QM/MM Calculations Reveal the Important Role of α‑Heteroatom Substituents in Controlling Selectivity of Mononuclear Nonheme HppE-Catalyzed Reactions

The nonheme iron enzyme, (S)-2-hydroxypropylphosphonate [(S)-2-HPP] epoxidase (HppE), uses H₂O₂ as the oxidizing cosubstrate to catalyze the epoxidation of (S)-2-HPP to fosfomycin, as well as other oxidative processes (including dehydrogenation and 1,2-phosphono-migration) of structural isomers of its native substrate. The HppE-catalyzed 1,2-phosphono-migration of (R)-1-HPP, which involves the pro-R hydrogen abstraction from C2, was proposed to proceed via the formation of a C2 carbocation intermediate. Here, we show that such an intermediate does not transpire during the reaction. The reorientation of the singly occupied orbital at the C2-radical center, through out-of-plane torsion of the P–C1–C2–C3 dihedral angle, triggers the phosphono-migration and precludes the attack of the Fe-bound OH on the C2 radical (hydroxylation) as well as the C2–O coupling (epoxidation). For the substrate (1R)-1-hydroxyl-2-aminopropyl phosphonate, the lone-pair electrons on the amino group enable a fast proton-coupled electron transfer process, wherein the proton transfer from the amino group to phosphonate is coupled to the electron transfer from the C2 radical to the iron center, leading to a C2-iminium cation rather than the phosphono-migration. Finally, the C2-iminium intermediate is converted to a ketone product and NH₃. These results demonstrate that the α-heteroatom substituent is vital for the generation of the cationic species, as well as the selectivity control in the HppE-catalyzed reactions.