Schmitz Oliver, Boison Gudrun, Salzmann Heike, Bothe Hermann, Schütz Kathrin, Wang Shu-hua, Happe Thomas
Botanisches Institut der Universität Köln, Gyrhofstr. 15, D-50931 Cologne, Germany.
Biochim Biophys Acta. 2002 Apr 22;1554(1-2):66-74. doi: 10.1016/s0005-2728(02)00214-1.
NAD(P)(+)-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 micromol H(2) evolved (min mg protein)(-1) and 15 micromol H(2) evolved (min mg protein)(-1), respectively. H(2)-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 degrees C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)(2).
NAD(P)(+)还原氢化酶被认为由一个递氢酶（HoxFU）和一个氢化酶（HoxYH）部分组成。本研究首次提供了实验证据，表明在蓝细菌中，第五个亚基HoxE是这种双向氢化酶的一部分。HoxE与大肠杆菌呼吸复合体I的NuoE具有序列同源性。对蓝细菌双向氢化酶的亚基组成进行了研究。在厌氧条件下，从集胞藻属PCC 6803和聚球藻属PCC 6301中纯化出了氧不稳定的酶复合物，纯度接近均一。纯化后的酶分别富集了647倍和1290倍，比活性分别为46 μmol H₂释放/（分钟·毫克蛋白）⁻¹和15 μmol H₂释放/（分钟·毫克蛋白）⁻¹。集胞藻属PCC 6803纯化酶的H₂释放量在60℃和pH 6.3时最高。使用多克隆抗HoxE抗体进行的免疫印迹实验表明，在聚球藻属PCC 6301中，HoxE与氢化酶活性共同纯化。纯化酶的SDS-PAGE凝胶显示有六种蛋白质，除了一种非氢化酶成分外，部分测序并鉴定为HoxF、HoxU、HoxY、HoxH，值得注意的是，还有HoxE。天然蛋白的分子量（375 kDa）表明该酶复合物为二聚体组装形式，即Hox(EFUYH)₂。
