Colbeau A, Elsen S, Tomiyama M, Zorin N A, Dimon B, Vignais P M
CEA/Grenoble, Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR CEA/CNRS no. 314), Département de Biologie Moléculaire et Structurale, France.
Eur J Biochem. 1998 Jan 15;251(1-2):65-71. doi: 10.1046/j.1432-1327.1998.2510065.x.
The photosynthetic bacterium Rhodobacter capsulatus contains a membrane-bound [NiFe]hydrogenase encoded by the hupSL genes. We show in this study that hypF mutants are devoid of hydrogenase activity and lack the HupL protein. We also observed that, in contrast to the wild-type strain B10, transcription of the hupSL genes was not stimulated by H2 in the hypF mutants RS13 and BSE19. Complementation of the hypF mutants with the plasmid borne hypF gene restored hydrogenase activity to wild-type levels and inducibility by H2. The R. capsulatus hupU and hupV gene products share significant similarities with the small (HupS) and the large (HupL) hydrogenase subunits, respectively. Active HupUV proteins can catalyze the hydrogen-deuterium exchange reaction. In whole cells, this H-D exchange is distinguishable from the H-D exchange catalyzed by the membrane-bound HupSL proteins by its insensitivity to O2 and to acetylene. By measuring the formation of H2 and HD in exchange with D2 uptake, we demonstrated that the hypF mutants have no active HupUV nor HupSL proteins. H-D exchange activity, of both HupUV and HupSL, was restored by hypF gene complementation. These data indicate that the HypF protein participates not only in the maturation of HupSL, but also in the maturation of the HupUV proteins and that the latter are involved in the cellular response to H2.
光合细菌荚膜红细菌含有一种由hupSL基因编码的膜结合[NiFe]氢化酶。我们在本研究中表明，hypF突变体缺乏氢化酶活性且缺少HupL蛋白。我们还观察到，与野生型菌株B10不同，在hypF突变体RS13和BSE19中，hupSL基因的转录不受H2刺激。用携带hypF基因的质粒对hypF突变体进行互补，可将氢化酶活性恢复到野生型水平，并恢复对H2的诱导性。荚膜红细菌的hupU和hupV基因产物分别与小（HupS）和大（HupL）氢化酶亚基有显著相似性。活性HupUV蛋白可催化氢-氘交换反应。在全细胞中，这种H-D交换与膜结合的HupSL蛋白催化的H-D交换的区别在于它对O2和乙炔不敏感。通过测量与D2摄取交换时H2和HD的形成，我们证明hypF突变体没有活性HupUV和HupSL蛋白。hypF基因互补恢复了HupUV和HupSL的H-D交换活性。这些数据表明，HypF蛋白不仅参与HupSL的成熟，还参与HupUV蛋白的成熟，并且后者参与细胞对H2的反应。
