ubiquinone and menaquinol-6

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

Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.

Topics: Anilides; Dinitrophenols; Electron Transport Complex II; Enzyme Inhibitors; Escherichia coli; Eukaryotic Cells; Fumarates; Hydrogen-Ion Concentration; Hydroxyquinolines; Kinetics; Multienzyme Complexes; Naphthols; Oxidoreductases; Pentachlorophenol; Prokaryotic Cells; Succinate Dehydrogenase; Succinic Acid; 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