The identification of cytochromes involved in the transfer of electrons to the periplasmic NO⁻₃ reductase of Rhodobacter capsulatus and resolution of a soluble NO⁻₃ ‐reductase − cytochrome‐c₅₅₂ redox complex

The involvement of cytochromes in the electron‐transport pathway to the periplasmic NO⁻₃ reductase of Rhodobacter capsulatus was studied in cells grown photoheterotrophically in the presence of nitrate with butyrate as carbon source. The specific rate of NO⁻₃ reduction by such cells was five times higher than when malate was carbon source. Reduced minus NO⁻₃‐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 NO⁻₃. The total ferricyanide‐oxidizable cytochrome that was also oxidized in the steady state by NO⁻₃‐was greater in cells grown with butyrate rather than malate. Low concentrations of cyanide inhibited NO⁻₃ reduction. Neither CN⁻, nor a previously characterized inhibitor of NO⁻₃ reduction, 2‐n‐heptyl‐4‐hydroxyquinoline N‐oxide, prevented the oxidation of the cytochromes by NO⁻₃. 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 NO⁻₃ 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 NO⁻₃ to a periplasmic fraction. The NO⁻₃‐oxidizable cytochrome co‐purified with the periplasmic NO⁻₃ reductase through fractionation procedures that included ammonium sulphate precipitation, gel filtration at low and high salt concentrations, and ion‐exchange chromatography. A NO⁻₃‐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.