Studies on the carbon monoxide dehydrogenase enzyme complex present in acetate-grown Methanosarcina thermophila strain TM-1
| dc.contributor.author | Terlesky, Katherine C. | en |
| dc.contributor.committeechair | Ferry, James G. | en |
| dc.contributor.committeecochair | Gregory, E.M. | en |
| dc.contributor.committeemember | Ebel, Richard E. | en |
| dc.contributor.committeemember | Wilkins, Tracy D. | en |
| dc.contributor.committeemember | Johnson, J.L. | en |
| dc.contributor.department | Microbiology | en |
| dc.date.accessioned | 2015-07-10T20:00:06Z | en |
| dc.date.available | 2015-07-10T20:00:06Z | en |
| dc.date.issued | 1989 | en |
| dc.description.abstract | The carbon monoxide dehydrogenase complex was purified from acetate-grown Methanosarcina thermophila. This complex made up greater than 10% of the cellular protein and the native enzyme formed aggregates with a Mr of approximately 1,000,000. The enzyme contained five subunits of different molecular weight suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The electron paramagnetic resonance spectrum of CO-reduced enzyme at 113K contained g values of 2.073, 2.049, and 2.028. Isotopic substitution with ⁶¹Ni, ⁵⁷Fe, or ¹³Co resulted in broadening of the spectrum consistent with a Ni-Fe-C spin-coupled complex. Acetyl-CoA caused a perturbation of the signal that was not caused by acetyl-phosphate or mercaptoethanol indicating acetyl-CoA is a physiological substrate. Cell extracts from acetate-grown M. thermophila contained CO-oxidizing:H₂-evolving activity 16-fold greater than extracts of methanol-grown cells. CO-oxidizing:H₂-evolving activity was reconstituted upon combination of: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase and b-type cytochrome. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The molecular weight of the isolated protein was 16,400, and the apparent minimum molecular weight was 4,900. The ferredoxin contained 2.8 ± 0.56 Fe atoms and 1.98 ± 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with a A₃₉₅/A₂₉₅ ratio range of 0.80 to 0.88. The N-terminal amino acid sequence revealed a 4-cysteine cluster, similar to other Fe:S centers that coordinate a Fe:S center. A CH₃-B₁₂:HS-CoM methyltransferase activity was characterized in extracts of acetate- and methanol-grown cells. The activity from extracts of acetate-grown M. thermophila was stable at 70°C for 30 minutes. The activity in cell extracts of acetate- and methanol-grown cells was fractionated with ammonium sulfate treatment and FPLC phenyl superose chromatography. Two peaks of methyltransferase activity were observed in each cell extract sample following phenyl superose fractionation. | en |
| dc.description.degree | Ph. D. | en |
| dc.format.extent | xiv, 135 leaves | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.uri | http://hdl.handle.net/10919/54440 | en |
| dc.language.iso | en_US | en |
| dc.publisher | Virginia Polytechnic Institute and State University | en |
| dc.relation.isformatof | OCLC# 19840835 | en |
| dc.rights | In Copyright | en |
| dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
| dc.subject.lcc | LD5655.V856 1989.T474 | en |
| dc.subject.lcsh | Carbon monoxide | en |
| dc.subject.lcsh | Dehydrogenases | en |
| dc.subject.lcsh | Enzymes | en |
| dc.subject.lcsh | Methanobacteriaceae | en |
| dc.title | Studies on the carbon monoxide dehydrogenase enzyme complex present in acetate-grown Methanosarcina thermophila strain TM-1 | en |
| dc.type | Dissertation | en |
| dc.type.dcmitype | Text | en |
| thesis.degree.discipline | Microbiology | en |
| thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
| thesis.degree.level | doctoral | en |
| thesis.degree.name | Ph. D. | en |
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