Inhibitory analogs of ubiquinol act anti-cooperatively on the Yeast cytochrome bc1 complex. Evidence for an alternating, half-of-the-sites mechanism of ubiquinol oxidation.
The cytochrome bc(1) complex is a dimeric enzyme that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is re-reduced at a second center, referred to as center N. To understand better the mechanism of ubiquinol oxidation, we have examined the interaction of several inhibitory analogs of ubiquinol with the yeast cytochrome bc(1) complex. Stigmatellin and methoxyacrylate stilbene, two inhibitors that block ubiquinol oxidation at center P, inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex, indicating that one molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme. This stoichiometry was obtained when the inhibitors were titrated in cytochrome c reductase assays and in reactions of quinol with enzyme in which the inhibitors block pre-steady state reduction of cytochrome b. As an independent measure of inhibitor binding, we titrated the red shift in the optical spectrum of ferrocytochrome b with methoxyacrylate stilbene and thus confirmed the results of the inhibition of activity titrations. The titration curves also indicate that the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. Because these inhibitors bind to the ubiquinol oxidation site in the bc(1) complex, we propose that the yeast cytochrome bc(1) complex oxidizes ubiquinol by an alternating, half-of-the-sites mechanism.
Gutierrez-Cirlos
Emma Berta
EB
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Trumpower
Bernard L
BL
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000968:Antimycin A / Q000031:analogs & derivatives* / Q000494:pharmacology; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011090:Polyenes / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; D011090:Polyenes; D013267:Stilbenes; C032456:antimycin; D014451:Ubiquinone; D000968:Antimycin A; C041573:stigmatellin; D014450:Electron Transport Complex III; C003741:ubiquinol