Defluorinationof Fluorophenols by a Heme Dehaloperoxidase:Insights into the Defluorination Mechanism

Fluorinated compounds are extensively used in industry and materials science due to their high stability and physical properties, however, they are poorly biodegradable and lead to environmental accumulation. In this work, we explore heme dehaloperoxidases for the defluorination of fluorinated aromatic compounds. Heme dehaloperoxidases are efficient enzymes that utilize H₂O₂ on a heme-active site to dehalogenate aromatic compounds. They usually operate by dechlorination or debromination, but recent evidence suggests defluorination is also possible; however, details of the mechanism are elusive and remain controversial. To establish the mechanism and feasibility of aromatic defluorination by dehaloperoxidase enzymes for environmental remediation and particularly the detoxification of fluorinated compounds, we performed detailed molecular dynamics (MD) and quantum mechanics studies. A complete enzyme model was created based on the dehaloperoxidase crystal structure and several fluorinated arenes were inserted. Despite the fact that the substrate binding pocket and heme active site are solvent-exposed, actually during the MD simulation, the substrates are locked inside the substrate-binding pocket through tight hydrogen bonding interactions and π-stacking interactions with amino acid residues, including several Phe amino acids and the Tyr₃₈ and His₅₅ side chains. We then created a large cluster model of 324 atoms of a Compound I model with 2,4,6-trifluorophenol bound and included the second-coordination sphere and studied the oxygen activation and defluorination reaction of the substrate via several possible reaction pathways. The calculations reveal a mechanism that starts with a hydrogen atom abstraction from the phenol group followed by OH rebound to the <i>ortho</i>- or <i>para</i>-positions to form 2,4,6-trifluoro-4-hydroxycyclohexadienone and 2,4,6-trifluoro-6-hydroxycyclohexadienone. These products either react inside the protein with the assistance of a proton by fluorine release or escape into the solution where the defluorination happens and the final benzoquinone products are formed. The calculations show that the hydrogen atom abstraction and OH rebound steps have small free energies of activation of <Δ<i>G</i> = 10 kcal mol^–1, while the defluorination is rate-determining. Consequently, our studies predict that heme haloperoxidases have the potential for environmental remediation of fluorophenols from water, although a mixture of <i>ortho</i>- and <i>para</i>-activation will occur.