Mechanistic Insights Into Substrate Positioning Across Non-heme Fe(II)/alpha-ketoglutarate-dependent Halogenases and Hydroxylases

Abstract

Non-heme iron halogenases and hydroxylases activate inert C-H bonds to selectively catalyze the functionalization of a diversity of biological products under physiological conditions. To better understand the differences in substrate positioning key to their divergent reactivities, we compiled available crystallographic and spectroscopic data, which revealed that hydroxylases prefer an acute oxo-Fe-H target angle, while halogenases prefer a more obtuse angle. With molecular dynamics simulations guided by this experimental information, we simulated the representative hydroxylases TauD and VioC and the halogenases BesD and WelO5 with both acute and obtuse harmonic restraints. We identified key native interactions that maintain the angle of approach in the respective enzymes, such as Asp94 in TauD and His127 in BesD. Moreover, our simulations reveal that the protein environment in halogenases prevents the sampling of acute angles observed in hydrogenases and vice versa. To validate these classical observations, we optimized the structure with large-scale quantum mechanical (QM) simulations and confirmed QM-derived substrate-enzyme hydrogen bond strengths were higher in the native configurations. To understand the effect of positioning on reactivity, we confirmed that reaction barriers for the rate-limiting hydrogen atom transfer reaction was slightly lower from an acute angle regardless of the enzyme-substrate complex. Analysis of the halogenase reaction coordinates reveals the formation of hydrogen bonding networks between the Fe(II)-hydroxyl, monodentate succinate, and a member of the second coordination sphere that may inhibit the hydroxyl rebound.

Why Nonheme Iron Halogenases Do Not Fluorinate C–H Bonds: A Computational Investigation

“…Specifically, it is slightly lower than the HAT reaction barriers of ca. 30 kcal/mol observed for BesD in prior work 73 owing to additional constraints used in that study. In comparison to another study, 62 we find that the HAT barriers we observe are higher by ca.…”

Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

“…Specifically, the mutation of E119 to an Ala was shown to result in a loss of activity, and W137 was found to impose the proper substrate angle for halogenation reactivity. 7,38 Our simulations show that the H-bond network formed by these residues in 6c-diaqua complex I undergoes a dramatic reorganization upon binding chloride and lysine to form the 5c-Cl-Lys complex IV (Figures 3B and S8).…”

“…31−33 This motivated several experimental and computational studies to explore the factors determining the chemoselectivity of NHFe-Hals and engineered NHFe-Hyds. These factors include the configuration of the haloferryl isomer 34−37 and the oxo-Fe−H angle that it forms with the target C−H group, 38 the optimal positioning of the substrate radical in relation to the chloride ligand of the Cl−Fe III −OH intermediate, 16,39,40 as well as protein pocket electrostatics that can facilitate chloride transfer. 41,42 While most of these studies have focused on the oxygen activation, C−H abstraction, and chloride transfer stages of the catalytic cycle (Figure S1), how catalytic pocket electrostatics can control the active site assembly when working with charged and untethered substrates like free lysine and functionalizing anions like chloride, along with their implications for the overall catalytic performance and product yields, remains unexplored.…”