A Spectroscopically Validated Computational Investigation of Viable Reaction Intermediates in the Catalytic Cycle of...

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.