Conformational Isomerization of the Fe(III)-OH Species Enables Selective Halogenation in Non-Heme Iron Halogenase BesD and Hydroxylase-Evolved Halogenase

Non-heme iron(II)/2-oxoglutarate (Fe/2OG)-dependent enzymes catalyze a large number of C-H bond activation and functionalization. Especially, non-heme iron halogenases catalyze the selective C-H halogenation, representing an attractive strategy for the biosynthesis of halogenated compounds. Despite extensive endeavors from experiments and computations, how non-heme iron halogenases dictate the reaction toward the thermodynamically unfavored halogenation is still elusive. Here, we have investigated the chlorination versus hydroxylation selectivity in both halogenase BesD and hydroxylase-evolved halogenase Chi-14, using extensive MD simulations and QM/MM calculations. In BesD, our calculations have shown that 2OG-assisted O2 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 the conformational isomerization to the equatorial one. This can remove the steric effects between the axial Fe(III)-OH and the substrate radical, thereby facilitating the migration of substrate radical toward Cl-ligand during the Cl-rebound. Notably, the second-sphere residue Asn (Asn219 in BesD or Asn225 in Chi-14) can form persistent hydrogen bond interactions with succinate, which is vital to maintain the unsaturated five-coordination shell of Fe, thereby facilitating the conformational flip of Fe(III)-OH from the axial orientation to the equatorial one. Our findings agree with the available experimental information, highlighting the key role of the coordination dynamics of iron in dictating the catalysis of non-heme enzymes.