Mechanistic Insights into Substrate Positioning That Distinguish Non-heme Fe(II)/α-Ketoglutarate-Dependent Halogenases and Hydroxylases

Abstract

Non-heme iron halogenases and hydroxylases activate inert C−H bonds to selectively catalyze the functionalization of diverse 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 substrate interaction partners 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 hydroxylases and vice versa. To validate these classical observations, we optimized the structure with large-scale quantum mechanical (QM) simulations and confirmed that QM-derived substrate−enzyme hydrogen bond strengths were higher in the native configurations. We computed reaction barriers for the rate-limiting hydrogen atom transfer step and found them to be slightly lower from an acute angle regardless of the enzyme−substrate complex. Analysis of the halogenase reaction coordinate reveals the formation of hydrogen bonding networks between the Fe(III)-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 owing to additional constraints used in that study. In comparison to another study, we find that the HAT barriers we observe are higher by ca.…”

“…We study the rate-determining hydrogen atom transfer (HAT) and rebound reactions to determine if these reaction barriers could explain why C–H chlorination and bromination are preferred over C–H fluorination. Because isomerization to alter relative substrate positioning ,,− has been invoked to rationalize selectivity of some ,,, albeit not all , nonheme iron halogenases, we also study isomer energetics and isomerization reaction coordinates (RCs) to understand the isomer energetic favorability of key intermediates such as Fe­(IV)=O and Fe­(III)–OH formed during the catalytic cycle with all three halides.…”

Electrostatically Regulated Active Site Assembly Governs Reactivity in Nonheme Iron Halogenases

“…Initial attempts to reprogram NHFe-Hyds into halogenases by this single amino acid substitution of Glu/Asp to Ala/Gly were unsuccessful in achieving the desired chemoselectivity, indicating a more complex molecular mechanism at play. − 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 − and the oxo-Fe–H angle that it forms with the target C–H group, the optimal positioning of the substrate radical in relation to the chloride ligand of the Cl–Fe III –OH intermediate, ,, as well as protein pocket electrostatics that can facilitate chloride transfer. , 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. Thus, a more rigorous understanding of the entire catalytic cycle and how NHFe-Hals facilitate binding, activation, and reactivity with specific substrates and functionalizing anions is needed.…”

PyConSolv: A Python Package for Conformer Generation of (Metal-Containing) Systems in Explicit Solvent

“…We would like to stress here that the force-field parameters are generated in such a way that the QM optimized structure is retained. While the use of a bonded model, as implemented in MCPB, provides accurate results for many different (transition-)­metal-containing complexes, ,,,− the generation of parameters is a tedious and error prone process, due to the amount of user intervention that is required.…”