Atomic and Electronic Structure Determinants Distinguish between Ethylene Formation and l‑Arginine Hydroxylation Reaction Mechanisms in the Ethylene-Forming Enzyme

The ethylene-forming enzyme (EFE) is a non-heme Fe­(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that catalyzes dual reactions: the generation of ethylene from 2OG and the C5 hydroxylation of l-Arg. Using an integrated molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) approach that references previous experimental studies, we tested the hypothesis that synergy between the conformation of l-Arg and the coordination mode of 2OG directs the reaction toward ethylene formation or l-Arg hydroxylation. The dynamics of EFE·Fe­(III)·OO•–·2OG·l-Arg show that l-Arg can exist in conformation A (productive for hydroxylation) and conformation B (unproductive for hydroxylation). QM/MM calculations show that when 2OG is bound in an off-line mode and l-Arg is present in conformation A, the Fe­(III)-OO•– intermediate undergoes the standard O2 activation mechanism involving ferryl-dependent hydroxylation. With the same off-line 2OG coordination, but with conformation B of l-Arg, a unique pathway produces a half-bond ferric-bicarbonate intermediate that decomposes to ethylene, two CO2, and a ferrous-bicarbonate species. The results demonstrate that when 2OG is coordinated in off-line mode to the Fe center, the l-Arg conformation acts as a switch that directs the reaction toward ethylene formation or hydroxylation. Analysis of the electronic structure shows that the l-Arg conformation defines the precise location of an unpaired β electron in the Fe­(III)-OO– complex, either in a π*∥ orbital that triggers ethylene formation or a π*⊥ orbital that cascades to l-Arg hydroxylation. A change in 2OG coordination from off-line to in-line reduces stabilization of the 2OG C1 carboxylate such that neither conformation of l-Arg produces the ethylene-forming half-bond ferric-bicarbonate intermediate. Thus, l-Arg conformation-dependent changes in the electronic structure of the Fe­(III)-OO•– orbitals, together with the 2OG binding mode-associated stabilization of the C1-carboxylate, distinguish whether the EFE reaction proceeds via the ethylene-forming pathway or catalyzes a hydroxylation mechanism.