Ubiquinone at center N is responsible for triphasic reduction of cytochrome b in the cytochrome bc(1) complex

We have examined the pre-steady state reduction kinetics of the Saccharomyc es cerevisiae cytochrome bc(1) complex by menaquinol in the presence and ab sence of endogenous ubiquinone to elucidate the mechanism of triphasic cyto chrome b reduction. With cytochrome bc(1) complex from wild type yeast, cyt ochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase. Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this w as caused by a bona fide reoxidation of cytochrome b and not by any negativ e spectral contribution from cytochrome c(1). With cytochrome bc(1) complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduct ion by either menaquinol or ubiquinol was rapid and monophasic, Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased. When reduction of the cytochrome bc(1) complex through center P was blocked , cytochrome b reduction through center N was biphasic and was slowed by th e addition of exogenous ubiquinone. These results show that ubiquinone resi ding at center N in the oxidized cytochrome bc(1) complex is responsible fo r the triphasic reduction of cytochrome b.