Conformational Isomerization of the Fe(III)–OH Species Enables Selective Halogenation in Carrier-Protein-Independent Halogenase BesD and Hydroxylase-Evolved Halogenase

Abstract

Despite extensive studies, how carrier-protein-independent BesD dictates the reaction toward thermodynamically unfavored halogenation is still elusive. Here, we investigated the chlorination versus hydroxylation selectivity in both carrier-protein-independent halogenase BesD and hydroxylase-evolved halogenase Chi-14, employing extensive MD simulations and QM/MM calculations. In BesD, our calculations have shown that 2OGassisted O 2 activation affords the axial Fe(IV)-oxo species that is responsible for the substrate C−H activation. To facilitate the following Cl-rebound reaction, the nascent axial Fe(III)−OH species has to undergo conformational isomerization to the equatorial one. This can remove the steric effects between the axial Fe(III)−OH and the substrate radical, thereby enhancing the migration of the substrate radical toward the Cl − ligand during the Clrebound. Notably, the hydrogen-bond interactions with second-sphere residue Asn are vital to maintain the unsaturated five-coordination shell of the Fe center. This maintenance is essential for enabling the conformational transition of Fe(III)−OH from an axial to an equatorial orientation. Our results are in concordance with existing experimental findings, underscoring the pivotal influence of iron coordination dynamics in governing the catalytic processes of nonheme enzymes.

Probing Ferryl Reactivity in a Nonheme Iron Oxygenase Using an Expanded Genetic Code

“…However, performing structure–function relationships to probe the role of the conserved bis-histidine coordination environment has proven challenging, as mutation of either of the cis-disposed ligands abolishes catalytic function. If we were able to make functional ligand replacements, we could begin to address important questions such as the effect of ligand strength on Fe IV O reactivity or positioning (“in-line” vs “off-line” , ) across family members. Previously, our group and others have shown that the axial ligands of heme enzymes can be replaced by noncanonical analogues of cysteine and histidine, creating a new way to probe structure–activity relationships directly in enzyme active sites. − , Here, we show that a similar approach can be used to modulate the primary coordination sphere of nonheme iron enzymes, providing new opportunities to probe and augment the catalytic mechanisms of this enzyme superfamily.…”