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

David J. Richardson, Alastair G. McEwan, M. Dudley Page, J. Baz Jackson, Stuart J. Ferguson

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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 α‐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 χ‐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.

Original languageEnglish
Pages (from-to)263-270
Number of pages8
JournalEuropean Journal of Biochemistry
Issue number1
Publication statusPublished - Nov 1990

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