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Heckel, B. ; Phillips, E.* ; Edwards, E.* ; Sherwood Lollar, B.* ; Elsner, M. ; Manefield, M.J.* ; Lee, M.*

Reductive dehalogenation of trichloromethane by two different dehalobacter restrictus strains reveal opposing dual element isotope effects.

Environ. Sci. Technol. 53, 2332-2343 (2019)

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Trichloromethane (TCM) is a frequently detected and persistent groundwater contaminant. Recent studies have reported that two closely related Dehalobacter strains (UNSWDHB and CF) transform TCM to dichloromethane, with inconsistent carbon isotope effects (e(13)C(UNSWDHB) = -4.3 +/- 0.45 parts per thousand; e13C(CF) = -27.5 +/- 0.9 parts per thousand). This study uses dual element compound specific isotope analysis (C; Cl) to explore the underlying differences. TCM transformation experiments using strain CF revealed pronounced normal carbon and chlorine isotope effects (e13C(CF) = -27.9 +/- 1.7 parts per thousand; e37Cl(CF) = -4.2 +/- 0.2 parts per thousand). In contrast, small carbon and unprecedented inverse chlorine isotope effects were observed for strain UNSWDHB (e13C(UNSWDHB) = -3.1 +/- 0.5 parts per thousand; e37Cl(UNSWDHB) = 2.5 +/- 0.3 parts per thousand) leading to opposing dual element isotope slopes (?CF = 6.64 +/- 0.14 vs lambda(UNSWDHB) = -1.20 +/- 0.18). Isotope effects of strain CF were identical to experiments with TCM and Vitamin B12 (e13C(Vitamin B12) = -26.0 +/- 0.9 parts per thousand, e37Cl(Vitamin B12) = -4.0 +/- 0.2 parts per thousand, lambda(Vitamin) (B12) = 6.46 +/- 0.20). Comparison to previously reported isotope effects suggests outer-sphere-single-electron transfer or S(N)2 as possible underlying mechanisms. Cell suspension and cell free extract experiments with strain UNSWDHB were both unable to unmask the intrinsic KIE of the reductive dehalogenase (TmrA) suggesting that enzyme binding and/or mass-transfer into the periplasm were rate-limiting. Nondirected intermolecular interactions of TCM with cellular material were ruled out as reason for the inverse isotope effect by gas/water and gas/hexadecane partitioning experiments indicating specific, yet uncharacterized interactions must be operating prior to catalysis.

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