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 be, 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 c(1) at 1-ms intervals after a dead time of 2 ms, and menaquinol fully reduces both cytochromes b(H) and c(1) and a portion of cytochrome b(L). 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 be, 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 bc(1) 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. (C) 1998 Elsevier Science B.V.