title: Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS.
abstract: Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.
authors: †Departments of Chemical Engineering and Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States.
authors: Anutthaman
authors: A
authors: Parthasarathy
authors: ‡Department of Chemistry, University of California, Davis, Davis, California 95616, United States.
authors: Troy A
authors: TA
authors: Stich
authors: †Departments of Chemical Engineering and Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States.
authors: Svenja T
authors: ST
authors: Lohner
authors: †Departments of Chemical Engineering and Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States.
authors: Ann
authors: A
authors: Lesnefsky
authors: ‡Department of Chemistry, University of California, Davis, Davis, California 95616, United States.
authors: R David
authors: RD
authors: Britt
authors: †Departments of Chemical Engineering and Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States.
authors: Alfred M
authors: AM
authors: Spormann
mesh_terms: D041862:Chloroflexi / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D054879:Halogenation; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D014752:Vinyl Chloride / Q000378:metabolism*
chemical_list: D006867:Hydrolases; D014752:Vinyl Chloride