Can ferric-oxyl excited states explain elongated iron-oxygen bonds in heme peroxidase catalytic intermediates?
- Lewis J. Williams
- Jos J.A.G. Kamps
- Adrian M. V. Brânzanic
- Maria Lehene
- Kristoffer J. M. Lundgren
- Ulf Ryde
- Kuntal Chatterjee
- Margaret D. Doyle
- Philipp S. Simon
- Hiroki Makita
- Amy J. Thompson
- Aaron S. Brewster
- Tiankun Zhou
- Marina Lučić
- Michael T. Wilson
- Pierre Aller
- Juan Sanchez-Weatherby
- Leland Gee
- Sebastian Dehe
- Sandra Mous
- Junko Yano
- Vittal K. Yachandra
- Michael A. Hough
- Allen M. Orville
- Jan F. Kern
- Radu L. Silaghi-Dumitrescu
- Jonathan A. R. Worrall
2026-02-03
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 H 2 O 2 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 (Fe IV -OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (Fe III –O •– ) character. Such states offer insight into the nature of ferryl heme.