How Fe(II)/2-Oxoglutarate Oxygenase Chooses Chlorination over Hydroxylation: Electric Field-Driven Ligand Exchange Governs C–Cl Formation

Non-heme Fe(II)/2-oxoglutarate (2OG)-dependent halogenases catalyze highly selective C–H halogenation. BesD is a non-heme Fe(II)/2OG halogenase that performs regio- and stereoselective chlorination of the l-lysine (l-Lys) substrate. Understanding the mechanism by which halogenation is favored over canonical hydroxylation is essential for guiding enzyme engineering efforts aimed at converting hydroxylases into halogenases. Here, we combine molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the origin of chlorination selectivity in BesD and variants derived from a homologous hydroxylase. Our results indicate that, although the initial inline Cl–Fe(III)–OH intermediate is inherently predisposed toward hydroxylation, it undergoes a two-step isomerization in which the −Cl and −OH ligands exchange coordination positions. This rearrangement positions the chloride ligand trans to H207, where the protein’s intrinsic electric field (IEF) enhances Fe–Cl bond polarization and promotes C–Cl bond formation, ultimately enabling the selective chlorination of l-Lys. Comparative analysis of the homologous hydroxylase and two halogenation-competent variants (Hydrox-3R and Chimera14) reveals halogenase-specific correlated motions between substituted second coordination sphere, long-range residues with the active-site, particularly the coordinated succinate, and the substrate. Notably, these collective motions mirror those observed in the native halogenase BesD. Furthermore, the Hydrox-3R and Chimera14 variants employ a two-step isomerization strategy analogous to that of BesD, enabling efficient chlorination through IEF alignment along the Fe–Cl bond. These results highlight the critical roles of collective correlated motions, the second coordination sphere, and long-range interactions, as well as enzyme-generated electric fields, in determining halogenation selectivity in non-heme Fe(II)/2OG-dependent oxygenases. Ultimately, halogenation is achieved not due to a new catalytic mechanism, but rather due to subtle electronic, geometric, and dynamic perturbations of a hydroxylase scaffold. These insights provide a mechanistic framework for engineering hydroxylases into halogenases with enhanced activity.