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Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Lewis, Russell D. and Garcia-Borràs, Marc and Chalkley, Matthew J. and Buller, Andrew R. and Houk, K. N. and Kan, S. B. Jennifer and Arnold, Frances H. (2018) Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase. Proceedings of the National Academy of Sciences of the United States of America, 115 (28). pp. 7308-7313. ISSN 0027-8424. PMCID PMC6048479.

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Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c. High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

Item Type:Article
Related URLs:
URLURL TypeDescription Information CentralArticle
Lewis, Russell D.0000-0002-5776-7347
Garcia-Borràs, Marc0000-0001-9458-1114
Chalkley, Matthew J.0000-0002-0484-7335
Buller, Andrew R.0000-0002-9635-4844
Houk, K. N.0000-0002-8387-5261
Kan, S. B. Jennifer0000-0001-6371-8042
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2018 National Academy of Sciences. Published under the PNAS license. Contributed by Frances H. Arnold, May 31, 2018 (sent for review April 26, 2018; reviewed by Kara L. Bren and Ryan G. Hadt). We thank J. M. Bollinger Jr., K. Chen, X. Huang, C. Krebs, C. J. Pollock, and R. K. Zhang for helpful discussions and A. Tang for experimental assistance. This work was supported by National Science Foundation Division of Chemistry Grant CHE-1361104 (to K.N.H.); the Rothenberg Innovation Initiative (RI2) Program (S.B.J.K. and F.H.A.); the Jacobs Institute for Molecular Engineering for Medicine at Caltech (S.B.J.K. and F.H.A.); National Science Foundation Division of Molecular and Cellular Biosciences Grant MCB-1513007 (to F.H.A.); and Office of Chemical, Bioengineering, Environmental and Transport Systems SusChEM Initiative Grant CBET-1403077 (to F.H.A.). R.D.L. is supported by NIH National Research Service Award Training Grant 5 T32 GM07616. M.G.-B. thanks the Ramón Areces Foundation for a postdoctoral fellowship. M.J.C. thanks the Center for Environmental Microbial Interactions at Caltech for a fellowship. Crystallography experiments were supported by J. Kaiser and the Caltech Molecular Observatory. EPR experiments were performed with the assistance of P. Oyala and supported by National Science Foundation Grant NSF-1531940 (to the Caltech EPR Facility). Computational resources were provided by the University of California, Los Angeles Institute for Digital Research and Education and the Extreme Science and Engineering Discovery Environment, which is supported by National Science Foundation Grant OCI-1053575. R.D.L. and M.G.-B. contributed equally to this work. Author contributions: R.D.L., M.G.-B., K.N.H., S.B.J.K., and F.H.A. designed research; R.D.L., M.G.-B., M.J.C., and S.B.J.K. performed research; R.D.L., M.G.-B., M.J.C., A.R.B., K.N.H., S.B.J.K., and F.H.A. analyzed data; and R.D.L., M.G.-B., K.N.H., S.B.J.K., and F.H.A. wrote the paper. Reviewers: K.L.B., University of Rochester; and R.G.H., Argonne National Laboratory. The authors declare no conflict of interest. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, (PDB ID codes 6CUK and 6CUN). This article contains supporting information online at
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Rothenberg Innovation Initiative (RI2)UNSPECIFIED
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
NIH Predoctoral Fellowship5 T32 GM07616
Ramón Areces FoundationUNSPECIFIED
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
Subject Keywords:carbene; reactive intermediate; heme; metalloenzyme; carbene transferase
Issue or Number:28
PubMed Central ID:PMC6048479
Record Number:CaltechAUTHORS:20180626-131945645
Persistent URL:
Official Citation:Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase Russell D. Lewis, Marc Garcia-Borràs, Matthew J. Chalkley, Andrew R. Buller, K. N. Houk, S. B. Jennifer Kan, Frances H. Arnold Proceedings of the National Academy of Sciences Jul 2018, 115 (28) 7308-7313; DOI: 10.1073/pnas.1807027115
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:87351
Deposited By: George Porter
Deposited On:26 Jun 2018 20:55
Last Modified:27 Feb 2020 20:05

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