound at center N of the yeast bc1 complex. The EPR-detectable signal, which reports semiquinone bound in the vicinity of reduced bH 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 semiquinone bound close to oxidized bH, we calculated an Em ~90 mV for all bound semiquinone. Considering the Em values for bH and free quinone, this result indicates that center N preferentially stabilizes semiquinone:bH3+ complexes that favor recycling of the electron coming from center P, and also implies a >2.5-fold higher affinity for quinol than for quinone at center N, which would potentially inhibit bH oxidation by quinone. Using pre-steady state kinetics we show that quinone does not inhibit the initial rate of bH reduction by quinol through center N but does decrease the extent of reduction, indicating that quinone binds only when bH is reduced, whereas quinol binds when bH is oxidized. Kinetic modeling of these results suggests that formation of semiquinone at one center N in the dimer allows stabilization of semiquinone in the other monomer by quinone reduction after intra-dimer electron transfer. This model allows maximal semiquinone:bH3+ formation without inhibition of quinone binding by quinol.
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