Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
Department of Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany.
J Biol Chem. 2022 Aug;298(8):102216. doi: 10.1016/j.jbc.2022.102216. Epub 2022 Jun 30.
Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H-dependent Fd reduction and Fd-dependent H production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N'-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na for activity and did not translocate Na into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H-translocating, H-reducing enzyme.
能量转换氢化酶（Ech）是古老的膜结合酶，它使用还原型铁氧还蛋白（Fd）作为电子供体将质子还原为分子氢。使用完整细胞、膜和囊泡级分的实验表明，质子还原与质膜质子跨膜转运偶联，但这从未用纯化酶证明过。为此，我们在嗜热厌氧细菌 Thermoanaerobacter kivui 中生产了带有 His 标签的 Ech 复合物。该酶可以通过亲和层析从溶解的膜中进行纯化，保留其所有八个亚基以及完整的生理活性，即 H 依赖性 Fd 还原和 Fd 依赖性 H 产生。我们发现纯化的酶含有 34.2±12.2 mol 铁/摩尔蛋白质，与七个预测的[4Fe-4S]-簇和一个[Ni-Fe]-中心一致。最适 pH 和温度分别为 7 至 8 和 66°C。值得注意的是，我们发现酶活性被 N,N'-二环己基碳二亚胺抑制，该试剂已知与埋藏在质膜中的离子转运谷氨酸或天冬氨酸结合，从而抑制离子转运。为了证明 Ech 复合物在离子转运中的功能，我们进一步建立了一种将酶复合物以活性状态掺入脂质体的程序。我们表明该酶不需要 Na 即可发挥活性，并且不会将 Na 转运到蛋白脂质体腔中。相反，Ech 活性导致 pH 梯度和质膜电位在蛋白脂质体膜两侧产生，表明 T. kivui 的 Ech 复合物是一种 H 转运、H 还原酶。
