menaquinone-6 and ubiquinol

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography

Topics: Bacteriochlorophylls; Binding Sites; Chlorophyll; Crystallography; Cytoplasm; Electron Transport; Electrons; Hyphomicrobiaceae; Lasers; Models, Molecular; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protons; Ubiquinone; Vitamin K 2

Cytochrome

Topics: Binding Sites; Cytochrome b Group; Escherichia coli; Escherichia coli Proteins; Kinetics; Plastoquinone; Protein Binding; Ubiquinone; Vitamin K 2

Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).

Topics: Bacillus subtilis; Benzoquinones; Chromatography, High Pressure Liquid; Electrochemistry; Electron Spin Resonance Spectroscopy; Electron Transport Complex IV; Escherichia coli; Hydrogen Bonding; Models, Chemical; Mutagenesis, Site-Directed; Nitrogen; Photosystem I Protein Complex; Ubiquinone; Vitamin K; Vitamin K 2

A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske iron-sulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c(1). A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol-cytochrome c reductase kinetics of the bc(1) complex include both a pH dependence and a dependence on Rieske iron-sulfur protein midpoint potential.An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc(1) complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc(1) complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and 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. An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc(1) activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.

Topics: Antimycin A; Binding Sites; Cytochrome b Group; Dimerization; Electron Transport; Electron Transport Complex III; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Kinetics; Models, Molecular; NADH Dehydrogenase; Proton-Motive Force; Thermodynamics; Ubiquinone; Vitamin K 2

A quinol-oxidase activity was detected in crude extracts of the thermoacidophilic archaebacterium Thermoplasma acidophilum. The activity was optimal at pH 5.4 and 50 degrees C. The Km for ubiquinol-10 was 18 microM. The enzyme was inhibited by 2n-heptyl-4-hydroxyquinoline N-oxide with a Ki of 150 nM. Ubiquinols with different side-chain lengths were oxidized at similar rates, whereas menaquinols were converted at 6-12-fold higher rates compared to ubiquinols. Membranes from T. acidophilum contain cytochromes of b, d and a1 types, as shown by optical spectroscopy. CO difference spectroscopy suggests the existence of a cytochrome o. A b-type cytochrome with an apparent molecular mass of 18 kDa was purified in the presence of high detergent concentrations. It readily forms dimers on SDS/PAGE. This cytochrome also contains Cu, as shown by atomic-absorption spectroscopy. Redox titration suggests that the 18-kDa cytochrome may contain 2 heme groups; b558 with a midpoint potential of 75 mV and b562/553 with a midpoint potential of -150 mV.

Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Cytochrome b Group; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Hydroxyquinolines; Kinetics; Molecular Weight; Oxidoreductases; Substrate Specificity; Temperature; Thermoplasma; Ubiquinone; Vitamin K; Vitamin K 2