Magnani P, Doussiere J, Lissolo T
Laboratoire TEPE, ESIGEC, Université de Savoie, Le Bourget Du Lac, France.
Biochim Biophys Acta. 2000 Jul 20;1459(1):169-78. doi: 10.1016/s0005-2728(00)00145-6.
The photosynthetic bacterium Rhodobacter capsulatus synthesises a membrane-bound [NiFe] hydrogenase encoded by the H2 uptake hydrogenase (hup)SLC structural operon. The hupS and hupL genes encode the small and large subunits of hydrogenase, respectively; hupC encodes a membrane electron carrier protein which may be considered as the third subunit of the uptake hydrogenase. In Wolinella succinogenes, the hydC gene, homologous to hupC, has been shown to encode a low potential cytochrome b which mediates electron transfer from H2 to the quinone pool of the bacterial membrane. In whole cells of R. capsulatus or intact membrane preparation of the wild type strain B10, methylene blue but not benzyl viologen can be used as acceptor of the electrons donated by H2 to hydrogenase; on the other hand, membranes of B10 treated with Triton X-100 or whole cells of a HupC- mutant exhibit both benzyl viologen and methylene blue reductase activities. We report the effect of diphenylene iodonium (Ph2I), a known inhibitor of mitochondrial complex I and of various monooxygenases on R. capsulatus hydrogenase activity. With H2 as electron donor, Ph2I inhibited partially the methylene blue reductase activity in an uncompetitive manner, and totally benzyl viologen reductase activity in a competitive manner. Furthermore, with benzyl viologen as electron acceptor, Ph2I increased dramatically the observed lagtime for dye reduction. These results suggest that two different sites exist on the electron donor side of the membrane-bound [NiFe] hydrogenase of R. capsulatus, both located on the small subunit. A low redox potential site which reduces benzyl viologen, binds Ph2I and could be located on the distal [Fe4S4] cluster. A higher redox potential site which can reduce methylene blue in vitro could be connected to the high potential [Fe3S4] cluster and freely accessible from the periplasm.
光合细菌荚膜红细菌合成一种由吸氢氢化酶（hup）SLC结构操纵子编码的膜结合[NiFe]氢化酶。hupS和hupL基因分别编码氢化酶的小亚基和大亚基；hupC编码一种膜电子载体蛋白，可被视为吸氢氢化酶的第三个亚基。在产琥珀酸沃林氏菌中，与hupC同源的hydC基因已被证明编码一种低电位细胞色素b，它介导电子从H2转移到细菌膜的醌池。在荚膜红细菌的全细胞或野生型菌株B10的完整膜制剂中，亚甲基蓝而非苄基紫精可作为H2向氢化酶供电子的受体；另一方面，用 Triton X-100处理的B10膜或HupC-突变体的全细胞同时表现出苄基紫精和亚甲基蓝还原酶活性。我们报道了二亚苯基碘鎓（Ph2I），一种已知的线粒体复合体I和各种单加氧酶抑制剂，对荚膜红细菌氢化酶活性的影响。以H2作为电子供体时，Ph2I以非竞争性方式部分抑制亚甲基蓝还原酶活性，并以竞争性方式完全抑制苄基紫精还原酶活性。此外，以苄基紫精作为电子受体时，Ph2I显著增加了观察到的染料还原延迟时间。这些结果表明，荚膜红细菌膜结合[NiFe]氢化酶的电子供体侧存在两个不同的位点，均位于小亚基上。一个低氧化还原电位位点可还原苄基紫精，结合Ph2I，可能位于远端[Fe4S4]簇上。一个较高氧化还原电位位点在体外可还原亚甲基蓝，可能与高电位[Fe3S4]簇相连，可从周质自由接近。
