Title page for ETD etd-07282008-134014


Type of Document Dissertation
Author Jablonski, Peter Edward
URN etd-07282008-134014
Title Studies on two nickel-containing enzymes from Methanosarcina thermophila TM-1
Degree PhD
Department Anaerobic Microbiology
Advisory Committee
Advisor Name Title
Ferry, James G. Committee Chair
Dean, Dennis R. Committee Member
Johnson, John L. Committee Member
Krieg, Noel R. Committee Member
Larson, Timothy J. Committee Member
Keywords
  • Metalloenzymes
Date of Defense 1992-08-15
Availability restricted
Abstract
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.

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