The nitric oxide reductase of Paracoccus denitrificans.
The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.
Carr
G J
GJ
Department of Biochemistry, University of Oxford, U.K.
Ferguson
S J
SJ
D001205:Ascorbic Acid / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D003574:Cytochrome c Group / Q000378:metabolism; D003902:Detergents; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology*; D012995:Solubility; D013053:Spectrophotometry; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D003574:Cytochrome c Group; D003902:Detergents; D005960:Glucosides; D009243:NAD; C005056:isoascorbic acid; D009569:Nitric Oxide; C040358:dodecyl maltoside; D010088:Oxidoreductases; C034747:nitric-oxide reductase; D013772:Tetramethylphenylenediamine; D001205:Ascorbic Acid