Abstract
The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.
Highlights
The bc1 complex transfers two electrons from ubiquinol to two molecules of cytochrome c, while translocating four protons from the negative to the positive side of the membrane via the protonmotive Q cycle mechanism [5]
In this study we wanted to shed some light on the Qn site by applying the novel center N inhibitor ilicicolin H to isolate cytochrome b mutants in S. cerevisiae
We know that antimycin displaces semiquinone from center N in the bovine bc1 complex [15], and more recently we have found that all three of the center N inhibitors eliminate the EPR detectable signal attributed to the stable semiquinone at center N in the yeast enzyme
Summary
Resistance conferring mutations might identify amino acids that contribute to semiquinone stability at center N. Seven cytochrome b mutations, differing from the wild-type protein by only one amino acid exchange, were obtained. The sequenced mutations were confirmed and assigned to the cytochrome b gene using genetic complementation techniques. Six of the seven mutations obtained in this study have not been described before. The effects of these mutations on the activity of the enzyme, as well as the sensitivity of the altered cytochrome b proteins toward ilicicolin H, were determined enzymatically. We checked the mutant yeast strains for crossresistance using antimycin and funiculosin
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