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Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme

Nature Catalysis volume 1, pages 578–584 (2018)Cite this article

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Abstract

Non-canonical amino acid ligands are useful for fine-tuning the catalytic properties of metalloenzymes. Here, we show that recombinant replacement of the histidine ligand proximal to haem in myoglobin with Nδ-methylhistidine enhances the protein’s promiscuous carbene-transfer chemistry, enabling efficient styrene cyclopropanation in the absence of reductant, even under aerobic conditions. The increased electrophilicity of the modified Fe(iii) centre, combined with subtle structural adjustments at the active site, allows direct attack of ethyl diazoacetate to produce a reactive carbenoid adduct, which has an unusual bridging Fe(iii)–C–N(pyrrole) configuration as shown by X-ray crystallography. Quantum chemical calculations suggest that the bridged complex equilibrates with the more reactive end-on isomer, ensuring efficient cyclopropanation. These findings underscore the potential of non-canonical ligands for extending the capabilities of metalloenzymes by opening up new reaction pathways and facilitating the characterization of reactive species that would not otherwise accumulate.

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Fig. 1: Mb variant containing a non-canonical NMH ligand.
Fig. 2: Styrene cyclopropanation catalysed by Mb containing a non-canonical NMH ligand.
Fig. 3: UV–Vis of Mb*(NMH) and its EDA adduct.
Fig. 4: EPR spectra of Mb*(NMH) and its EDA adduct.
Fig. 5: Crystallographic analysis of the Mb*(NMH) haem-iron–carbenoid complex.
Fig. 6: Free-energy landscape for the reaction of end-on and bridged Fe(iii) carbene complexes with styrene.

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Acknowledgements

We thank the Swiss Light Source team at the Paul Scherrer Institute for technical assistance, the Service for Mass Spectrometry (ETH Zürich) for carrying out the HRMS measurements, and T. Sandmeier for assistance with the SFC measurements. We are grateful to E. Carreira and P. Chen for access to the SFC and gas chromatography instruments, and A. Mezzetti and P. Pregosin for helpful discussions. This work was generously supported by ETH Zürich and the Swiss National Science Foundation.

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  1. These authors contributed equally: Takahiro Hayashi, Matthias Tinzl.

Authors and Affiliations

  1. Laboratory of Organic Chemistry, ETH Zürich, Zürich, Switzerland

    Takahiro Hayashi, Matthias Tinzl, Takahiro Mori & Donald Hilvert

  2. Department of Chemistry, University of Oslo, Oslo, Norway

    Ute Krengel

  3. Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland

    Jonny Proppe, Janne Soetbeer, Daniel Klose, Gunnar Jeschke & Markus Reiher

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Contributions

T.H., M.T. and D.H. designed the research. T.H., M.T. and T.M. carried out the experiments. U.K. provided guidance for the X-ray analysis and validated the crystal structures. J.P. and M.R. designed the computational methodology. J.P. carried out the calculations, and J.P. and M.R. then analysed the data obtained. J.S. and D.K. carried out the EPR measurements under the guidance of G.J. All authors contributed to writing the manuscript.

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Correspondence to Donald Hilvert.

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Hayashi, T., Tinzl, M., Mori, T. et al. Capture and characterization of a reactive haem–carbenoid complex in an artificial metalloenzyme. Nat Catal 1, 578–584 (2018). https://doi.org/10.1038/s41929-018-0105-6

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