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Mechanistic and structural characterization of an iridium-containing cytochrome reveals kinetically relevant cofactor dynamics

Nature Catalysis volume 6, pages 39–51 (2023)Cite this article

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Abstract

Artificial metalloenzymes (ArMs), which contain non-native, typically synthetic, metal cofactors, are a flourishing class of biocatalyst for unnatural reactions. Although the number of these reactions is rapidly increasing, multi-faceted mechanistic studies of ArMs comprising structural, kinetic, computational and cofactor binding data to reveal detailed mechanistic information on the effects of the protein scaffold on the structure and reactivity of ArMs are more limited. Here we report the structure of an unnatural P450 analogue using X-ray diffraction. We also report the kinetic analysis of its reaction, catalyst activation during an induction period, and the origins of the stereoselectivity for the cyclopropanation of a terpene catalysed by the iridium-containing P450 variant (Ir(Me)–CYP119). Our data reveal a mechanism initiated by the flip of the cofactor from an inactive to an active conformation. This change in conformation is followed by thousands of turnovers occurring by rate-determining formation of an iridium–carbene intermediate, thereby highlighting the influence of cofactor dynamics within a single active site on an ArM-catalysed reaction.

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Fig. 1: The structural details of Ir(Me)–CYP119.
Fig. 2: Potential kinetic mechanisms of cyclopropanation catalysed by Ir(Me)–CYP119.
Fig. 3: Kinetic data on the cyclopropanation of carvone with EDA catalysed by Ir(Me)–CYP119.
Fig. 4: Putative mechanism for cyclopropanation involving a flip of the cofactor to generate the active catalyst.
Fig. 5: Kinetics of cofactor and ligand binding to apo-CYP119 and Ir(Me)–CYP119.
Fig. 6: Proposed mechanism of cyclopropanation catalysed by Ir(Me)–CYP119.
Fig. 7: DFT calculations of cyclopropanation and MD simulations of Ir(Me)–CYP119.

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Data availability

Atomic coordinates and structure factors for Ir(Me)–CYP119 have been deposited in the PDB with the PDB code 7UOR. The data on gas evolution that support the findings of this study are accessible through Dryad (https://doi.org/10.6078/D10Q6S). The other relevant kinetics data supporting the findings of this study are available within the Supplementary Information.

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Acknowledgements

B.J.B., S.N.N. and J.F.H. acknowledge support from the Director, Office of Science, 271 US Department of Energy, under Contract No. DE-AC02-272 05CH1123. S.N.N. also thanks the NIH (F32-GM126652) and the Burroughs Wellcome fund (PDEP). K.N.H. thanks the National Institute of General Medical Sciences of the NIH (grant R01GM124480) for support. M.G.-B. acknowledges support from the Spanish Ministerio de Ciencia e Innovación (projects PID2019-111300GA-I00 and RyC2020-028628-I). The work conducted by the Joint BioEnergy Institute is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under contract no. DE-AC02-05CH11231 between LBNL and the US Department of Energy. We would like to thank R. G. Bergman, F. D. Toste, J. Y. Wang and E. D. Kalkman for valuable discussions and suggestions. We would like to thank A. Quest for helpful insight on figures.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, CA, USA

    Brandon J. Bloomer, Sean N. Natoli, Derek B. Hu & John F. Hartwig

  2. Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Brandon J. Bloomer & John F. Hartwig

  3. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Marc Garcia-Borràs & K. N. Houk

  4. Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, Girona, Spain

    Marc Garcia-Borràs

  5. Joint BioEnergy Institute, Emeryville, CA, USA

    Jose H. Pereira & Paul D. Adams

  6. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jose H. Pereira, Paul D. Adams & Douglas S. Clark

  7. Department of Bioengineering, University of California, Berkeley, CA, USA

    Paul D. Adams

  8. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA

    Douglas S. Clark

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Contributions

B.J.B. and J.F.H. designed and B.J.B. conducted the chemical kinetics and fluorimetry experiments and analysed the data. D.B.H. assisted with protein expression and purification. S.N.N. and J.H.P. designed and conducted the protein crystallography experiments. J.H.P. and P.D.A. analysed the crystallographic data. B.J.B., D.S.C. and J.F.H. interpreted the experimental data and prepared the experimental portion of the manuscript and Supplementary Information. M.G.-B. and K.N.H. designed the computational studies and interpreted the data. M.G.-B. conducted the computations, analysed the data and prepared the computational section of the manuscript and Supplemenary Information. All authors contributed to discussions, commented on and edited the manuscript.

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Correspondence to John F. Hartwig.

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Supplementary Methods, Tables 1–10 and Figs. 1–23.

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Bloomer, B.J., Natoli, S.N., Garcia-Borràs, M. et al. Mechanistic and structural characterization of an iridium-containing cytochrome reveals kinetically relevant cofactor dynamics. Nat Catal 6, 39–51 (2023). https://doi.org/10.1038/s41929-022-00899-9

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