title: The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex.
abstract: The involvement of cytochromes in the electron-transport pathway to the periplasmic NO3- reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO3- reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO3(-)-oxidized spectra of cells had peaks in the alpha-band region for cytochromes at 552 nm and 559 nm, indicating the involvement of c- and b-type cytochromes in the electron-transport pathway to NO3-. The total ferricyanide-oxidizable cytochrome that was also oxidized in the steady state by NO3- was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO3- reduction. Neither CN-, nor a previously characterized inhibitor of NO3- reduction, 2-n-heptyl-4-hydroxyquinoline N-oxide, prevented the oxidation of the cytochromes by NO3-. This suggested a site of action for these inhibitors on the reducing side of the b- and c-type cytochromes involved in electron transport to the NO3- reductase. The predominant cytochrome in a periplasmic fraction prepared from cells of R. capsulatus grown on butyrate medium was cytochrome c2 but a c-type cytochrome with an alpha-band reduced absorbance maximum at 552 nm could also be identified. The reduced form of this latter cytochrome, but not that of cytochrome c2, was oxidized upon addition of NO3- to a periplasmic fraction. The NO3(-)-oxidizable cytochrome co-purified with the periplasmic NO3- reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion-exchange chromatography. A NO3(-)-reductase-cytochrome-c552 redox complex that comprised two types of polypeptide, a nitrate reductase subunit and a c-type cytochrome subunit, was purified. The polypeptides were separated when the complex was chromatographed on a phenyl-Sepharose hydrophobic chromatography column.
authors.affiliation: School of Biochemistry, University of Birmingham, England.
authors.forename: D J
authors.initials: DJ
authors.lastname: Richardson
authors.forename: A G
authors.initials: AG
authors.lastname: McEwan
authors.forename: M D
authors.initials: MD
authors.lastname: Page
authors.forename: J B
authors.initials: JB
authors.lastname: Jackson
authors.forename: S J
authors.initials: SJ
authors.lastname: Ferguson
mesh_terms: D002458:Cell Fractionation; D003486:Cyanides / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D006912:Hydroxyquinolines / Q000494:pharmacology; D046911:Macromolecular Substances; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D013057:Spectrum Analysis; D014451:Ubiquinone / Q000378:metabolism / Q000031:analogs & derivatives
chemical_list: D003486:Cyanides; D003574:Cytochrome c Group; D006912:Hydroxyquinolines; D046911:Macromolecular Substances; D009566:Nitrates; C083951:cytochrome c553; D014451:Ubiquinone; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; C022302:cytochrome C-552; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C003741:ubiquinol