Chlorinated Ethene Reactivity with Vitamin B₁₂ Is Governed by Cobalamin Chloroethylcarbanions as Crossroads of Competing Pathways

Abstract

Chlorinated ethenes are toxic groundwater contaminants. Although they can be dechlorinated by microorganisms, reductive dehalogenases, and their corrinoid cofactor, biochemical reaction mechanisms remain unsolved. This study uncovers a mechanistic shift revealed by contrasting compound-specific carbon (ε13C) and chlorine (ε37Cl) isotope effects between perchloroethene, PCE (ε37Cl = −4.0‰) and cis-dichloroethene, cis-DCE (ε37Cl = −1.5‰), and a pH-dependent shift for trichloroethene, TCE (from ε37Cl = −5.2‰ at pH 12 to ε37Cl = −1.2‰ at pH 5). Different pathways are supported also by pH-dependent reaction rates, TCE product distribution, and hydrogen isotope effects. Mass balance deficits revealed reversible and irreversible cobalamin-substrate association, whereas high-resolution mass spectrometry narrowed down possible structures to chloroalkyl and chlorovinyl cobalamin complexes. Combined experimental evidence is inconsistent with initial electron transfer or alkyl or vinyl complexes as shared intermediates of both pathways. In contrast, it supports cobalamin chlorocarbanions as key intermediates from which Cl– elimination produces vinyl complexes (explaining rates and products of TCE at high pH), whereas protonation generates less reactive alkyl complexes (explaining rates and products of TCE at low pH). Multielement isotope effect analysis holds promise to identify these competing mechanisms also in real dehalogenases, microorganisms, and even contaminated aquifers.

Mechanistic Dichotomy in Bacterial Trichloroethene Dechlorination Revealed by Carbon and Chlorine Isotope Effects

“…lovleyi KB-1 = 3.1 ±0.1, see Table 1 and Figure S2) for all three experiments, indicating that TCE was dechlorinated via a similar chemical mechanism, irrespective of the type of RDase (Geo-PceA vs. VcrA vs. TceA). The three slopes agree with those at high pH in the Vitamin B12 study 14 , suggesting that in all three cases a sequence of addition-elimination was the predominant reaction pathway.…”

“…3 below). Our Vitamin B12 study demonstrated that the slope ΛC/Cl can provide a sensitive indicator of the underlying reaction mechanisms in reductive chlorinated ethene dehalogenation with Vitamin B12 14 . Values of ΛC/Cl were much larger in the addition-protonation mechanism, reflecting the fact that no C-Cl bond was cleaved in the initial step so that chlorine isotope effects were small.…”

“…With reduced Vitamin B12 as a chemical model system, we recently achieved a breakthrough in understanding reaction mechanisms in vitro 14 . Our evidence suggests that cob(I)alamin acts as a supernucleophile and adds to the double bond in chlorinated ethenes so that a carbanion complex is formed.…”

“…Scheme 1. Reaction mechanisms for reductive dehalogenation of chlorinated ethenes via addition protonation or addition elimination (adapted from Heckel et al 14 ).…”

Diversity and Niche of Archaea in Bioremediation

“…The ability to dehalogenate is likely from the high concentrations of corrinoids, such as cobalamin, in methanogens which are needed for methanogenesis [ 148 , 149 ]. Corrinoids are able to dehalogenate organics abiotically [ 150 , 151 ].…”