Abiological catalysis by artificial haem proteins containing noble metals in place of iron

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Abstract

Enzymes that contain metal ions—that is, metalloenzymes—possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and substrate-selectivity of the system1. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations2,3. However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites1,4,5. The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein6,7,8. This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes9,10. In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C–H bonds to form C–C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C–H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.

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Figure 1: Strategy for expedient preparation of [M]-PIX-proteins.
Figure 2: Evaluation of artificial [M]-PIX mOCR-myoglobins as catalysts.
Figure 3: Summary of activities and selectivities for reactions catalysed by Ir(Me)-mOCR-Myo.
Figure 4: Directed evolution strategy used to obtain Ir(Me)-mOCR-Myo mutants capable of producing either enantiomer of the products of C–H insertion reactions of varied substrates.

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Acknowledgements

This work was supported by the Director, Office of Science, of the US Department of Energy under contract no. DE-AC02- 05CH11231, by the NSF (graduate research fellowship to H.M.K.), and the NWO Netherlands Organization for Scientific Research (Rubicon postdoctoral fellowship no. 680-50-1306 to P.D.). We thank the QB3 MacroLab facility (sub-cloning), the UC Berkeley DNA Sequencing Facility (plasmid sequencing), T. Iavarone and the QB3 Mass Spectrometry Facility (supported by NIH grant 1S10RR022393-01) for native NS-ESI-MS data and analysis, and H. Zhao (University of Illinois-Champaign Urbana) for the P411-CIS gene.

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H.M.K., P.D., and J.F.H. conceived the work, designed the initial experiments, and interpreted the data. All authors discussed the results to design subsequent experiments. H.M.K. and P.D. performed all the experiments. H.M.K., P.D., and J.F.H. wrote the manuscript and all authors reviewed the manuscript.

Corresponding author

Correspondence to John F. Hartwig.

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Competing interests

Competing Interests: The authors have filed through the Lawrence Berkeley National Laboratory a provisional patent application (U.S. Patent Application Ser. No: 62/241,487) that is based on the results presented here.

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Key, H., Dydio, P., Clark, D. et al. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016). https://doi.org/10.1038/nature17968

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