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

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.