Substrate redox potential controls superoxide production kinetics in the cytochrome bc complex.

The Q-cycle mechanism of the cytochrome bc(1) complex maximizes energy conversion during the transport of electrons from ubiquinol to cytochrome c (or alternate physiological acceptors), yet important steps in the Q-cycle are still hotly debated, including bifurcated electron transport, the high yield and specificity of the Q-cycle despite possible short-circuits and bypass reactions, and the rarity of observable intermediates in the oxidation of quinol. Mounting evidence shows that some bypass reactions producing superoxide during oxidation of quinol at the Q(o) site diverge from the Q-cycle rather late in the bifurcated reaction and provide an additional means of studying initial reactions of the Q-cycle. Bypass reactions offer more scope for controlling and manipulating reaction conditions, e.g., redox potential, because they effectively isolate or decouple the Q-cycle initial reactions from later steps, preventing many complications and interactions. We examine the dependence of oxidation rate on substrate redox potential in the yeast cytochrome bc(1) complex and find that the rate limitation occurs at the level of direct one-electron oxidation of quinol to semiquinone by the Rieske protein. Oxidation of semiquinone and reduction of cyt b or O(2) are subsequent, distinct steps. These experimental results are incompatible with models in which the transfer of electrons to the Rieske protein is not a distinct step preceding transfer of electrons to cytochrome b, and with conformational gating models that produce superoxide by different rate-limiting reactions from the normal Q-cycle.