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

Abstract

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.

Hybrid radical-polar pathway for excision of ethylene from 2-oxoglutarate by an iron oxygenase

“…3A). Similarly, the D191E variant protein, previously shown to produce only ~7% as much ethylene as wild-type EFE (6), converted 2OA almost exclusively to glutarate; 4-hydroxybutanoate was barely detectable (Fig. 3C).…”

“…Microbial ethylene-forming enzyme (EFE) conforms to this functional paradigm only in its minor reaction. In ~30% of its turnovers, the canonical ferryl complex abstracts hydrogen from C5 of L-arginine (6) to initiate its oxidative fragmentation to guanidine and pyrroline-5-carboxylate (Fig. 1A, bottom) (7)(8)(9).…”

“…1A, bottom) (7)(8)(9). The ferryl complex that initiates L-arginine oxidation (RO) is not an intermediate in the major (~70%) ethylene-forming (EF) reaction; the pathways branch from an earlier state (6). The EF reaction is a four-electron oxidation that cleaves three of the four carbon-carbon bonds in 2OG (C1-C2, C2-C3, and C4-C5) to yield three CO 2 equivalents and excise the C3-C4 unit intact.…”

“…Indeed, one recent study showed that substitution of cofactor ligand aspartate 191 with glutamate (D191E) severely compromises ethylene formation but has minimal impact on L-arginine oxidation, thus shifting the partition ratio (EF:RO) from ~2.5 to ~0.05 (Fig. 1A) (6).…”

Use of Non-canonical Tyrosine Analogs to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)

“…An isocratic elution spanning 5 minutes was then performed with a flow rate of 0.4 mL/min. 28 2OG and succinate were detected at the m/z values listed in Table S4 by electrospray ionization in the negative-ion mode (ESI-) using the HPLC-MS instrument.…”