title: Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis.
abstract: 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).
authors.affiliation: Departments of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
authors.forename: Sophia M
authors.initials: SM
authors.lastname: Yi
authors.forename: Kuppala V
authors.initials: KV
authors.lastname: Narasimhulu
authors.forename: Rimma I
authors.initials: RI
authors.lastname: Samoilova
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Dikanov
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D016227:Benzoquinones / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000737:chemistry; D045331:Photosystem I Protein Complex / Q000737:chemistry; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000737:chemistry; D014812:Vitamin K / Q000737:chemistry; D024482:Vitamin K 2 / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D016227:Benzoquinones; D045331:Photosystem I Protein Complex; D024482:Vitamin K 2; D014812:Vitamin K; D014451:Ubiquinone; C025232:semiquinone radicals; C062629:menaquinone 7; D003576:Electron Transport Complex IV; C003741:ubiquinol; D009584:Nitrogen