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


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.


  1. 1.

    Schwizer, F. et al. Artificial metalloenzymes: reaction scope and optimization strategies. Chem. Rev. 118, 142–231 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. 2.

    Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Wang, J. Z., Peck, N. E., Renata, H. & Arnold, F. H. Cytochrome P450-catalyzed insertion of carbenoids into N–H bonds. Chem. Sci. 5, 598–601 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Sreenilayam, G. & Fasan, R. Myoglobin-catalyzed intermolecular carbene N–H insertion with arylamine substrates. Chem. Commun. 51, 1532–1534 (2015).

    Article  CAS  Google Scholar 

  5. 5.

    Tyagi, V., Bonn, R. B. & Fasan, R. Intermolecular carbene S–H insertion catalysed by engineered myoglobin-based catalysts. Chem. Sci. 6, 2488–2494 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. 7.

    Bordeaux, M., Tyagi, V. & Fasan, R. Highly diastereoselective and enantioselective olefin cyclopropanation using engineered myoglobin-based catalysts. Angew. Chem. Int. Ed. 54, 1744–1748 (2015).

    Article  CAS  Google Scholar 

  8. 8.

    Coelho, P. S. et al. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol. 9, 485–487 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Key, H. M., Dydio, P., Clark, D. S. & Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. 10.

    Sreenilayam, G., Moore, E. J., Steck, V. & Fasan, R. Metal substitution modulates the reactivity and extends the reaction scope of myoglobin carbene transfer catalysts. Adv. Synth. Catal. 359, 2076–2089 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Sreenilayam, G., Moore, E. J., Steck, V. & Fasan, R. Stereoselective olefin cyclopropanation under aerobic conditions with an artificial enzyme incorporating an iron-chlorin e6 cofactor. ACS Catal. 7, 7629–7633 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Oohora, K. et al. Catalytic cyclopropanation by myoglobin reconstituted with iron porphycene: acceleration of catalysis due to rapid formation of the carbene species. J. Am. Chem. Soc. 139, 17265–17268 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413–444 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Lewis, J. C. Metallopeptide catalysts and artificial metalloenzymes containing unnatural amino acids. Curr. Opin. Chem. Biol. 25, 27–35 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Pott, M. et al. A non-canonical proximal heme ligand affords an efficient peroxidase in a globin fold. J. Am. Chem. Soc. 140, 1535–1543 (2018).

    Article  CAS  PubMed  Google Scholar 

  16. 16.

    Green, A. P., Hayashi, T., Mittl, P. R. E. & Hilvert, D. A chemically programmed proximal ligand enhances the catalytic properties of a heme enzyme. J. Am. Chem. Soc. 138, 11344–11352 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Bhagi-Damodaran, A., Petrik, I. D., Marshall, N. M., Robinson, H. & Lu, Y. Systematic tuning of heme redox potentials and its effects on O2 reduction rates in a designed oxidase in myoglobin. J. Am. Chem. Soc. 136, 11882–11885 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Xiao, H. et al. Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase. ACS Chem. Biol. 9, 1092–1096 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Springer, B. A. & Sligar, S. G. High-level expression of sperm whale myoglobin in Escherichia coli. Proc. Natl Acad. Sci. USA 84, 8961–8965 (1987).

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Gurd, F. R., Falk, K. E., Malmström, B. G. & Vänngård, T. A magnetic resonance study of sperm whale ferrimyoglobin and its complex with 1 cupric ion. J. Biol. Chem. 242, 5724–5730 (1967).

    CAS  PubMed  Google Scholar 

  21. 21.

    Mbuvi, H. M. & Woo, L. K. Catalytic C–H insertions using iron(III) porphyrin complexes. Organometallics 27, 637–645 (2008).

    Article  CAS  Google Scholar 

  22. 22.

    Baumann, L. K., Mbuvi, H. M., Du, G. & Woo, L. K. Iron porphyrin catalyzed N–H insertion reactions with ethyl diazoacetate. Organometallics 26, 3995–4002 (2007).

    Article  CAS  Google Scholar 

  23. 23.

    Li, Y., Huang, J. S., Zhou, Z. Y., Che, C. M. & You, X. Z. Remarkably stable iron porphyrins bearing nonheteroatom-stabilized carbene or (alkoxycarbonyl)carbenes: isolation, X-ray crystal structures, and carbon atom transfer reactions with hydrocarbons. J. Am. Chem. Soc. 124, 13185–13193 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. 24.

    Liu, Y. et al. Electronic configuration and ligand nature of five-coordinate iron porphyrin carbene complexes: an experimental study. J. Am. Chem. Soc. 139, 5023–5026 (2017).

    Article  CAS  Google Scholar 

  25. 25.

    Smith, D. A., Heeg, M. J., Heineman, W. R. & Elder, R. C. Direct determination of Fe–C bond lengths in iron(II) and iron(III) cyanide solutions using EXAFS spectroelectrochemistry. J. Am. Chem. Soc. 106, 3053–3054 (1984).

    Article  CAS  Google Scholar 

  26. 26.

    Simonneaux, G. & Le Maux, P. Carbene complexes of heme proteins and iron porphyrin models. Top. Organomet. Chem. 17, 83–122 (2006).

    Article  CAS  Google Scholar 

  27. 27.

    Latos-Grazynski, L., Cheng, R. J., La Mar, G. N. & Balch, A. L. Reversible migration of an axial carbene ligand into an iron–nitrogen bond of a porphyrin. Implications for high oxidation states of heme enzymes and heme catabolism. J. Am. Chem. Soc. 103, 4270–4272 (1981).

    Article  CAS  Google Scholar 

  28. 28.

    Chevrier, B., Weiss, R., Lange, M., Chottard, J. C. & Mansuy, D. An iron(III)–porphyrin complex with a vinylidene group inserted into an iron–nitrogen bond: relevance to the structure of the active oxygen complex of catalase. J. Am. Chem. Soc. 103, 2899–2901 (1981).

    Article  CAS  Google Scholar 

  29. 29.

    Yeung, N. et al. Rational design of a structural and functional nitric oxide reductase. Nature 462, 1079–1082 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Jentzen, W., Ma, J.-G. & Shelnutt, J. A. Conservation of the conformation of the porphyrin macrocycle in hemoproteins. Biophys. J. 74, 753–763 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Renata, H. et al. Identification of mechanism-based inactivation in P450-catalyzed cyclopropanation facilitates engineering of improved enzymes. J. Am. Chem. Soc. 138, 12527–12533 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Callot, H. J. & Tschamber, T. Rearrangement of N-substituted porphyrins. Preparation and structure of homoporphyrins. J. Am. Chem. Soc. 97, 6175–6178 (1975).

    Article  CAS  Google Scholar 

  33. 33.

    Khade, R. L. & Zhang, Y. Catalytic and biocatalytic iron porphyrin carbene formation: effects of binding mode, carbene substituent, porphyrin substituent, and protein axial ligand. J. Am. Chem. Soc. 137, 7560–7563 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Dzik, W. I., Xu, X., Zhang, X. P., Reek, J. N. H. & de Bruin, B. ‘Carbene radicals’ in CoII(por)-catalyzed olefin cyclopropanation. J. Am. Chem. Soc. 132, 10891–10902 (2010).

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Carminati, D. M. et al. Designing ‘totem’ C2-symmetrical iron porphyrin catalysts for stereoselective cyclopropanations. Chem. Eur. J. 22, 13599–13612 (2016).

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Kan, S. B. J., Lewis, R. D., Chen, K. & Arnold, F. H. Directed evolution of cytochrome c for carbon–silicon bond formation: bringing silicon to life. Science 354, 1048–1051 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Kan, S. B. J., Huang, X., Gumulya, Y., Chen, K. & Arnold, F. H. Genetically programmed chiral organoborane synthesis. Nature 552, 132–136 (2017).

    CAS  PubMed  Google Scholar 

  38. 38.

    Rioz-Martínez, A., Oelerich, J., Ségaud, N. & Roelfes, G. DNA-accelerated catalysis of carbene-transfer reactions by a DNA/cationic iron porphyrin hybrid. Angew. Chem. Int. Ed. 55, 14136–14140 (2016).

    Article  CAS  Google Scholar 

  39. 39.

    Nicolas, I., Maux, P., Le & Simonneaux, G. Synthesis of chiral water-soluble metalloporphyrins (Fe, Ru,): new catalysts for asymmetric carbene transfer in water. Tetrahedron Lett. 49, 5793–5795 (2008).

    Article  CAS  Google Scholar 

  40. 40.

    Intrieri, D. et al. Highly diastereoselective cyclopropanation of α-methylstyrene catalysed by a C2-symmetrical chiral iron porphyrin complex. Chem. Commun. 50, 1811–1813 (2014).

    Article  CAS  Google Scholar 

  41. 41.

    Zeldin, O. B., Gerstel, M. & Garman, E. F. RADDOSE-3D: time- and space-resolved modelling of dose in macromolecular crystallography. J. Appl. Crystallogr. 46, 1225–1230 (2013).

    Article  CAS  Google Scholar 

  42. 42.

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  43. 43.

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Vojtěchovský, J., Chu, K., Berendzen, J., Sweet, R. M. & Schlichting, I. Crystal structures of myoglobin–ligand complexes at near-atomic resolution. Biophys. J. 77, 2153–2174 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  CAS  Google Scholar 

  46. 46.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

    Article  CAS  PubMed  Google Scholar 

  47. 47.

    Terwilliger, T. C. et al. Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias. Acta Crystallogr. D 64, 515–524 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. 48.

    Liebschner, D. et al. Polder maps: improving OMIT maps by excluding bulk solvent. Acta Crystallogr. D 73, 148–157 (2017).

    Article  CAS  Google Scholar 

  49. 49.

    Berry, E. A. & Trumpower, B. L. Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem. 161, 1–15 (1987).

    Article  CAS  PubMed  Google Scholar 

  50. 50.

    Williams, J. N. A method for the simultaneous quantitative estimation of cytochromes a, b, c1, and c in mitochondria. Arch. Biochem. Biophys. 107, 537–543 (1964).

    Article  CAS  PubMed  Google Scholar 

  51. 51.

    Efimov, I. et al. A simple method for the determination of reduction potentials in heme proteins. FEBS Lett. 588, 701–704 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Stoll, S. & Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. 53.

    Frisch, M. J. E. A. et al. Gaussian 09, revision D.01 (Gaussian, Inc., 2009).

  54. 54.

    Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 77, 3865–3868 (1996).

    Article  CAS  Google Scholar 

  55. 55.

    Weigend, F. & Ahlrichs, R. Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297–3305 (2005).

    Article  CAS  Google Scholar 

  56. 56.

    Weigend, F. Accurate Coulomb-fitting basis sets for H to Rn. Phys. Chem. Chem. Phys. 8, 1057–1065 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. 57.

    Adamo, C. & Barone, V. Toward reliable density functional methods without adjustable parameters: the PBE0 model. J. Chem. Phys. 110, 6158–6170 (1999).

    Article  CAS  Google Scholar 

  58. 58.

    Reiher, M., Salomon, O. & Hess, B. A. Reparameterization of hybrid functionals based on energy differences of states of different multiplicity. Theor. Chem. Acc. 107, 48–55 (2001).

    Article  CAS  Google Scholar 

  59. 59.

    Peng, C. & Bernhard Schlegel, H. Combining synchronous transit and quasi-Newton methods to find transition states. Isr. J. Chem. 33, 449–454 (1993).

    Article  CAS  Google Scholar 

  60. 60.

    Fukui, K. The path of chemical reactions—the IRC approach. Acc. Chem. Res. 14, 363–368 (1981).

    Article  CAS  Google Scholar 

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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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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).

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