menaquinol-6 and ubiquinol

The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 A of resolution. We have shown that pentachlorophenol is a potent inhibitor of quinol:nitrate oxidoreductase activity and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI). This new structural information together with site-directed mutagenesis data, biochemical analyses, and molecular modeling provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI. A possible proton conduction pathway linked to electron transfer reactions has also been defined, providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.

Topics: Binding Sites; Cell Membrane; Crystallography, X-Ray; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Escherichia coli; Heme; Histidine; Hydroxyquinolines; Kinetics; Lysine; Models, Chemical; Models, Molecular; Mutation; Naphthols; Nitrate Reductase; Nitrate Reductases; Oxidoreductases; Oxygen; Pentachlorophenol; Plasmids; Protein Binding; Protons; Terpenes; Ubiquinone

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.

Topics: Animals; Cattle; Electron Transport Complex III; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Mutagenesis, Site-Directed; Naphthols; Oxidation-Reduction; Saccharomyces cerevisiae; Terpenes; Ubiquinone