Nuclear Quantum Effects Matter: Zero-Point Energy Governs the Chemoselectivity of Desaturation over Hydroxylation in Nonheme Iron Enzyme AusE

The nonheme iron dioxygenase AusE from the austinol biosynthesis of Aspergillus nidulans catalyzes the regioselective oxidative desaturation of its substrate preaustinoid A1, producing preaustinoid A2 through C=C bond formation, rather than hydroxylated products which are far more common across iron dioxygenases. To elucidate the reaction mechanism underlying AusE-mediated formation of preaustinoid A2, we performed combined computational studies using molecular dynamics, QM/MM, and multireference methods. The results indicated that the reaction proceeds via two sequential hydrogen atom transfer reactions by an Fe(IV)=O species, i.e., C2–H abstraction from the α carbon of an ester group, followed by C1–H abstraction. The latter is more kinetically and thermodynamically favorable over hydroxyl rebound to the C2 radical intermediate (formed after C2–H abstraction), which is consistent with the experimental observation of the desaturation product as the sole product. Our study elucidates multiple critical factors that govern the desaturation selectivity of AusE. Nuclear quantum effects, namely, zero-point energy contributions associated with the C–H bond cleavage, make the dominant contribution to the desaturation selectivity. In addition, (1) substrate orientation relative to the iron center, (2) hydrogen bonding interactions, and (3) the long-range intrinsic electric fields within the enzyme, all contribute positively to the observed selectivity. The present findings are expected to shed light on the origins of chemoselectivity in other iron α-ketoglutarate (αKG)-dependent enzymes.