An Iron(IV)–Oxo Intermediate Initiating l‑Arginine Oxidation but Not Ethylene Production by the 2‑Oxoglutarate-Dependent Oxygenase, Ethylene-Forming Enzyme

• Published in: Journal of the American Chemical Society Vol. 143; no. 5; pp. 2293 - 2303
• Main Authors: Copeland, Rachelle A, Davis, Katherine M, Shoda, Tokufu Kent C, Blaesi, Elizabeth J, Boal, Amie K, Krebs, Carsten, Bollinger, J. Martin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 10.02.2021; American Chemical Society (ACS)
• Subjects: Absorption; Chemical reactions; Hydrocarbons; Hydrogen isotopes; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Peptides and proteins
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.0c10923

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.