Architecture of the QoSite of the Cytochromebc1Complex Probed by Superoxide Production

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Abstract
Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc1 complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Qo) site. In this study, the interaction of molecular oxygen with the reactive intermediate Qo semiquinone is used to probe the Qo site. It has been known for some time that partial turnover of the cyt bc1 complex in the presence of antimycin A, a Qi site inhibitor, results in accumulation of a semiquinone at the Qo site, which can reduce O2 to superoxide (O2•-). It was more recently shown that myxothiazol, which binds close to the cyt bL heme in the proximal Qo niche, also induces O2•- production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Qo inhibitors [including (E)-β-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O2•- production in the isolated yeast cyt bc1 complex, at ∼50% of the Vmax observed in the presence of antimycin A. It is proposed that proximal Qo site inhibitors induce O2•- production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Qo site pocket. The apparent Km for ubiquinol at the Qo site in the presence of proximal Qo site inhibitors suggests that the “distal niche” of the Qo pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron−sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Qo site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Qo site is possible even with bound proximal Qo site inhibitors is consistent with the participation of two separate functional Qo binding niches, occupied simultaneously or sequentially.