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 CS 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 center dot Fe(III)center dot OO center dot-center dot 2OG center dot 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 LArg is present in conformation A, the Fe(III)-OO center dot- intermediate undergoes the standard O-2 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 beta electron in the Fe(III)-OO center dot- complex, either in a pi(parallel to) orbital that triggers ethylene formation or a pi*(perpendicular to) 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(center dot-)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.