Revisiting theMechanism of Fluoroacetate Dehalogenase-CatalyzedDegradation of Fluorocarboxylic Acid via Cluster Models

Abstract Fluoroacetate dehalogenase (FAcD) is a unique, nonmetal-dependent enzyme capable of cleaving the inert C–F bond of fluoroacetate (FA) under mild conditions, making it an important enzymatic model for organofluorine degradation. This study investigated the catalytic degradation mechanism of FA by FAcD using density functional theory calculations with an active-site cluster model. The model included the FA substrate, two crystallographic water molecules, and the key amino acid residues surrounding the active site. An integrated energy profile connecting defluorination and hydrolysis was obtained within a single computational framework. The calculated reaction pathway consists of four elementary steps: (I) C–F bond activation, (II) nucleophilic attack, (III) C–O bond cleavage, and (IV) proton transfer. Structural analyses showed that Arg111, Arg114, and Tyr219 stabilize the carboxylate group of FA and His155, Trp156, and Tyr219 stabilize the fluoride anion during C–F bond cleavage. Furthermore, His155 was suggested to activate the hydrolytic water molecule and participate directly in the nucleophilic attack step. These findings provide a unified quantum-chemical description of FAcD-catalyzed defluorination and hydrolysis and offer insights into the design of engineered enzymes and biomimetic catalysts for organofluorine degradation.