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Dynamic metal coordination controls chemoselectivity in a radical halogenase

Nature Chemical Biology volume 22, pages 491–500 (2026)Cite this article

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A preprint version of the article is available at bioRxiv.

Abstract

The activation of inert C(sp3)–H bonds by nonheme Fe enzymes provides a powerful biocatalytic platform for the chemical synthesis of molecules with increased sp3 complexity. In this context, FeII/α-ketoglutarate-dependent radical halogenases are uniquely capable of carrying out transfer of a diverse array of bound anions following C–H activation. Here, we provide experimental evidence that bifurcation of radical rebound after H-atom abstraction can be driven both by the ability of a dynamic metal coordination sphere to reorganize and by a second-sphere hydrogen-bonding network where only two residues are sufficient. In addition, we present crystallographic data supporting the existence of an early peroxyhemiketal intermediate in the O2 activation pathway of FeII/α-ketoglutarate-dependent enzymes. These data provide a paradigm for understanding the evolution of catalytic plasticity in these enzymes and yields insight into the design principles by which to expand their reaction scope.

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Fig. 1: Radical halogenases exert precise control over chemoselectivity.
Fig. 2: Probing the structure of HalA intermediates by crystallography.
Fig. 3: Spectroscopic validation of VIV–oxo isomerism in HalA.
Fig. 4: Proposed mechanism of chemoselectivity.
Fig. 5: Investigation of second-sphere residues validates the DFT model.

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Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. Should any raw data files be needed in another format, they are available from the corresponding author upon request. Crystallographic data for structures were deposited to the Protein Data Bank (PDB) under accession numbers 9OER, 9OEU, 9OES, 9OET, 9OEW and 9OEV. Source data are provided with this paper.

Code availability

The code used to produce the TD-DFT fits of the XAS data is provided as Supplementary Data 2.

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Acknowledgements

This work was funded by generous support from the National Institutes of Health (NIH; R01 GM134271 to M.C.Y.C. and R01 GM138580 to J.M.B.) and by DOE/LBL DEAC02-05CH11231 FWP CH030201. E.N.K. acknowledges the support of an NIH National Research Service Award training grant (T32 GM066698). I.K. is supported by the Miller Institute for Basic Research in Science (University of California, Berkeley). E.A.S. acknowledges support from the Jane Coffin Childs Fund for Medical Research. J.W.S. acknowledges support of the National Institute of General Medical Sciences of the NIH (F32 GM136156). J.Y. acknowledges support of the NIH (R01 GM110501) for the XAS data collection. We thank E. I. Solomon and C. Krebs for helpful discussions and access to their EPR and Mössbauer spectrometers, respectively. We also thank P. Jeffrey for his helpful feedback regarding the analysis of crystallography data. We acknowledge L. Nocka and the J. Kuriyan laboratory for assistance using their size-exclusion chromatography multiangle light scattering instrument. X-ray diffraction data were collected at the Advanced Light Source beamline 8.3.1, which is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives (MR-15-328599), the NIH (R01 GM124149 and P30 GM124169), Plexxikon and the Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research. The Advanced Light Source is a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the US Department of Energy under contract number DEAC02-05CH11231. XAS data were collected at SSRL beamline 9-3. Use of the SSRL, Stanford Linear Accelerator Center National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DEAC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the Department of Energy Office of Biological and Environmental Research and by the NIH National Institute of General Medical Sciences (P30 GM133894).

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Author notes

  1. Elizabeth A. Stone

    Present address: Department of Chemistry and Biochemistry, Fairfield University, Fairfield, CT, USA

Authors and Affiliations

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

    Elijah N. Kissman, Ioannis Kipouros, Elizabeth A. Stone, Avery Y. Yang, Andrew M. Whitten & Michelle C. Y. Chang

  2. Miller Institute for Basic Research in Science, University of California, Berkeley, CA, USA

    Ioannis Kipouros

  3. Department of Chemistry, The Pennsylvania State University, University Park, PA, USA

    Jeffrey W. Slater & J. Martin Bollinger Jr.

  4. Department of Chemistry, Stanford University, Stanford, CA, USA

    Augustin Braun

  5. Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, USA

    Alder R. Ensberg & Michelle C. Y. Chang

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

    Kuntal Chatterjee, Isabel Bogacz & Junko Yano

  7. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA

    J. Martin Bollinger Jr.

  8. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Michelle C. Y. Chang

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Contributions

E.N.K. designed the study, performed the enzyme characterization experiments, led the protein crystallography studies and analyzed the SF-Abs kinetics. I.K. designed the study, performed the DFT calculations and contributed to the analysis of spectroscopic data. J.W.S. assisted with the enzyme kinetics, EPR and Mössbauer data collection and analysis. E.A.S. assisted with the protein crystallography. A.Y.Y. and A.R.E. assisted with the enzyme characterization and protein crystallography studies. A.B. assisted with the spectroscopic data analysis. A.M.W. assisted with the protein crystallography. K.C., I.B. and J.Y. performed the XAS experiments. J.M.B. assisted with the design and analysis of SF-Abs, EPR and Mössbauer experiments. M.C.Y.C. designed the study, assisted with the data analysis and managed the project. E.N.K., I.K. and M.C.Y.C. wrote the paper with contributions from all other authors.

Corresponding author

Correspondence to Michelle C. Y. Chang.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Crystal structure of HalA with the peroxyhemiketal intermediate bound.

Analysis of the assignment of the electron density found in the in crystallo reaction of anaerobic HalA with O2 as the peroxyhemiketal intermediate. See Supplementary Table 15 for additional information.

Extended Data Fig. 2 Analysis and comparison of the Fe-peroxyhemiketal(PHK)/Criegee intermediate observed in HalA.

Analysis of the geometry of the HalA peroxyhemiketal intermediate and its comparison to a structure of homoprotocatechuate 2,3-dioxygenase from Bulbophyllum fuscum (2,3-HPCD) containing a similar intermediate (PDB: 2IGA). See Supplementary Table 15 for additional information.

Extended Data Fig. 3 Crystal structure of Hydrox with VIV-oxo bound.

A crystal structure of Hydrox substituted with VIV-oxo was also solved to compare to HalA. See Supplementary Table 15 for additional information.

Extended Data Fig. 4 EPR spectroscopy of HalA and Hydrox with VIV-oxo.

Details of the analysis of VIV-oxo speciation in HalA and Hydrox based on EPR spectroscopy. See Supplementary Table 15 for additional information.

Source data

Extended Data Fig. 5 DFT-optimized structures and relative energies for VIV-oxo and FeIV-oxo isomers of HalA.

DFT was used to analyze the energetics of various configurational isomers of VIV-oxo and FeIV-oxo intermediates for HalA with varying coordination numbers (5-coordinate with succinate monodentate vs 6-coordinate with succinate bidentate), oxo/Cl isomerism (in-line vs off-line), and axial metal ligands (that is trans to the oxo ligand; succinate vs histidine). See Supplementary Table 15 for additional information.

Extended Data Fig. 6 Mössbauer spectroscopy of HalA.

Mössbauer spectroscopy of HalA shows evidence of only one FeIV-oxo species, however, the in-line and off-line isomers of FeIV-oxo HalA are pseudo-enantiomers with indistinguishable Mössbauer parameters. See Supplementary Table 15 for additional information.

Source data

Extended Data Fig. 7 SF-Abs kinetics of wild-type HalA and proposed model.

Pre-steady state kinetic analysis of HalA was carried out to measure the rate of H-atom abstraction by rapid mixing of anaerobic HalA samples with O2 and measurement of the rate of decay of the signature FeIV-oxo peak. See Supplementary Table 15 for additional information.

Source data

Extended Data Fig. 8 Thr226 tunes the active site H-bonding network.

Analysis of the role of Thr226 in altering the partitioning between halogenation and hydroxylation. Although no effect is observed in the T226 single mutants, mutation in a HalA N224V background does show an effect on this ratio. See Supplementary Table 15 for additional information.

Source data

Supplementary information

Supplementary Information (download PDF )

Supplementary Methods, Supplementary Figs. 1–9, Supplementary Tables 1–15 and Supplementary References.

Reporting Summary (download PDF )

Supplementary Data 1 (download XLSX )

Analytical size-exclusion chromatography data for Supplementary Fig. 1.

Supplementary Data 2 (download XLSX )

XAS and TD-DFT data for Supplementary Fig. 4.

Supplementary Data 3 (download XLSX )

Chloride-dependent kinetics data for Supplementary Fig. 6.

Supplementary Data 4 (download XLSX )

Steady-state kinetics of HalA variants for Supplementary Fig. 7.

Supplementary Data 5 (download XLSX )

Stopped-flow kinetics of HalA variants for Supplementary Fig. 8.

Supplementary Data 6 (download TXT )

DFT model coordinates.

Supplementary Code 1 (download TXT )

Code to generate TD-DFT fit.

Source data

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Kissman, E.N., Kipouros, I., Slater, J.W. et al. Dynamic metal coordination controls chemoselectivity in a radical halogenase. Nat Chem Biol 22, 491–500 (2026). https://doi.org/10.1038/s41589-025-02077-x

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