Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 148-161). Also available via the Internet.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 53-69). Also available via the Internet.

The iron-sulfur flavoprotein (Isf) from the acetate utilizing methanoarchaeon Methanosarcina thermophila was heterologously produced in Escherichia coli, purified to homogeneity, and characterized to determine the properties of the iron-sulfur cluster and FMN. Chemical and spectroscopic analyses indicated that Isf contained one 4Fe-4S cluster and one FMN per monomer. The midpoint potentials of the [4Fe-4S]2+/1+ center and FMN/FMNH2 redox couple were -394 and -277 mV respectively. The deduced amino acid sequence of Isf revealed high identity with Isf homologues from the CO2 reducing methanoarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum. Extracts of H2-CO2-grown M. thermoautotrophicum cells were able to reduce Isf from M. thermophila using either H2 or CO as the reductant. Addition of ferredoxin A to the reaction further stimulated the rate of Isf reduction. These results suggest that Isf homologues are coupled to ferredoxin in electron transfer chains in methanoarchaea with diverse metabolic pathways. Reconstituted systems containing carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS), ferredoxin A, Isf, and the designated electron carriers (NAD, NADP, F420, and 2-hydroxyphenazine) were used in an attempt to determine the electron acceptor for Isf. Isf was unable to reduce any of these compounds. Furthermore, 2-hydroxyphenazine competed with Isf to accept electrons from ferredoxin A indicating that ferredoxin A is a more favorable electron partner for 2-hydroxyphenazine. Thus, the physiological electron acceptor for Isf is unknown. Amino acid sequence alignment of Isf sequences revealed a conserved atypical cysteine motif with the potential to ligate the 4Fe-4S cluster. Site-directed mutagenesis of the cysteine residues in this motif, and the two additional cysteines in the sequence, was used to investigate these cysteine residue as ligands for coordinating the 4Fe-4S center of Isf. Spectroscopic and biochemical analyses were consistent with the conserved cysteine motif functioning as ligating the 4Fe-4S center. Redox properties of the 4Fe-4S and FMN centers revealed a role for the 4Fe-4S center in the transfer of electrons from ferredoxin A to FMN. / Ph. D.

The CO dehydrogenase~linked ferredoxin from acetate-grown <i>Methanosarcina thermophiIa</i> was characterized to determine the structure and biochemical properties of the iron-sulfur clusters. Chemical and spectroscopic analyses indicated that the ferredoxin contained two [4Fe-4S] clusters per monomer of 6,790 Da, although a [3Fe-4S] species was also detected in the oxidized protein. The midpoint potentials of the [4Fe-4S] and [3Fe~4S] clusters at pH 7 were -407 m V and + 103 m V, respectively. Evidence from biochemical and spectroscopic studies indicated that the [3Fe-4S] species may have been formed from [4Fe-4S] clusters when ferredoxin was oxidized. The gene encoding the CO dehydrogenase-linked ferredoxin (<i>fdxA</i>) in <i>Ms. thermophila</i> had the coding capacity for a 6,230-Da protein which contained eight cysteines with spacings typical of 2[4Fe-4S] ferredoxins. A second open reading frame (ORF1) was also identified which had the potential to encode a 2[4Fe-4S] bacterial-like ferredoxin (5,850 Da). The deduced proteins from <i>fdxA</i> and ORF1 were 62% identical. <i>fdxA</i> and ORFI were present as single copies in the genome and each was transcribed on a monocistronic mRNA. Both <i>fdxA</i> and ORF1 were transcribed in cells grown on methanol and trimethylamine, but only the <i>fdxA</i> -specific transcript was detected in acetate-grown cells. The apparent transcriptional start sites of <i>fdxA</i> and ORFI were downstream of sequences which had high identity with the consensus methanogen promoter. The heterodisulfide of two cofactors unique to the methanogenic microorganisms, HS-HTP and HS-CoM, was enzymatically reduced in cell extracts of <i>Ms. thermophila</i> using electrons from the oxidation of either H₂ or CO. The homodisulfides of either HS-HTP or HS-CoM were not reduced under the same conditions. The results indicated that methane is formed by reductive demethylation of CH₃-S-CoM using HS-HTP as a reductant in <i>Ms. thermophila</i>. Coupling of CO oxidation with reduction of the heterodisulfide suggested that the CO dehydrogenase-linked ferredoxin may be involved, although the details of electron flow are not known. / Ph. D.

The genes encoding the acetate kinase and phosphotransacetylase enzymes from <i>Methanosarcina thermophila</i> were isolated from a genomic library on a fifteen kilobase fragment The genes are located adjacent to one another, with the phosphotransacetylase gene (<i>pta</i>) directly upstream of the acetate kinase gene (<i>ack</i>). The two genes were sequenced, along with a third Open Reading Frame (designated <i>orfY</i>). The <i>orfY</i> gene appears to encode a novel protein whose physiological function has yet to be determined. / Ph. D.

The cell extract protein content of acetate- and methanol-grown Methanosarcina thermophila was examined by two-dimensional polyacrylamide gel electrophoresis to determine the extent of regulation by the growth substrate. More than 100 mutually-exclusive spots were present in acetate- and methanol-grown cells suggesting a high degree of regulation. Spots corresponding to acetate kinase, phosphotransacetylase, and the five subunits of the nickel-containing carbon monoxide dehydrogenase (CODH) complex were identified in acetate-grown cells. The nickel-containing methyl coenzyme M methylreductase from acetate-grown M. thermophila was purified 16-fold from a cell extract to apparent homogeneity. The enzyme had a native molecular weight of between 132,000 and 141,000 and contained three subunits with a configuration of a1B1y1-. The as-isolated enzyme was inactive, but could be reductively reactivated by either titanium (III) citrate or reduced ferredoxin. Reactivation with ferredoxin was a simplification over previously reported reactivation systems. ATP stimulated, but was not required for reactivation. The CO dehydrogenase enzyme complex from M. thermophila was purified and separated into its respective components: the CO-oxidizing nickel/iron-sulfur (Ni/Fe-S) component and the cobalt-containing corrinoid/iron sulfur (Co/Fe-S) component. EPR spectroscopy and spectroelectrochemical titration of the Fe-S centers of the Ni/Fe-S component indicated the presence of two low-potential [4Fe-4S]2+/1+ centers and third high-potential center whose Fe-S configuration is unknown. When reduced with CO, the NilFe-S component exhibited a previously unobserved Ni-Fe-C EPR signal. The Co/Fe-S component contained one [4Fe-4S]2+/1+ cluster, and the as-isolated corrinoid in the component was in the base-off conformation suggesting that modulation of the electron density of the cobalt ion may result in a modified reactivity of the active site of the corrin. The CODH enzyme complex and isolated Co/Fe-S component reductively dechlorinated trichloroethylene to cis-dichloroethylene, trans-dichloroethylene, 1,1-dichloroethylene, vinyl chloride, and ethylene. Factor III also catalyzed the dechlorination of trichloroethylene when in the presence of titanium (III) citrate. Reconstitution of the Co/Fe-S component with the CO-reduced NilFe-S component also allowed dechlorination demonstrating an electron transfer from the reduced Ni/Fe-S component to the Co/Fe-S component. / Ph. D.

Methanosarcina are methanogens capable of growth and methanogenesis from H₂/CO₂, formate, methanol, methylamines, and acetate. Methanosarcina conserve energy by coupling electron transport and methyl transfer to the generation of ion gradients during acetoclastic growth. This work focuses on cytochrome b and heterodisulfide reductase, two proteins involved in energy conservation by electron transport. A procedure was developed for mass cultivation of Methanosarcina thermophila strain TM-1 in 12-liter fermentations which produced up to 10 grams wet weight/liter, in order to facilitate biochemical studies. Cytochromes occurring in Methanosarcina thermophila were characterized spectrophotometrically using chemical and physiological reactants. This analysis revealed two heme centers, one of which was only reduced by Na₂S₂O₄ or carbon monoxide. Partially purified cytochromes were found to be present in a complex and were characterized by electrophoretic and spectrophotometric analysis. The cytochrome-containing protein was found to contain two hemes and had an M_r of 28,000 Da. Heterodisulfide reductase was isolated from the soluble fraction by anion exchange chromatography and assayed using methyl viologen as an artificial electron donor. Electron transport from CO to the heterodisulfide of 2-mercaptoethanesulfonic acid (HS-CoM) and 7- mercaptoheptanoylthreonine phosphate (HS-HTP) was reconstituted using carbon monoxide dehydrogenase, ferredoxin, membranes, and heterodisulfide reductase. Both membranes and ferredoxin were required for reduction of the heterodisulfide. / Master of Science

Protein phosphorylation constitutes an important mechanism for cellular regulation in both Eucarya and Bacteria. All living organisms evolved from a common progenitor; this implies that protein phosphorylation as a means of regulation also exists in Archaea. Previously, in the sulfur-dependent archaeon Sulfolobus solfataricus a gene was cloned encoding a protein-serine/threonine phosphatase that was similar to eucaryal protein-serine/threonine phosphatases type 1, 2A, and 2B. To identify protein phosphatases in other archaeons, oligonucleotides encoding conserved regions of eucaryal protein-serine/threonine phosphatases were used in the polymerase chain reaction to amplify genomic DNA from the methanogenic archaeon Methanosarcina thermophila. From the PCR reaction a fragment of DNA was isolated that encoded a portion of a protein phosphatase. Using this DNA fragment as a probe, the entire phosphatase gene was isolated. The amino acid sequence of the phosphatase encoded by this gene displayed greater than 30% identity with eucaryal protein-serine/threonine phosphatase type 1. The gene encoding the Methanosarcina phosphatase was expressed in Escherichia coli. The expressed protein exhibited protein serine phosphatase activity that was sensitive to inhibitors of eucaryal phosphatases such as okadaic acid, microcystin, calyculin, and tautomycin. In order to identify potential endogenous substrates of archaeal protein-serine/threonine phosphatases and kinases, a study was initiated to characterize the most prominent phosphoproteins in S. solfataricus. Cell extracts were incubated with [γ-³²P] ATP, MgCl₂, and MnCl₂, and the proteins in the extracts were separated by SDS-PAGE. Autoradiography of the gels revealed four prominent phosphoproteins with apparent molecular masses of 35, 46, and 50 kDa. N-terminal sequence analysis and enzymatic assays of the 35 kDa phosphoprotein identified this phosphoprotein as the a-subunit of succinyl-CoA synthetase. N-terminal sequence analysis and enzymatic assays revealed that the 50 kDa phosphoprotein was a hexosephosphate mutase. Neither the 50 kDa nor the 35 kDa phosphoprotein appeared to be the target of protein kinases or phosphatases. Therefore, while protein-serine phosphatases exist in Archaea, the targets of these phosphatases have yet to be determined. / Ph. D.