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Frontier Molecular Orbital Contributions to Chlorination versus Hydroxylation Selectivity in the Non-Heme Iron Halogenase SyrB2.

ABSTRACT: The ability of an FeIV?O intermediate in SyrB2 to perform chlorination versus hydroxylation was computationally evaluated for different substrates that had been studied experimentally. The ?-trajectory for H atom abstraction (FeIV?O oriented perpendicular to the C-H bond of substrate) was found to lead to the S = 2 five-coordinate HO-FeIII-Cl complex with the C• of the substrate, ?-oriented relative to both the Cl- and the OH- ligands. From this ferric intermediate, hydroxylation is thermodynamically favored, but chlorination is intrinsically more reactive due to the energy splitting between two key redox-active d?* frontier molecular orbitals (FMOs). The splitting is determined by the differential ligand field effect of Cl- versus OH- on the Fe center. This makes chlorination effectively competitive with hydroxylation. Chlorination versus hydroxylation selectivity is then determined by the orientation of the substrate with respect to the HO-Fe-Cl plane that controls either the Cl- or the OH- to rebound depending on the relative ?-overlap with the substrate C radical. The differential contribution of the two FMOs to chlorination versus hydroxylation selectivity in SyrB2 is related to a reaction mechanism that involves two asynchronous transfers: electron transfer from the substrate radical to the iron center followed by late ligand (Cl- or OH-) transfer to the substrate.

SUBMITTER: Srnec M

PROVIDER: S-EPMC5310988 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Frontier Molecular Orbital Contributions to Chlorination versus Hydroxylation Selectivity in the Non-Heme Iron Halogenase SyrB2.

Srnec Martin M Solomon Edward I EI

Journal of the American Chemical Society 20170202 6

The ability of an FeIV═O intermediate in SyrB2 to perform chlorination versus hydroxylation was computationally evaluated for different substrates that had been studied experimentally. The π-trajectory for H atom abstraction (FeIV═O oriented perpendicular to the C-H bond of substrate) was found to lead to the S = 2 five-coordinate HO-FeIII-Cl complex with the C• of the substrate, π-oriented relative to both the Cl- and the OH- lig ...[more]

PMID: 28095695

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Project description:We present here a computational study of reactions at a model complex of the SyrB2 enzyme active site. SyrB2, which chlorinates L-threonine in the syringomycin biosynthetic pathway, belongs to a recently discovered class of alpha-ketoglutarate (alphaKG), non-heme Fe(II)-dependent halogenases that share many structural and chemical similarities with hydroxylases. Namely, halogenases and hydroxylases alike decarboxylate the alphaKG co-substrate, facilitating formation of a high-energy ferryl-oxo intermediate that abstracts a hydrogen from the reactant complex. The reaction mechanisms differ at this point, and mutation of active site residues (Asp for the hydroxylase to Ala or Ala to Asp/Glu for halogenase) fails to reproduce hydroxylating activity in SyrB2 or halogenating activity in similar hydroxylases. Using a density functional theory approach with a recently implemented Hubbard U correction for accurate treatment of transition-metal chemistry, we explore probable reaction pathways and mechanisms via a model complex consisting of only the iron center and its direct ligands. We show that the first step, alphaKG decarboxylation, is barrierless and exothermic, but the subsequent hydrogen abstraction step has an energetic barrier consistent with that accessible under biological conditions. In the model complex we use, radical chlorination is barrierless and exothermic, whereas the analogous hydroxylation is found to have a small energetic barrier. The hydrogen abstraction and radical chlorination steps are strongly coupled: the barrier for the hydrogen abstraction step is reduced when carried out concomitantly with the exothermic chlorination step. Our work suggests that the lack of chlorination in mutant hydroxylases is most likely due to poor binding of chlorine in the active site, whereas mutant halogenases do not hydroxylate for energetic reasons. Although secondary shell residues undoubtedly modulate the overall reactivity and binding of relevant substrates, we show that a small model compound consisting exclusively of the direct ligands to the metal can help explain reactivity heretofore not yet understood in the halogenase SyrB2.

Project description:Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical β-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Project description:Non-heme iron based halogenase enzymes promote selective halogenation of the sp3-C-H bond through iron(iv)-oxo-halide active species. During halogenation, competitive hydroxylation can be prevented completely in enzymatic systems. However, synthetic iron(iv)-oxo-halide intermediates often result in a mixture of halogenation and hydroxylation products. In this report, we have developed a new synthetic strategy by employing non-heme iron based complexes for selective sp3-C-H halogenation by overriding hydroxylation. A room temperature stable, iron(iv)-oxo complex, [Fe(2PyN2Q)(O)]2+ was directed for hydrogen atom abstraction (HAA) from aliphatic substrates and the iron(ii)-halide [FeII(2PyN2Q)(X)]+ (X, halogen) was exploited in conjunction to deliver the halogen atom to the ensuing carbon centered radical. Despite iron(iv)-oxo being an effective promoter of hydroxylation of aliphatic substrates, the perfect interplay of HAA and halogen atom transfer in this work leads to the halogenation product selectively by diverting the hydroxylation pathway. Experimental studies outline the mechanistic details of the iron(iv)-oxo mediated halogenation reactions. A kinetic isotope study between PhCH3 and C6D5CD3 showed a value of 13.5 that supports the initial HAA step as the RDS during halogenation. Successful implementation of this new strategy led to the establishment of a functional mimic of non-heme halogenase enzymes with an excellent selectivity for halogenation over hydroxylation. Detailed theoretical studies based on density functional methods reveal how the small difference in the ligand design leads to a large difference in the electronic structure of the [Fe(2PyN2Q)(O)]2+ species. Both experimental and computational studies suggest that the halide rebound process of the cage escaped radical with iron(iii)-halide is energetically favorable compared to iron(iii)-hydroxide and it brings in selective formation of halogenation products over hydroxylation.

Dataset Information

Frontier Molecular Orbital Contributions to Chlorination versus Hydroxylation Selectivity in the Non-Heme Iron Halogenase SyrB2.

Publications

Frontier Molecular Orbital Contributions to Chlorination versus Hydroxylation Selectivity in the Non-Heme Iron Halogenase SyrB2.

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