Michel R, Massanz C, Kostka S, Richter M, Fiebig K
Institut für Pflanzenphysiologie und Mikrobiologie, Freien Universität Berlin, Germany.
Eur J Biochem. 1995 Nov 1;233(3):727-35. doi: 10.1111/j.1432-1033.1995.727_3.x.
The membrane-associated coenzyme F420-reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95-fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F420-reducing activity (82.8 mumol.min-1.mg protein-1 versus 11.5 mumol.min-1.mg protein-1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79-88]. The predominant coenzyme F420-reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non-identical subunits with apparent molecular masses of 48 (alpha), 33 (beta), and 30 kDa (gamma), apparently in a stoichiometry of alpha 2 beta 2 gamma 1. This minimal coenzyme F420-reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198-kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28-32 mol non-heme iron, and 34 mol acid-labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N-termini of the putative nickel-binding subunits of most [NiFe]- and [NiFeSe]hydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F420-reducing activity was obtained with reductively reactivated enzyme at 55 degrees C in the pH range 6.5-7.25. The Km values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3 microM and 4 microM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5 x 10(7) M-1.s-1 with coenzyme F420 and 6.9 x 10(7) M-1.s-1 with methylviologen.
厌氧产甲烷古菌巴氏甲烷八叠球菌Fusaro的膜相关辅酶F420反应性氢化酶已被纯化95倍，达到表观均一性。一种新的纯化方法和改变的储存条件使产量（13.4%对4.3%）和特定的辅酶F420还原活性（82.8 μmol·min⁻¹·mg蛋白⁻¹对11.5 μmol·min⁻¹·mg蛋白⁻¹）比之前报道的[Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79 - 88]显著更高。氢化酶的主要辅酶F420反应形式的表观分子量为198 kDa，由三个不同的亚基组成，表观分子量分别为48 kDa（α）、33 kDa（β）和30 kDa（γ），化学计量比明显为α₂β₂γ₁。这种最小的辅酶F420还原氢化酶形成了表观分子量约为845 kDa的聚集体。1摩尔198 kDa形式的氢化酶含有2摩尔FAD、2摩尔镍、28 - 32摩尔非血红素铁和34摩尔酸不稳定硫；此外，检测到0.2摩尔硒。其等电点为5.30。氨基酸序列PXXRXEGH（其中X为任意氨基酸）在产甲烷古菌和细菌的大多数[NiFe]-和[NiFeSe]氢化酶的假定镍结合亚基的N端被发现是保守的。然而，在[Fe]氢化酶的蛋白质序列中未检测到该基序。在55℃、pH范围6.5 - 7.25条件下，用还原再活化的酶可获得最大的辅酶F420还原活性。以辅酶F420或甲基紫精作为电子受体时，纯化酶对H₂的Km值极低，分别为3 μM和4 μM。两种可还原共底物的H₂催化效率系数（kcat/Km）都很高：以辅酶F420时为2.5×10⁷ M⁻¹·s⁻¹，以甲基紫精时为6.9×10⁷ M⁻¹·s⁻¹。
