A Spectroscopically Validated Computational Investigation of Viable Reaction Intermediates in the Catalytic Cycle of the Reductive Dehalogenase PceA

Organisms that produce reductive
dehalogenases utilize halogenated
aromatic and aliphatic substances as terminal electron acceptors in
a process termed organohalide respiration. These organisms can couple
the reduction of halogenated substances with the production of ATP.
Tetrachloroethylene reductive dehalogenase (PceA) catalyzes the reductive
dehalogenation of per- and trichloroethylenes (PCE and TCE, respectively)
to primarily cis-dichloroethylene (DCE). The enzymatic
conversion of PCE to TCE (and subsequently DCE) could potentially
proceed via a mechanism in which the first step involves a single-electron
transfer, nucleophilic addition followed by chloride elimination or
protonation, or direct attack at the halogen. Difficulties with producing
adequate quantities of PceA have greatly hampered direct experimental
studies of the reaction mechanism. To overcome these challenges, we
have generated computational models of resting and TCE-bound PceA
using quantum mechanics/molecular mechanics (QM/MM) calculations and
validated these models on the basis of experimental data. Notably,
the norpseudo-cob­(II)­alamin [Co­(II)­Cbl*] cofactor remains five-coordinate
upon binding of the substrate to the enzyme, retaining a loosely bound
water on the lower face. Thus, the mechanism for the thermodynamically
challenging Co­(II) → Co­(I)­Cbl* reduction used by PceA differs
fundamentally from that utilized by adenosyltransferases, which generate
four-coordinate Co­(II)­Cbl species to facilitate access to the Co­(I)
oxidation state. The same QM/MM computational methodology was then
applied to viable reaction intermediates in the catalytic cycle of
PceA. The intermediate predicted to possess the lowest energy is that
resulting from electron transfer from Co­(I)­Cbl* to the substrate to
yield Co­(II)­Cbl*, a chloride ion, and a vinylic radical.