Mechanism of ubiquinol oxidation by the cytochrome bc1 complex: pre-steady-state kinetics of cytochrome bc1 complexes containing site-directed mutants of the Rieske iron-sulfur protein.
To facilitate characterization of mutated cytochrome bc1 complexes in S. cerevisiae we have developed a new approach using a rapid scanning monochromator to examine pre-steady-state reduction of the enzyme with menaquinol. The RSM records optical spectra of cytochromes b and c1 at 1-ms intervals after a dead time of 2 ms, and menaquinol fully reduces both cytochromes bH and c1 and a portion of cytochrome bL. The rapid-mixing, rapid-scanning monochromator methodology obviates limitations inherent in previous rapid kinetics methods and permits measurements of rates exceeding 200 s-1. To document the validity of this methodology we have examined the reduction kinetics of the cytochrome bc1 complexes from wild-type yeast and yeast that lack ubiquinone. The results establish that menaquinol reacts via the Q cycle pathway both in the presence and absence of ubiquinone. From analyzing bc1 complexes containing Rieske proteins in which the midpoint potential of the iron-sulfur cluster has been altered from +280 to +105 mV, we propose a mechanism in which the protonated quinol displaces a proton from the imidazole nitrogen of one of the histidines that is a ligand to the iron-sulfur cluster and forms a quinol-imidazolate complex that is the electron donor to the redox active iron.
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
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D000818:Animals; D002417:Cattle; D014450:Electron Transport Complex III / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009284:Naphthols / Q000378:metabolism; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae; D013729:Terpenes / Q000378:metabolism; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D007506:Iron-Sulfur Proteins; D009284:Naphthols; C037989:Rieske iron-sulfur protein; D013729:Terpenes; D014451:Ubiquinone; C066500:menaquinol 6; D014450:Electron Transport Complex III; C003741:ubiquinol