Enzymes of hydrogen metabolism in Pyrococcus furiosus
Enzymes of hydrogen metabolism in Pyrococcus furiosus
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DOI：
10.1046/j.1432-1327.2000.01745.x
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发表时间：
2000-11-01
期刊：
影响因子：
--
通讯作者：
Hagen, WR
中科院分区：
文献类型：
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作者：
Silva, PJ;van den Ban, ECD;Hagen, WR
狂热杆菌基因组中含有一个与镍铁氢酶相关的14亚基跨膜复合体的操纵子。10个ORF(MbhA-I和MbhM)编码疏水性的跨膜亚基。4个ORF(mbhJKL和mbhN)编码可能的可溶性蛋白。其中两个对应于典型的镍铁氢酶小亚基和大亚基，但小亚基只能配位一个铁硫簇，对应于近端的[4Fe-4S]立方烷。在基因组中，大亚基和小亚基的结构基因由第三个开放阅读框MbhK分开，该ORF编码一种功能未知的蛋白质，不与铁和S结合。第四个ORF，mbhN，编码一个2[4Fe-4S]蛋白。以P.Furiosus可溶性[4Fe-4S]铁氧还蛋白为电子供体，膜产生H-2，在温和的条件下溶解和部分纯化时，这种活性保留在MBH操纵子的抽提核心复合体中。这种膜结合氢酶的性质是独一无二的。它对一氧化碳的抑制作用相当强。与其他氢酶相比，它还表现出极高的放氢活性与吸氢活性的比率。其活性对NADH脱氢酶抑制剂二环己基碳二亚胺(络合物I)的抑制很敏感。还原核心复合体的EPR是铁-硫簇与E-m约为-0.33V相互作用的特征。基因组包含第二个可能的操纵子mbxABCDFGHH‘MJKLN，用于一个与MBH操纵子同源性很高的多亚单位跨膜复合体，然而，在大的氢酶亚基中，Ni-Fe-簇具有非常不寻常的结合基序。通过对膜结合氢酶、可溶性氢酶和硫化物脱氢酶活性的动力学研究，提出了一个全面的工作假说，即通过能量传递、转移、回收和安全阀门装置连接的三个还原当量(铁氧还蛋白、NADPH、H-2)，来描述赤潮假单胞菌的H-2代谢。
The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni-Fe hydrogenase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. furiosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes produce H-2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H-2 evolution to H-2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with E-m approximate to -0.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H-2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H-2) connected by devices for transduction, transfer, recovery and safety-valving of energy.