Vignais P M, Dimon B, Zorin N A, Tomiyama M, Colbeau A
CEA/Grenoble, Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR CEA/CNRS/UJF no. 5092), Département de Biologie Moléculaire et Structurale, 38054 Grenoble Cedex 9, France.
J Bacteriol. 2000 Nov;182(21):5997-6004. doi: 10.1128/JB.182.21.5997-6004.2000.
Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4. 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.
荚膜红细菌能合成两种同源蛋白质复合物，一种是与呼吸链相连的膜结合[NiFe]氢化酶（HupSL），能够激活分子氢（H₂），另一种是由hupUV基因编码的H₂传感器。比较研究了在D₂和H₂O存在的情况下，由hupSL编码的酶和hupUV编码的酶催化的氢-氘（H-D）交换活性。HupSL存在于膜中，而HupUV的活性定位于可溶性细胞质部分。由于荚膜红细菌的氢化酶基因簇包含一个与hoxH同源的基因，hoxH编码与NAD相连的四聚体可溶性氢化酶的大亚基，因此使染色体上的hoxH基因失活，并使用hoxH突变体来证明细胞质HupUV蛋白质复合物的H-D交换活性。由HupSL氢化酶催化的H-D交换反应在pH 4.5时最大，并受到乙炔和氧气的抑制，而由HupUV蛋白质复合物催化的H-D交换对乙炔和氧气不敏感，并且在pH 4至pH 11之间没有显著变化。基于这些特性，已证明辅助hypD基因的产物是合成活性HupUV酶所必需的。HupUV与D₂交换形成HD和H₂的动力学表明质子和气体进入活性位点受到限制。通过质谱法测量D₂、HD和H₂的浓度变化表明，除了H-D交换反应外，HupUV还能将H₂与苄基紫精氧化，用还原的甲基紫精产生H₂，并表现出真正的氢化酶活性。因此，不仅就其在细胞中的H₂信号功能而言，而且就其催化特性而言，HupUV酶代表了一类独特的氢化酶。
