Abstract
The use of X-ray structures to determine and interpret the ferryl iron-oxygen bond order in molecular oxygen-activating heme enzymes has, in the past, been controversial. This has mainly stemmed from the susceptibility of ferryl species to X-ray-induced electronic state changes. In this work we establishe using time-resolved serial femtosecond X-ray crystallography (tr-SFX) on a dye-decolourising peroxidase that the ferryl intermediate species (Compounds I and II) captured following in situ mixing of microcrystals with H2O2 have single, rather than the double bond character expected. X-ray emission validated tr-SFX data with quantum refinement, time-dependent-DFT calculations and QM/MM geometry optimizations together support the concept that the single iron-oxygen bond character is not an indication of ferryl reduction or a protonated form (FeIV-OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (FeIII–O•–) character. Such states offer insight into the nature of ferryl heme.
Data availability
The atomic coordinates and structure factors generated in this study for the DtpAa Y389F variant time series have been deposited in the Protein Data Bank (PDB) under the accession codes; 9S5O t = 0 [https://doi.org/10.2210/pdb9S5O/pdb]; 9S5P t = 0.5 s [https://doi.org/10.2210/pdb9S5P/pdb]; 9S5Q t = 1 s [https://doi.org/10.2210/pdb9S5Q/pdb]; 9S5R t = 5 s [https://doi.org/10.2210/pdb9S5R/pdb]; 9S5S t > 20 min [https://doi.org/10.2210/pdb9S5S/pdb]. The source data underlying Figs. 1, 4, 5, 7A, B, and Supplementary Figs. 1, 5, and 8 are provided as a Source Data file. Source data are provided with this paper.
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Acknowledgements
Data were collected at the LCLS, SLAC National Accelerator Laboratory (proposal no. L10057), supported by the DOE Office of Science, OBES under contract no. DE-AC02-76SF00515. SFX data processing was performed in part at the National Energy Research Scientific Computing Center, supported by the DOE Office of Science, contract no. DEAC02-05CH11231. The Rayonix detector used at LCLS was supported by the NIH grant S10 OD023453. Data processing was supported by the US DIALS National Resource (R24GM154040). Experiments at the LCLS were supported by the NIH grant P41GM139687, and additional assistance and technical support from Dr Vandana Tiwari and Dr Humberto Sanchez is acknowledged. The ID29 beamline at the European Synchrotron Radiation Facility (ESRF) is acknowledged for early sample testing using a tape-drive delivery setup under BAG number MX2438. L.J.W. was supported by a joint studentship award (STU0436) from Diamond Light Source and the University of Essex. J.A.R.W. and M.A.H. acknowledge support from the BBSRC (BB/W001950/1). A.M.O. is supported by the Wellcome Trust (210734/Z/18/Z) and a Royal Society Wolfson Fellowship (RSWF\R2\182017). We acknowledge the XFEL hub at Diamond Light Source for travel assistance to LCLS to perform experiments. Support from the Romanian Ministry for Education and Research and the European Union—NextGenerationEU for the Romanian Government, under the National Recovery and Resilience Plan for Romania and the European Fund of Regional Development through the Competitiveness Operational Programme 2014-2020 (projects PNRR-III-C9-2023-I8-CF76, POC/398/1/1/124155, 390005/23.10.2024-INSPIRE-II, 235/2020-CLOUDUT) is gratefully acknowledged. U.R. acknowledges support of grants from the Swedish research council (projects 2020-06176 and 2022-04978). The computations were enabled by resources provided by LUNARC, the Centre for Scientific and Technical Computing at Lund University. Support from the U.S. Department of Energy, Office of Science (OS), Office of Basic Energy Sciences (BES), Chemical Sciences, Geosciences, and Biosciences Division, contract no. DE-AC02-05CH11231 (J.Y., V.K.Y., and J.F.K.) and by the National Institutes of Health (NIH) Grants GM149528 (V.K.Y.), GM110501 (J.Y.), GM126289 (J.F.K.), is acknowledged.
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L.J.W., M.A.H., R.L.S-D., J.A.R.W.: conceptualization. L.J.W., J.J.A.G.K., A.M.V.B., Maria L., K.J.M.L., K.C., M.A.D., P.S.S., H.M., Marina L., M.T.W., P.A., J.S.W., L.G., S.D., S.M., U.R., A.M.O., J.F.K., R.L.S.-D., and J.A.R.W.: investigation. A.J.T., A.S.B., T.Z., K.C., and M.A.D.: formal analysis. J.Y., V.K.Y., J.F.K., A.M.O., K.J.M.L., and U.R.: methodology. L.J.W., M.T.W., R.L.S-D., and J.A.R.W.: writing—original draft. J.F.K., U.R., M.A.H., K.C., L.J.W., M.T.W., R.L.S-D., and J.A.R.W.: writing—review and editing.
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Williams, L.J., Kamps, J.J., Brânzanic, A.M.V. et al. Can ferric-oxyl excited states explain elongated iron-oxygen bonds in heme peroxidase catalytic intermediates?. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69192-8
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DOI: https://doi.org/10.1038/s41467-026-69192-8
