Biochemical and EPR-Spectroscopic Investigation into Heterologously Expressed Vinyl Chloride Reductive Dehalogenase(VcrA) from Dehalococcoides mccartyi Strain VS

摘要

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] duster 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.