menaquinone-7 and ubiquinol

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 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