Substrate Redox Potential Controls Superoxide Production Kinetics in the Cytochrome <i>bc</i> Complex
2009-11-17T00:00:00Z (GMT) by
The Q-cycle mechanism of the cytochrome <i>bc</i><sub>1</sub> complex maximizes energy conversion during the transport of electrons from ubiquinol to cytochrome <i>c</i> (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<sub>o</sub> 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 <i>bc</i><sub>1</sub> 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 <i>b</i> or O<sub>2</sub> 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 <i>b</i>, and with conformational gating models that produce superoxide by different rate-limiting reactions from the normal Q-cycle.