On the mechanism of quinol oxidation in the bc1 complex

The question of whether significant levels of a semiquinone can be generated in the Qo site of the bc1 complex under conditions of oxidant-induced reduction is relevant to the mechanism of bifurcation of electron transfer in this site. It has already been reported that beef heart submitochondrial particles under such conditions exhibit an EPR-detectable semiquinone, which is distinct from Q-i and which was attributed to a semiquinone in the Qo site (de Vries, S., Albracht, S. P. J., Berden, J. A., and Slater, E. C. (1981) J. Biol. Chem. 256, 11996-11998). However, we show here that this signal, which can be generated to a level of around 0.1 per bc1 monomer, is insensitive to the Qo site inhibitors myxothiazol, E-beta-methoxyacrylate-stilbene, and stigmatellin, indicating that it does not arise from a Q-o species. Based on sensitivities to inhibitors of other Q sites, up to 60% of the signal may arise from semiquinones of complexes I and II. We further show that the iron-sulfur center remains EPR silent under oxidant-induced reduction conditions. Overall, the results indicate that, under conditions of oxidant-induced reduction, the Qo site is occupied primarily by quinol with the iron-sulfur center oxidized, or, possibly, by an antiferromagnetically coupled semiquinone/reduced iron-sulfur center pair, which are EPR silent. This is discussed in relation to proposed mechanisms of quinol oxidation in the Qo site, and we describe a minimal intermediate-controlled bifurcation model based on rate constants by which bifurcated electron transfer at the Qo site might occur.