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. 2013 Jul 18;499(7458):320-3.
doi: 10.1038/nature12304.

Elucidation of the Fe(IV)=O intermediate in the catalytic cycle of the halogenase SyrB2

Shaun D Wong  1 , Martin Srnec, Megan L Matthews, Lei V Liu, Yeonju Kwak, Kiyoung Park, Caleb B Bell 3rd, E Ercan Alp, Jiyong Zhao, Yoshitaka Yoda, Shinji Kitao, Makoto Seto, Carsten Krebs, J Martin Bollinger Jr, Edward I Solomon
Affiliations

Elucidation of the Fe(IV)=O intermediate in the catalytic cycle of the halogenase SyrB2

Shaun D Wong et al. Nature. .

Abstract

Mononuclear non-haem iron (NHFe) enzymes catalyse a broad range of oxidative reactions, including halogenation, hydroxylation, ring closure, desaturation and aromatic ring cleavage reactions. They are involved in a number of biological processes, including phenylalanine metabolism, the production of neurotransmitters, the hypoxic response and the biosynthesis of secondary metabolites. The reactive intermediate in the catalytic cycles of these enzymes is a high-spin S = 2 Fe(IV)=O species, which has been trapped for a number of NHFe enzymes, including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe(IV)=O intermediate are limited in applicability owing to the paucity of experimental knowledge about its geometric and electronic structure. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes involving Fe on the nature of the Fe(IV)=O active site. Here we present NRVS structural characterization of the reactive Fe(IV)=O intermediate of a NHFe enzyme, namely the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae. This intermediate reacts via an initial hydrogen-atom abstraction step, performing subsequent halogenation of the native substrate or hydroxylation of non-native substrates. A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate directs the orientation of the Fe(IV)=O intermediate, presenting specific frontier molecular orbitals that can activate either selective halogenation or hydroxylation.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Catalytic cycle of αKG-dependent NHFe enzymes. αKG and substrate binding induces a 6-coordinate → 5-coordinate conversion (top), providing a site for O2 to bind and form an FeIV—peroxo species that nucleophilically attacks αKG, producing a peroxo-bridged FeIV species (right). Decarboxylation of αKG leads to the reactive FeIV=O intermediate (bottom right).
Figure 2
Figure 2
NRVS PVDOS spectra of SyrB2–Cl and SyrB2–Br, with regions containing intense features indicated in brackets.
Figure 3
Figure 3
a, DFT-predicted PVDOS NRVS spectra of 5C TBP structural candidate 1Cpg–X for FeIV=O intermediate of SyrB2. Vertical bars represent relative calculated mode-composition factors of vibrational modes, and brackets correspond to energy regions from Fig. 2. (Inset) Peak intensity contributions (from three bracketed regions) to overall PVDOS envelope. b, Structure of 1Cpg–X (left), along with geometric parameters and Fe—oxo stretching frequencies (right).
Figure 4
Figure 4
DFT-predicted normal modes of 5C TBP FeIV=O structures 1Cpg–X, with corresponding frequencies. The Fe—oxo vector defines the z-axis.
Figure 5
Figure 5
H-atom abstraction reaction coordinates for π-trajectory (1Thr–Cl, green) and σ-trajectory (orange), with energies (ΔE/ΔG) given in kJ mol−1. Structures of FeIII(S=5/2)—OH products displayed on right (with distances in Å), showing 1Thr–FeIIIOH (with hydrogen-bonding interactions indicated) set up for chlorination and σ-product set up for hydroxylation. For additional structural details, see Supplementary Fig. 11.
See this image and copyright information in PMC

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