QM/MM calculations for a non-heme 2-oxoglutarate-dependent ethylene-forming enzyme reveal that the selectivity of ethylene formation versus succinate formation is determined by the relative energies of transition states for the competing CO2 insertion and O–O bond cleavage of the FeII-peroxysuccinate species.

Herein, we use in-protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the mechanism of dioxygen activation, oxygen atom exchange and substrate epoxidation processes by AsqJ, an Fe/α-ketoglutarate-dependent dioxygenase (α-KGD) using a 2-His-1-Asp facial triad. Our results demonstrated that the whole reaction proceeds through a quintet surface. The dioxygen activation by AsqJ leads to a quintet penta-coordinated Fe-oxo species, which has a square pyramidal geometry with the oxo group trans to His134. This penta-coordinated Fe-oxo species is not the reactive one in the substrate epoxidation reaction since its oxo group is pointing away from the target C[double bond, length as m-dash]C bond. Instead, it can undergo the oxo group isomerization followed by water binding or the water binding followed by oxygen atom exchange to form the reactive hexa-coordinated Fe-oxo species with the oxo group trans to His211. The calculated parameters of Mössbauer spectra for this hexa-coordinated Fe-oxo intermediate are in excellent agreement with the experimental values, suggesting that it is most likely the experimentally trapped species. The calculated energetics indicated that the rate-limiting step is the substrate C[double bond, length as m-dash]C bond activation. This work improves our understanding of the dioxygen activation by α-KGD and provides important structural information about the reactive Fe-oxo species.

The iron(IV)-oxo (ferryl) intermediate has been amply established as the principal oxidant in nonheme enzymes and the key player in C-H bond activations and functionalizations. In contrast to this status, our present QM/MM calculations of the mechanism of fosfomycin biosynthesis (a broad range antibiotic) by the nonheme HppE enzyme rule out the iron(IV)-oxo as the reactive species in the hydrogen abstraction (H-abstraction) step of the pro-R hydrogen from the (S)-2-hydroxypropylphosphonic substrate. Moreover, the study reveals that the ferryl species is bypassed in HppE, while the actual oxidant is an HO(•) radical hydrogen-bonded to a ferric-hydroxo complex, resulting via the homolytic dissociation of the hydrogen peroxide complex, Fe(II)-H2O2. The computed energy barrier of this pathway is 12.0 kcal/mol, in fair agreement with the experimental datum of 9.8 kcal/mol. An alternative mechanism involves the iron-complexed hydroxyl radical (Fe(III)-(HO(•))) that is obtained by protonation of the iron(IV)-oxo group via the O-H group of the substrate. The barrier for this pathway, 23.0 kcal/mol, is higher than the one in the first mechanism. In both mechanisms, the HO(•) radical is highly selective; its H-abstraction leading to the final cis-fosfomycin product. It appears that HppE is prone to usage of HO(•) radicals for C-H bond activation, because the ferryl oxidant requires a specific H-abstraction trajectory (∠FeOH ∼ 180°) that cannot be met for intramolecular H-abstraction. Thus, this work broadens the landscape of nonheme iron enzymes and makes a connection to Fenton chemistry, with implications on new potential biocatalysts that may harness hydroxyl radicals for C-H bond functionalizations.

The second coordination sphere constitutes adistinguishing factor in the active site to modulate enzymatic reactivity.T ou nravel the origin of NO-to-N 2 Or eduction activity of non-heme diiron enzymes,herein we report astrong second-coordination-sphere interaction between ac onserved Tyr 197 and the key iron-nitrosyl intermediate of Tm FDP (flavo-diiron protein), which leads to decreased reaction barriers towards N-N formation and N-O cleavage in NO reduction. This finding supports the direct coupling of diiron dinitrosyl as the N-N formation mode in our QM/MM modeling,a nd reconciles the mechanistic controversy of external reduction between FDPs and synthetic biomimetics of the iron-nitrosyls.T his work highlights the application of QM/MM 57 Fe Mçssbauer modeling in elucidating the structural features of not only first, but also second coordination spheres of the key transient species involved in NO/O 2 activation by non-heme diiron enzymes.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.