Carr G J, Ferguson S J
Department of Biochemistry, University of Oxford, U.K.
Biochem J. 1990 Jul 15;269(2):423-9. doi: 10.1042/bj2690423.
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.
反硝化副球菌细胞质膜的一氧化氮（NO）还原酶活性可用十二烷基麦芽糖苷溶解，并能很好地保留活性。溶解后的酶缺乏依赖NADH的活性，但可用异抗坏血酸加2,3,5,6-四甲基对苯二胺作为电子供体，并以马心细胞色素c作为介质进行测定。用电流型电极测量NO的还原情况。通过几步色谱法可将溶解后的酶与其他电子传递成分（包括细胞色素bc1复合物和亚硝酸还原酶）分离。纯化后的酶比活性为11 μmol·min⁻¹·mg蛋白质⁻¹，Km(NO)估计小于10 μM。该酶以预期的化学计量比由NO生成N₂O。这些观察结果支持了NO还原酶是一种参与反硝化过程的独立酶的观点。该酶含有b型和c型血红素。前者与表观分子量为37 kDa的多肽相关，后者与18 kDa的多肽相关。在纯化的蛋白质中还鉴定出了29 kDa和45 kDa的多肽，它们在聚丙烯酰胺凝胶电泳中表现出可变的行为。
