Epoxidation Catalyzed by the Nonheme Iron(II)- and 2‑Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate

Mechanisms of enzymatic
epoxidation via oxygen atom transfer (OAT)
to an olefin moiety is mainly derived from the studies on thiolate-heme
containing epoxidases, such as cytochrome P450 epoxidases. The molecular
basis of epoxidation catalyzed by nonheme-iron enzymes is much less
explored. Herein, we present a detailed study on epoxidation catalyzed
by the nonheme iron­(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase,
AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy)
to electron-withdrawing groups (e.g., trifluoromethyl) were used to
probe the mechanism. The results derived from transient-state enzyme
kinetics, Mössbauer spectroscopy, reaction product analysis,
X-ray crystallography, density functional theory calculations, and
molecular dynamic simulations collectively revealed the following
mechanistic insights: (1) The rapid O2 addition to the
AsqJ Fe­(II) center occurs with the iron-bound 2OG adopting an online-binding
mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial
triad, instead of assuming the offline-binding mode with the C1 carboxylate
group trans to the distal histidine (His211); (2)
The decay rate constant of the ferryl intermediate is not strongly
affected by the nature of the para substituents of
the substrate during the OAT step, a reactivity behavior that is drastically
different from nonheme Fe­(IV)-oxo synthetic model complexes; (3) The
OAT step most likely proceeds through a stepwise process with the
initial formation of a C­(benzylic)–O bond to generate an Fe-alkoxide
species, which is observed in the AsqJ crystal structure. The subsequent
C3–O bond formation completes the epoxide installation.