Direct Visualization of a Hydroperoxy Intermediate in Fluorescent Protein Chromophore Maturation in PAmCherry1
Abstract Despite the widespread use of fluorescent proteins in bioimaging, the mechanism of oxidative chromophore maturation to form the tyrosine Cα═Cβ double bond remains unclear. In the photoactivatable fluorescent protein PAmCherry1, this double bond has not yet been formed prior to photoactivation. Here, serial femtosecond crystallography (SFX) using an X-ray-free electron laser (XFEL) enabled us to capture the radiation damage-free structure of non-photoactivated PAmCherry1. This structure displayed a covalently bound hydroperoxy moiety (73% occupancy) at the sp3-hybridized Cα atom of the chromophore tyrosine residue within the imidazolinone ring. A unique hydrogen-bonding network surrounding the hydroperoxy moiety stabilizes this highly reactive chromophore species. Electrospray ionization mass spectrometry (ESI-MS) supported the coexistence of hydroperoxy and enolate chromophore forms. According to spectroscopic analysis, these species exist in dynamic equilibrium. MS analysis further demonstrated that photoactivation is accompanied by the formation of the mature chromophore containing a Cα═Cβ double bond together with Kolbe decarboxylation. Time-resolved SFX (TR-SFX) using a femtosecond pump laser further revealed decreasing electron density at 800 ps after photoexcitation, consistent with rapid oxidation and decarboxylation. Collectively, these results support a hydroperoxy intermediate as a key species in oxidative chromophore maturation, which is stabilized in non-photoactivated PAmCherry1.
Authors
- Jungmin Kang (ORCID: https://orcid.org/0000-0002-3506-6400)
- Nicolas Coquelle (ORCID: https://orcid.org/0000-0002-8501-4908)
- Kenichi Kamata (ORCID: https://orcid.org/0000-0003-4344-8473)
- Nobuhiko Miyasaka (ORCID: https://orcid.org/0000-0002-5225-9327)
- Eriko Nango (ORCID: https://orcid.org/0000-0001-9851-7355)
- Luc Van Meervelt (ORCID: https://orcid.org/0000-0003-2186-5209)
- Fangjia Luo (ORCID: https://orcid.org/0000-0003-0631-6236)
- Dohyun Im (ORCID: https://orcid.org/0000-0002-6939-7718)
- Eduard Fron (ORCID: https://orcid.org/0000-0003-2260-0798)
- Jeremy Noel Harvey (ORCID: https://orcid.org/0000-0002-1728-1596)
- Kaori Otsuki
- Kensuke Tono (ORCID: https://orcid.org/0000-0003-1218-3759)
- Shigeki Owada (ORCID: https://orcid.org/0000-0002-1451-7612)
- Masaya Usui (ORCID: https://orcid.org/0000-0001-8728-2167)
- Nipawan Nuemket (ORCID: https://orcid.org/0009-0005-5252-9488)
- Hideaki Mizuno (ORCID: https://orcid.org/0000-0002-6983-5255)
- Thi Yen Hang Bui (ORCID: https://orcid.org/0000-0001-6989-0839)
- Hidetsugu Asada (ORCID: https://orcid.org/0000-0001-6255-4728)
- Elke De Zitter (ORCID: https://orcid.org/0000-0001-6325-7354)
- Ronald Rios-Santacruz (ORCID: https://orcid.org/0000-0003-2087-4779)
- Janko Čivić (ORCID: https://orcid.org/0009-0002-9207-835X)
- Hanifa BiBi (ORCID: https://orcid.org/0009-0003-2897-0893)
- So Iwata (ORCID: https://orcid.org/0000-0003-1735-2937)
- Martin Weik (ORCID: https://orcid.org/0000-0001-9297-642X)
- Leonie De Vrieze (ORCID: https://orcid.org/0009-0005-5409-3251)
- olivia olislaegers (ORCID: https://orcid.org/0000-0002-0987-7808)
- Giorgio Schirò
- Gilles Garré
Institutions
- Nihon University (JP)
- Centre National de la Recherche Scientifique (FR)
- Hanoi National University of Education (VN)
- Tohoku University (JP)
- Kyoto University (JP)
- Commissariat à l'Énergie Atomique et aux Énergies Alternatives (FR)
- RIKEN Center for Brain Science (JP)
- CEA Grenoble (FR)
- SPring-8 (JP)
- Japan Synchrotron Radiation Research Institute (JP)
- Institut de Biologie Structurale (FR)
- KU Leuven (BE)
Publication Details
- Journal
- Journal of the American Chemical Society
- Published
- 2026-09-21
- DOI
- https://doi.org/10.1021/jacs.6c13605
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.00