Thermodynamics of Iron(II) and Substrate Binding to the Ethylene-Forming Enzyme

Li, Mingjie; Martinez, Salette; Hausinger, Robert P.; Emerson, Joseph P.

doi:10.1021/acs.biochem.8b00730

Cited by 16 publications

(9 citation statements)

References 30 publications

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“…Earlier experimental studies proposed that F283 might prevent the ferryl flip so that the off-line ferryl might lead to ethylene formation . Therefore, we further tested the proposal that intermediates along the standard O 2 activation mechanism (with l -Arg bound in conformation A and 2OG coordinated off-line), including the off-line ferryl, can produce ethylene. ,, We calculated the reaction paths for producing ethylene with a reaction coordinate combining an increase in the C2–C3 bond length and a reduction in the C3–C4 bond length (with numbering retained from 2OG), starting from each of the above intermediates (succinyl-peroxide AO-IM1 , half-bond AO-IM2 , and off-line ferryl AO-IM3 ). The activation energies required to decompose 2OG to form ethylene from AO-IM1 , AO-IM2 , and AO-IM3 are 68.1, 19.2, and 43.3 kcal/mol, respectively.…”

Section: Resultsmentioning

confidence: 99%

Atomic and Electronic Structure Determinants Distinguish between Ethylene Formation and l-Arginine Hydroxylation Reaction Mechanisms in the Ethylene-Forming Enzyme

et al. 2021

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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 C5 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•Fe(III)•OO •− •2OG•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 L-Arg is present in conformation A, the Fe(III)-OO •− intermediate undergoes the standard O 2 activation mechanism involving ferryldependent 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 CO 2 , 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 β electron in the Fe(III)-OO − complex, either in a π* ∥ orbital that triggers ethylene formation or a π* ⊥ 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 •− 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.

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Section: Resultsmentioning

confidence: 99%

Atomic and Electronic Structure Determinants Distinguish between Ethylene Formation and l-Arginine Hydroxylation Reaction Mechanisms in the Ethylene-Forming Enzyme

et al. 2021

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“…In addition to the most common type of hydroxylation reaction, − αKG-NHFe enzymes also catalyze many nonhydroxylation reactions, including desaturation, ring formation, decarboxylation-assisted desaturation, , sequential desaturation and epoxidation, − ring expansion, halogenation, carbon skeleton rearrangements, endoperoxidation, , and αKG decarboxylation to form ethylene. − Among the ∼60 X-ray crystal structures of αKG-NHFe enzymes in the Protein Data Bank (PDB), two subclasses exist (the distal- and proximal-types) . A vast majority of αKG-NHFe enzymes have a conserved His-X-Asp/Glu-X n -His (2-His-1-carboxylate) motif, in which His and Glu/Asp residues serve as the iron-coordinating ligands, , and αKG is a bidentate ligand in either the distal type (e.g., FtmOx1 with pdb entry 4Y5S in Scheme a) or the proximal type (e.g., NvfI with pdb entry 7ENB in Scheme b). , Based on phylogenetic tree analysis, FtmOx1 and NvfI belong to two different subclasses. ,,,− …”

Section: Introductionmentioning

confidence: 99%

Dissecting the Mechanism of the Nonheme Iron Endoperoxidase FtmOx1 Using Substrate Analogues

Zhu

Yan

Cheng

et al. 2022

JACS Au

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FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure–function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B ( 7 ) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•Co II •αKG• 7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•Fe II •αKG binary complex. Verruculogen ( 2 ) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.

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“…Thus, the ACC synthase converts S -adenosyl- l -methionine into 1-aminocyclopropane-1-carboxylic acid, whereas the ACC oxidase converts ACC into ethylene, CO 2 , and HCN (Scheme ). − Ethylene is also reported to be synthesized by plant microbes such as Pseudomonas syringae and Ralstonia solanacearum from the ethylene-forming enzyme (EFE). − EFE is a member of the superfamily consisting of mononuclear nonheme iron(II) and α-ketoglutarate (αKG, also called 2-oxoglutarate)-dependent oxygenases, which is a versatile family of nonheme iron dioxygenases that typically catalyze the hydroxylation of aliphatic groups. − In EFE, the enzyme produces ethylene from αKG on an iron(II) center using O 2 (Scheme ), while in the substrate-binding pocket, l -arginine is bound, which is hydroxylated through a side reaction.…”

Section: Introductionmentioning

confidence: 99%

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

et al. 2022

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Ethylene is an important signaling molecule in plants that triggers the growth of leaves, flowers, and fruits. One of the enzymes involved in the biosynthesis of ethylene is the ethylene-forming enzyme (EFE), which is an usual nonheme iron enzyme that biodegrades α-ketoglutarate into three CO 2 molecules and ethylene. As the detailed mechanism of EFE in the biosynthesis of ethylene remains controversial and particularly the function of the co-substrate L-arginine, we decided to pursue a density functional theory study on the possible pathways of the enzyme leading to ethylene biosynthesis and test many possible pathways and mechanisms. A large active site cluster model of 322 atoms was created, which contains all the features of the firstand second-coordination sphere of the active site and substrate (αketoglutarate) binding pockets. The calculations identify a persuccinate intermediate that triggers a bifurcation pathway in the enzyme and either react with a molecule of CO 2 to form a carbonate or forms a high-valent iron(IV)-oxo species through heterolytic dioxygen bond cleavage. Our studies show that both the bifurcation pathways converge to the same intermediate again and can lead to ethylene products, although the two pathways have different kinetics. Interestingly, our studies also show that the iron(IV)-oxo itself can form a carbonate and ethylene but through much higher barriers. As a matter of fact, these barriers are higher in energy than the typical aliphatic hydroxylation barriers and may not be competitive with arginine hydroxylation. Inclusion of the L-arginine co-substrate into the model leads to minor changes in the structure and fold, but its charge and dipole moment does not seem to affect the first stage of the catalytic cycle. Moreover, the key activation barriers seem less affected by the inclusion of L-arginine into the model. We, therefore, believe that the role of L-arginine is to lock α-ketoglutarate and its products into a tight binding pocket to enable its degradation and to prevent early release of CO 2 . Our studies show that due to the distinct differences in α-ketoglutarate positioning between different arginine activating nonheme iron dioxygenases in the co-substrate binding pocket and its tighter binding in EFE, we predict that the release of CO 2 is prevented in the first stage of the oxygen activation mechanism. This enables attack of CO 2 on a persuccinate complex to form carbonate products, leading to ethylene formation. This work gives suggestions on the engineering of EFE into a hydroxylase or improving the ethylene biosynthesis.

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Thermodynamics of Iron(II) and Substrate Binding to the Ethylene-Forming Enzyme

Cited by 16 publications

References 30 publications

Atomic and Electronic Structure Determinants Distinguish between Ethylene Formation and l-Arginine Hydroxylation Reaction Mechanisms in the Ethylene-Forming Enzyme

Atomic and Electronic Structure Determinants Distinguish between Ethylene Formation and l-Arginine Hydroxylation Reaction Mechanisms in the Ethylene-Forming Enzyme

Dissecting the Mechanism of the Nonheme Iron Endoperoxidase FtmOx1 Using Substrate Analogues

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

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