Time-resolved serial synchrotron and serial femtosecond crystallography of heme proteins using photocaged nitric oxide
Time-resolved X-ray crystallography is undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods for uniformly initiating time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions or signalling. A widely applicable approach is the use of photocaged substrates, where the photocage is soaked into the crystal in advance and then activated using a laser pulse to provide uniform initiation of the reaction throughout the crystal. This work characterizes photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c'-β and dye-decolourizing peroxidase B using a fixed target sample delivery system. Laser parameters for photoactivation are systematically explored, and time-resolved structures over timescales ranging from 100 µs to 1.4 s using synchrotron and XFEL beamlines are described. The effective use of this photocage for time-resolved crystallography is demonstrated and appropriate illumination conditions for such experiments are determined.
Authors
- Takehiko Tosha (ORCID: https://orcid.org/0000-0002-8971-0759)
- Sofia Jaho (ORCID: https://orcid.org/0000-0002-6271-3212)
- Kanji Shimba
- Amy J. Thompson (ORCID: https://orcid.org/0000-0003-2174-2751)
- Hiroaki Matsuura (ORCID: https://orcid.org/0000-0002-8778-7955)
- Motoyuki Kataoka
- Ann Fitzpatrick (ORCID: https://orcid.org/0000-0002-9748-3728)
- Kensuke Tono (ORCID: https://orcid.org/0000-0003-1218-3759)
- Marina Lučić (ORCID: https://orcid.org/0000-0002-8264-8251)
- Sinan Battah (ORCID: https://orcid.org/0000-0002-0452-1685)
- Shigeki Owada (ORCID: https://orcid.org/0000-0002-1451-7612)
- Jonathan A. R. Worrall (ORCID: https://orcid.org/0000-0002-1863-834X)
- Sam Horrell (ORCID: https://orcid.org/0000-0001-6355-8640)
- Michael A. Hough (ORCID: https://orcid.org/0000-0001-7377-6713)
- Danny Axford (ORCID: https://orcid.org/0000-0001-7694-8525)
- Robin Leslie Owen (ORCID: https://orcid.org/0000-0002-2104-7057)
- Hiroshi Sugimoto (ORCID: https://orcid.org/0000-0002-3140-8362)
- Γαβριήλ Καρράς (ORCID: https://orcid.org/0000-0002-9629-8336)
- L.J. Williams (ORCID: https://orcid.org/0009-0002-4066-3899)
- P. P. A. Smyth (ORCID: https://orcid.org/0000-0003-0917-3349)
- K. Ishihara
Institutions
- University of Essex (GB)
- Rutherford Appleton Laboratory (GB)
- University of Hyogo (JP)
- Diamond Light Source (GB)
- Research Complex at Harwell (GB)
- SPring-8 (JP)
- Japan Synchrotron Radiation Research Institute (JP)
Publication Details
- Journal
- IUCrJ
- Published
- 2025-08-22
- DOI
- https://doi.org/10.1107/s2052252525006645
- Citations
- 2
- Primary Topic
- Hemoglobin structure and function
- Type
- article
- Field-Weighted Citation Impact
- 2.30
Funders
- Engineering and Physical Sciences Research Council
- Biotechnology and Biological Sciences Research Council