Meuer J, Bartoschek S, Koch J, Künkel A, Hedderich R
Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany.
Eur J Biochem. 1999 Oct 1;265(1):325-35. doi: 10.1046/j.1432-1327.1999.00738.x.
Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.
最近研究表明，巴氏甲烷八叠球菌可产生一种多亚基膜结合[NiFe]氢化酶，命名为Ech（大肠杆菌氢化酶3）氢化酶。在本研究中，Ech氢化酶以高产率纯化至表观均一性。所获得的酶制剂仅含有先前已证明由ech操纵子编码的六种多肽。经测定，纯化后的酶每摩尔酶含有0.9摩尔镍、11.3摩尔非血红素铁和10.8摩尔酸不稳定硫。利用纯化后的酶测定了动力学参数。该酶在pH 7.0时催化依赖于H2的巴氏甲烷八叠球菌2[4Fe-4S]铁氧化还原蛋白还原，比活性为50 U·mg蛋白-1，对铁氧化还原蛋白的表观Km为1 μM。该酶还以还原型铁氧化还原蛋白作为电子供体，在pH 7.0时以90 U·mg蛋白-1的速率催化氢气生成。还原型铁氧化还原蛋白的表观Km为7.5 μM。铁氧化还原蛋白的还原或氧化速率分别与氧化型或还原型甲基紫精的还原或氧化速率相似。H2的表观Km为5 μM。动力学数据有力地表明，铁氧化还原蛋白是Ech氢化酶的生理电子供体或受体。根据定量蛋白质免疫印迹实验计算，在以乙酸盐、甲醇或H2/CO2培养的巴氏甲烷八叠球菌细胞中，Ech氢化酶约占细胞总蛋白的3%。Ech氢化酶的功能被认为是在乙酸盐生长过程中，与铁氧化还原蛋白相关的H2产生与乙酰辅酶A羰基氧化为CO2相偶联；在H2/CO2或甲醇生长过程中，与铁氧化还原蛋白相关的H2摄取与CO2还原为CO的氧化还原状态相偶联。
