Ethylene-forming enzyme (EFE) is an ambifunctional iron­(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase. In its major (EF) reaction, it converts carbons 1, 2, and 5 of 2OG to CO2 and carbons 3 and 4 to ethylene, a four-electron oxidation drastically different from the simpler decarboxylation of 2OG to succinate mediated by all other Fe/2OG enzymes. EFE also catalyzes a minor reaction, in which the normal decarboxylation is coupled to oxidation of l-arginine (a required activator for the EF pathway), resulting in its conversion to l-glutamate semialdehyde and guanidine. Here we show that, consistent with precedent, the l-Arg-oxidation (RO) pathway proceeds via an iron­(IV)–oxo (ferryl) intermediate. Use of 5,5-[2H2]-l-Arg slows decay of the ferryl complex by >16-fold, implying that RO is initiated by hydrogen-atom transfer (HAT) from C5. That this large substrate deuterium kinetic isotope effect has no impact on the EF:RO partition ratio implies that the same ferryl intermediate cannot be on the EF pathway; the pathways must diverge earlier. Consistent with this conclusion, the variant enzyme bearing the Asp191Glu ligand substitution accumulates ∼4 times as much of the ferryl complex as the wild-type enzyme and exhibits a ∼40-fold diminished EF:RO partition ratio. The selective detriment of this nearly conservative substitution to the EF pathway implies that it has unusually stringent stereoelectronic requirements. An active-site, like-charge guanidinium pair, which involves the l-Arg substrate/activator and is unique to EFE among four crystallographically characterized l-Arg-modifying Fe/2OG oxygenases, may serve to selectively stabilize the transition state leading to the unique EF branch.

Microbial ethylene-forming enzyme (EFE) converts the C3-C4 fragment of the ubiquitous primary metabolite, 2-oxoglutarate (2OG), to its namesake alkene product. This reaction is very different from the simple decarboxylation of 2OG to succinate promoted by related enzymes and has inspired disparate mechanistic hypotheses. We show that EFE produces stereochemically random (equal cis and trans) 1,2-[2H2]-ethylene from (3S,4R)-[2H2]-2OG, appends an oxygen from O2 upon the C1-derived (bi)carbonate, and can be diverted to ω-hydroxylated monoacid products by modifications to 2OG or the enzyme. These results implicate an unusual radical-polar hybrid mechanism involving iron(II)-coordinated acylperoxycarbonate and alkylcarbonate intermediates. The mechanism explains how EFE accesses a high-energy carboxyl radical to initiate its fragmentation cascade, and it hints at new capabilities of 2OG-dependent enzymes that may await discovery and exploitation.