Ubiquinone at Center N Is Responsible for Triphasic Reduction of Cytochrome b in the Cytochromebc 1 Complex

Abstract

We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc 1 complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction. With cytochrome bc 1 complex from wild type yeast, cytochrome 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 was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c 1 . With cytochrome bc 1 complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction 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 the addition of exogenous ubiquinone. These results show that ubiquinone residing at center N in the oxidized cytochrome bc 1 complex is responsible for the triphasic reduction of cytochrome b.Although the protonmotive Q cycle mechanism of the cytochrome bc 1 complex is generally well understood (1-3), the redox behavior of cytochrome b during pre-steady state reduction of the bc 1 complex is not fully understood. Cytochrome b reduction is triphasic, consisting of a rapid partial reduction phase, a partial reoxidation phase, and a slow rereduction phase. This behavior is puzzling, because the reoxidation phase occurs while reduced substrate is still available, and continued reduction of cytochrome b would be expected.Previous examinations of the pre-steady state reduction kinetics of the bc 1 complex were limited to single wavelength kinetics, and the spectral data, when collected, extended over time ranges that were long relative to the half-times of the reactions (4 -9). The substrates used in these studies, succinate, duroquinol, trimethylquinol, and ubiquinol, have relatively high redox potentials and reduce only a small percentage of cytochrome b. This is of concern because the high redox potential may predispose these substrates to oxidize cytochrome b and thus introduce artifacts into the pre-steady state kinetics in the absence of a low potential reductant.Several explanations for the triphasic reduction have been put forth. One proposal is that ubiquinone formed at center P is not in rapid equilibration with the quinone pool and oxidizes cytochrome b at center N (5, 10). Crystal structures of the mitochondrial cytochrome bc 1 complexes show a pear-shaped and dimeric integral membrane protein that extends ϳ80 Å into the matrix and ϳ30 Å into the intermembrane space (11, 12). There are two large cavities within the bc 1 dimer that lin...

Molecular Basis for Atovaquone Binding to the Cytochrome bc 1 Complex

“…The L275F mutation was introduced into the mitochondrial cytochrome b gene by biolistic transformation as described elsewhere (15). Cytochrome bc 1 complexes were isolated from yeast and bovine heart mitochondria as described previously (17,18) and concentrated by centrifugal filtration, using Amicon® Centriprep YM-30 filtration tubes.…”

Inhibition of the Yeast Cytochrome bc1 Complex by Ilicicolin H, a Novel Inhibitor That Acts at the Qn Site of the bc1 Complex

“…Purification of bc 1 Complexes-Yeast cytochrome bc 1 complex was isolated from yeast mitochondrial membranes as described previously (10,11). The bovine bc 1 complex was prepared from beef heart mitochondrial membranes using the same protocol for the yeast bc 1 complex, except that the membranes were extracted with 1.2 g of dodecyl maltoside per gram of protein, compared with 0.8 g of dodecyl maltoside per gram of protein used to extract the yeast enzyme.…”

“…4b. The effects of antimycin on the triphasic reduction have been described in detail elsewhere (11). Antimycin also slows the rate of cytochrome c 1 reduction through center P and the rate matches the slow phase of cytochrome b reduction.…”

Failure to Insert the Iron-Sulfur Cluster into the Rieske Iron-Sulfur Protein Impairs Both Center N and Center P of the Cytochrome bc 1 Complex

“…Purification of bc 1 Complexes-Yeast cytochrome bc 1 complexes were isolated as described previously (7,8). The CHS14 yeast mutant was obtained by site-directed mutagenesis as described by Denke et al (9).…”

“…Antimycin, DFP, phenylmethylsulfonyl fluoride, menaquinone, and dithionite were purchased from Sigma. Stigmatellin was purchased from Fluka Biochemica.Purification of bc 1 Complexes-Yeast cytochrome bc 1 complexes were isolated as described previously (7,8). The CHS14 yeast mutant was obtained by site-directed mutagenesis as described by Denke et al (9).…”