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

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 <i>cis</i>-dichloroethene, <i>cis</i>-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