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

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 metabolites1,2,3. 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 enzymes4,5,6,7,8, including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe(iv)=O intermediate9,10,11,12,13 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 site14,15,16. 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 substrates17. 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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Figure 1: Catalytic cycle of αKG-dependent NHFe enzymes.
Figure 2: NRVS PVDOS spectra of SyrB2–Cl and SyrB2–Br.
Figure 3: Computational spectra and structure of five-coordinate TBP structural candidate 1Cpg–X for the Fe(iv)=O intermediate of SyrB2.
Figure 4: DFT-predicted normal modes of
Figure 5: Hydrogen-atom abstraction reaction coordinates.

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Acknowledgements

Funding for this work was provided by the National Institutes of Health (GM-40392 to E.I.S. and GM-69657 to J.M.B. and C.K.) and the National Science Foundation (MCB-0919027 to E.I.S., and MCB-642058 and CHE-724084 to J.M.B. and C.K.). Work at the Advanced Photon Source was supported by the Department of Energy, Office of Science, under contract DE-AC-02-06CH11357. Synchrotron experiments at SPring-8 were performed with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; proposal no. 2010B1569). M.S. thanks the Rulíšek group at the IOCB, Prague, for use of their computational resources.

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Contributions

S.D.W. and M. Srnec contributed equally to this work. E.I.S., C.K. and J.M.B. designed the experiments. S.D.W., M. Srnec, M.L.M., L.V.L., Y.K., K.P. and C.B.B. performed the experiments. S.D.W., M. Srnec and E.I.S. analysed the data and wrote the manuscript. E.E.A., J.Z., Y.Y., S.K. and M. Seto provided technical assistance at the sychrotron beamlines.

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Correspondence to Carsten Krebs, J. Martin Bollinger or Edward I. Solomon.

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Wong, S., Srnec, M., Matthews, M. et al. Elucidation of the Fe(iv)=O intermediate in the catalytic cycle of the halogenase SyrB2. Nature 499, 320–323 (2013). https://doi.org/10.1038/nature12304

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