Schmidt H L, Stöcklein W, Danzer J, Kirch P, Limbach B
Eur J Biochem. 1986 Apr 1;156(1):149-55. doi: 10.1111/j.1432-1033.1986.tb09560.x.
An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.- is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made.
最近已报道过的一种来自粪链球菌的生成H₂O的NADH氧化酶[霍斯金斯，D.D.，怀特利，H.R.和麦克勒，B.（1962年）《生物化学杂志》237卷，2647 - 2651页]，通过两步亲和层析法被分离出来，得到一种均一的蛋白质，比活性为690 U/mg，总产率为50%。该酶不含金属，分子量约为51000 Da，可能由一条单一的多肽链组成。荧光滴定表明，辅基为1摩尔FAD/摩尔蛋白质。该酶的亲和行为证明存在一个能够结合NADH或FAD的二核苷酸结合结构域。该酶对NADH具有特异性（Km = 4.1×10⁻⁵ M），NADPH不被氧化。O₂是首选的电子受体，此外FAD以及非常缓慢地，单电子受体也会被还原。目前尚不清楚FAD的还原是通过二核苷酸结合位点进行还是通过辅基的交换进行。与O₂反应的化学计量关系对应于每摩尔O₂消耗2摩尔NADH，并且只生成H₂O（2 NADH + 2 H⁺ + O₂→2 NAD⁺ + 2 H₂O）。未检测到H₂O₂或超氧阴离子作为中间产物，并且H₂O₂不能替代O₂作为氧化剂。该酶主要在其还原状态下可被巯基试剂抑制。光谱数据没有提供还原过程中存在自由基中间体的证据。该酶显然每摩尔能够接受超过两个电子。基于这些数据讨论了两种可能的反应机制。还对该反应的生物学目的提出了一个建议。
