Double crossed? Structural and computational studies of an unusually crosslinked haem in <i>Methylococcus capsulatus</i> cytochrome P460
Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic <i>c</i>-type haem where the porphyrin is crosslinked to the protein <i>via</i> a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 haem oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460-containing hydroxylamine oxidoreductase utilises a tyrosine crosslink to haem and performs similar chemistry. Previous crystal structures of cytochrome P460 from <i>Nitrosomonas europaea</i> (NeP460) clearly show the existence of a single crosslink between the NZ atom of lysine and the haem porphyrin, with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the haem. Here we describe the evidence for a novel double crosslink between lysine and haem in the cytochrome P460 from <i>Methylococcus capsulatus</i> (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this, we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.
Authors
- Kakali Sen (ORCID: https://orcid.org/0000-0003-3135-3688)
- Takehiko Tosha (ORCID: https://orcid.org/0000-0002-8971-0759)
- Sofia Jaho (ORCID: https://orcid.org/0000-0002-6271-3212)
- Chin W. Yong (ORCID: https://orcid.org/0000-0002-6346-7383)
- Thomas W. Keal (ORCID: https://orcid.org/0000-0001-8747-3975)
- Aditya G. Rao (ORCID: https://orcid.org/0000-0001-6771-9157)
- Adrian John Mulholland (ORCID: https://orcid.org/0000-0003-1015-4567)
- Hannah R. Adams (ORCID: https://orcid.org/0000-0003-4715-6961)
- Ivo Tews (ORCID: https://orcid.org/0000-0002-4704-1139)
- James Beilsten‐Edmands (ORCID: https://orcid.org/0000-0003-1565-5328)
- Colin R. Andrew (ORCID: https://orcid.org/0000-0001-5908-012X)
- Hans E. Pfalzgraf (ORCID: https://orcid.org/0000-0003-0529-5429)
- 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)
- Marcus J. Edwards (ORCID: https://orcid.org/0000-0002-0243-3788)
- You Lü (ORCID: https://orcid.org/0000-0002-7524-4179)
- Robin Leslie Owen (ORCID: https://orcid.org/0000-0002-2104-7057)
- Hiroshi Sugimoto (ORCID: https://orcid.org/0000-0002-3140-8362)
Institutions
- University of Essex (GB)
- Rutherford Appleton Laboratory (GB)
- University of Hyogo (JP)
- Eastern Oregon University (US)
- Daresbury Laboratory (GB)
- Diamond Light Source (GB)
- University of Bristol (GB)
- Research Complex at Harwell (GB)
- SPring-8 (JP)
- University of Southampton (GB)
Publication Details
- Journal
- Chemical Science
- Published
- 2025-01-01
- DOI
- https://doi.org/10.1039/d5sc04213e
- Citations
- 1
- Primary Topic
- Hemoglobin structure and function
- Type
- article
- Field-Weighted Citation Impact
- 1.15
Funders
- National Science Foundation
- University of Essex
- Diamond Light Source
- Biotechnology and Biological Sciences Research Council
- Science and Technology Facilities Council