Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

Hydrogenotrophic methanococci specialize in utilizing H2 as an electron donor, and these organisms possess six different Ni-Fe hydrogenases. These enzymes include two F420--reducing hydrogenases, two non-F420-reducing hydrogenases, and two membrane-bound hydrogenases (Eha and Ehb [5]). The F420-reducing hydrogenases reduce coenzyme F420, which subsequently reduces methenyltetrahydromethanopterin and methylenetetrahydromethanopterin, intermediates in the pathway of methanogenesis. In Methanococcus voltae, the F420-reducing hydrogenase is also reported to reduce the 2-mercaptoethanesulfonate:7-mercaptoheptanoylthreonine phosphate heterodisulfide formed in the final step of methanogenesis (2). In contrast, Methanothermobacter marburgensis utilizes the non-F420-reducing hydrogenase to reduce the heterodisulfide (22, 25). The two membrane-bound hydrogenases couple the chemiosmotic energy of ion gradients to H2 oxidation and ferredoxin reduction. In the aceticlastic methanogen Methanosarcina barkeri, the homologous enzyme is called energy conserving hydrogenase or Ech and performs a variety of physiological functions, including the generation of a proton motive force during CO oxidation and concomitant proton reduction in aceticlastic methanogenesis and the generation of low potential electron donors for CO2 reduction to formylmethanofuran in the first step of methanogenesis and the reductive carboxylation of acetyl coenzyme A (acetyl-CoA) to pyruvate in carbon assimilation (11, 12). In the hydrogenotrophic methanogens, it is predicted that the two energy-conserving hydrogenases (Eha and Ehb) have distinct roles (26). The Ehb appears to reduce low potential electron carriers utilized in autotrophic CO2 fixation (16). Anabolic enzymes likely to be coupled to Ehb in this manner include (i) the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and the pyruvate oxidoreductase (Por), which catalyze the first two steps of carbon assimilation; (ii) the α-ketoglutarate oxidoreductase (Kor), which catalyzes the final step in the incomplete reductive tricarboxylic acid cycle; and (iii) the indolepyruvate oxidoreductase (Ior) and the 2-oxoisovalerate oxidoreductase (Vor), which are involved in amino acid biosynthesis from aryl and branched-chain acids, respectively. Support for these conclusions comes in large part from the phenotype of an M. maripaludis ehb gene replacement mutant S40, which was only capable of limited growth in the absence of acetate and amino acids (16). Furthermore, expression of CODH/ACS, Por, and Vor were significantly upregulated in the mutant, providing further evidence for a role of Ehb in these processes (16). In contrast, there is no direct evidence for the role of Eha. By analogy with the Methanosarcina Ech, it could be involved in generating reducing equivalents for the reduction of CO2 to formylmethanofuran. Alternatively, hydrogenotrophic methanogens may have an alternative method of CO2 reduction (27), and Eha could have another function entirely. In spite of some functional similarities between the Ech of the aceticlastic methanogens and Eha or Ehb of hydrogenotrophs, the structures of their operons are very different (Fig. ​(Fig.1).1). Based upon sequence comparisons, all of these membrane-bound hydrogenases possess conserved large and small hydrogenase subunits, a 2[4Fe-4S] ferredoxin, and an integral membrane ion translocator (3, 8, 26). Otherwise, the structures are very different. The purified Ech from Methanosarcina barkeri contains six polypeptides encoded by the six genes of the ech operon (8, 11). The Eha and Ehb hydrogenases have never been purified. The eha and ehb operons from the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus comprise 20 and 17 genes, respectively (23, 26). Most of these genes are predicted to encode transmembrane proteins, although there are also several polyferredoxins and hydrophilic proteins (26). Many of these genes are not homologous to the M. barkeri ech genes. The Methanococcus maripaludis genome contains homologs to the M. thermautotrophicus eha and ehb genes, although only nine of the ehb genes are contiguous on the genome (Fig. ​(Fig.1).1). In the present study, the Ehb from the hydrogenotrophic methanogen Methanococcus maripaludis was analyzed. M. maripaludis is a model organism that can be easily genetically modified. Furthermore, its genome has been sequenced, and many of its biochemical pathways have been characterized. FIG. 1. Genetic map of Methanosarcina barkeri ech (A), Methanothermobacter marburgensis ehb (MTH1235-1251) (B), and Methanococcus maripaludis ehb (MMP1631-1629) (C) operons. Genes encoding integral membrane proteins found only in Ehb are indicated in blue, integral ...