Reductive Dehalogenation of Trichloromethane by Two Different Dehalobacter restrictus Strains Reveal Opposing Dual Element Isotope Effects

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 (ϵ¹³C_UNSWDHB = -4.3 ± 0.45‰ ϵ¹³C_CF = -27.5 ± 0.9‰). 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 (ϵ¹³C_CF = -27.9 ± 1.7‰ ϵ³⁷Cl_CF = -4.2 ± 0.2‰). In contrast, small carbon and unprecedented inverse chlorine isotope effects were observed for strain UNSWDHB (ϵ¹³C_UNSWDHB = -3.1 ± 0.5‰ ϵ³⁷Cl_UNSWDHB = 2.5 ± 0.3‰) leading to opposing dual element isotope slopes (λ_CF = 6.64 ± 0.14 vs λ_UNSWDHB = -1.20 ± 0.18). Isotope effects of strain CF were identical to experiments with TCM and Vitamin B₁₂ (ϵ¹³C_{Vitamin B12} = -26.0 ± 0.9‰, ϵ³⁷Cl_{Vitamin B12} = -4.0 ± 0.2‰, λ_{Vitamin B12} = 6.46 ± 0.20). Comparison to previously reported isotope effects suggests outer-sphere-single-electron transfer or S_N2 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.