biostudies-literatureCopeland RABasic Energy SciencesNational Institute of General Medical SciencesNIGMS NIH HHS2293-2303https://www.ebi.ac.uk/biostudies/studies/S-EPMC7962147biostudies-literatureUnknown143(5)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.Journal of the American Chemical SocietyAn Iron(IV)-Oxo Intermediate Initiating l-Arginine Oxidation but Not Ethylene Production by the 2-Oxoglutarate-Dependent Oxygenase, Ethylene-Forming Enzyme.PMC79621474R00GM129460DE-SC0016255P30 GM124169R00 GM129460Bollinger JMShoda TKCDavis KMKrebs CCopeland RABlaesi EJBoal AKfalseAn Iron(IV)-Oxo Intermediate Initiating l-Arginine Oxidation but Not Ethylene Production by the 2-Oxoglutarate-Dependent Oxygenase, Ethylene-Forming Enzyme.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.2021-01-01T00:00:00Z2021 Feb2026-04-16T15:06:06.137Z2026-04-07T14:13:25.382ZS-EPMC79621473352281110.1021/jacs.0c10923