The role of the energy conserving hydrogenase b in autotrophy and the characterization of sulfur metabolism in Methanococcus maripaludis
Major, Tiffany Ann
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The energy-conserving hydrogenase b (Ehb) in hydrogenotrophic methanogens such as Methanococcus maripaludis couples the oxidation of H to the production of a proton motive force. The electrons and energy generated by the Ehb are used by enzymes involved in carbon fixation, which catalyze endergonic reactions requiring low-potential electrons. To characterize the Ehb function, several ehb genes were deleted. Most ehb deletion strains exhibited reduced growth phenotypes under autotrophic conditions (i.e., in minimal medium). Furthermore, growth of the Ehb large hydrogenase mutant strain S965 was inhibited by aryl acids. The indolepyruvate oxidoreductase (Ior) is a carbon assimilatory enzyme that converts aryl acids into amino acids. The lack of S965 growth on aryl acids provides the first physiological evidence that the Ehb activity is associated with the Ior function. Electron transport from the Ehb is performed by ferredoxins. The sequenced methanogen genomes contain a large number of open reading frames that contain CXCXCXC motifs, which bind 4Fe-4S clusters. Two proteins predicted to shuttle electrons between the Ehb and the pyruvate oxidoreductase are the PorE and PorF. Characterization of recombinant histidine-tagged PorE and PorF from E. coli was attempted; however, results were inconclusive due to protein insolubility during purification and the lack of PorE- or PorF-specific assays. Sulfur assimilation is necessary to produce both iron-sulfur clusters and cysteine. A pathway was proposed where cysteine biosynthesis occurs by tRNA-loading in the hydrogenotrophic methanogens. In vivo characterization of this pathway involved the deletion of the sepS gene in M. maripaludis, which encodes a protein that converts O-phosphoserine-CysCys tRNA to cysteine-tRNA in vitro. The sepS deletion strain S210 was a cysteine auxotroph, suggesting that no other mechanism of cysteine biosynthesis exists in this organism. Attempts to delete the gene encoding the second enzyme in this pathway were unsuccessful, suggesting that this gene may be essential. The quantity of cysteine necessary for S210 growth is less than the calculated value for the total thiols within M. maripaludis. As free cysteine is the sulfur source for thiol-containing cofactors and prosthetic groups in other organisms, this result suggests that this organism also uses an alternative method of sulfur assimilation. Current studies are underway to quantify cellular pools of free cysteine and homocysteine.