Jacobson F S, Daniels L, Fox J A, Walsh C T, Orme-Johnson W H
J Biol Chem. 1982 Apr 10;257(7):3385-8.
A coenzyme F430-reducing hydrogenase from Methanobacterium thermoautotrophicum has been purified 25-fold, to approximately 50% homogeneity. Following anaerobic preincubation in high salt under reducing conditions, the purified enzyme exhibits equal catalytic activity (turnover number = 725 s-1) toward the artificial 1-electron acceptor, methyl viologen, and the physiological 2-electron acceptor, 7,8-didemethyl-8-hydroxy-5-deazaflavin (F420). The enzyme had the following Km values (micromolarity) under the described assay conditions: 420 (methyl viologen, pH 9.0), 19 (F420, pH 7.2), 34 (Fo, 7.8-didemethyl-8-hydroxy-5-deazariboflavin, pH 7.2), 10 (H2, Fo as co-substrate, pH 7.2), 2 (H2, methyl viologen as co-substrate, pH 9.0). The native protein is oligomeric (apparent Mr greater than 500,000) and is composed of three distinct subunits with Mr - 40,000, 31,000, and 26,000 in the ratio of 2:2:1, leading to a minimum Mr = 170,000. In addition to 33 atoms of Fe and 24 atoms of acid-labile sulfur, the F420-hydrogenase contains 2.3 mol of FAD/mol of Mr - 170,000. This activity is chromatographically distinct from a smaller methanogen hydrogenase capable of rapid viologen reduction, but which only very slowly reduces 5-deazariboflavins.
来自嗜热自养甲烷杆菌的辅酶F430还原氢化酶已被纯化了25倍，纯度约为50%。在还原条件下于高盐中进行厌氧预孵育后，纯化后的酶对人工单电子受体甲基紫精和生理双电子受体7,8-二去甲基-8-羟基-5-脱氮黄素（F420）表现出相同的催化活性（周转数 = 725 s-1）。在所述测定条件下，该酶具有以下Km值（微摩尔浓度）：420（甲基紫精，pH 9.0）、19（F420，pH 7.2）、34（Fo，7,8-二去甲基-8-羟基-5-脱氮核黄素，pH 7.2）、10（H2，以Fo作为共底物，pH 7.2）、2（H2，以甲基紫精作为共底物，pH 9.0）。天然蛋白质是寡聚体（表观分子量大于500,000），由三个不同的亚基组成，分子量分别为40,000、31,000和26,000，比例为2:2:1，最小分子量 = 170,000。除了33个铁原子和24个酸不稳定硫原子外，F420-氢化酶每摩尔分子量为170,000还含有2.3摩尔FAD。这种活性在色谱上与一种较小的产甲烷菌氢化酶不同，后者能够快速还原紫精，但只能非常缓慢地还原5-脱氮黄素。
