Direct Visualization of a Hydroperoxy Intermediate in Fluorescent Protein Chromophore Maturation in PAmCherry1
12 affiliations
- 1Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200G bus 2403, 3001Leuven, Belgium.
- 2Université Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale , 38000Grenoble, France.
- 3Quantum Chemistry and Physical Chemistry Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200F bus 2404, 3001Leuven, Belgium.
- 4Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F bus 2404, 3001Leuven, Belgium.
- 5College of Science and Technology, Nihon University, 1-5-1 Kandasurugadai, Chiyoda-ku, Tokyo101-0062, Japan.
- 6Support Unit for Bio-Material Analysis, RIKEN Center for Brain Science, 2-1 Hirosawa, Wako-city, Saitama351-0198, Japan.
- 7Japan Synchrotron Radiation Research Institute , 1-1-1 Kouto, Sayo, Hyogo679-5198, Japan.
- 8RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo679-5148, Japan.
- 9Faculty of Chemistry, Hanoi National University of Education, 136 Xuan Thuy street, Cau giay district, Hanoi123105, Vietnam.
- 10Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto606-8501, Japan.
- 11Laboratory of Biomolecular Architecture, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200G bus 2403, 3001Leuven, Belgium.
- 12Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai980-8577, Japan.
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.
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BibTeX
@article{bibi2026direct,
title = {{Direct Visualization of a Hydroperoxy Intermediate in Fluorescent Protein Chromophore Maturation in PAmCherry1}},
author = {Hanifa Bibi and Leonie De Vrieze and Elke De Zitter and Nicolas Coquelle and Janko Civic and Eduard Fron and Kenichi Kamata and Masaya Usui and Kaori Otsuki and Gilles Garré and Ronald Rios-Santacruz and Giorgio Schirò and Fangjia Luo and Nipawan Nuemket and Jungmin Kang and Shigeki Owada and Kensuke Tono and Thi Yen Hang Bui and Olivia Olislaegers and Hidetsugu Asada and Dohyun Im and Luc Van Meervelt and So Iwata and Nobuhiko Miyasaka and Jeremy N Harvey and Eriko Nango and Martin Weik and Hideaki Mizuno},
journal = {Journal of the American Chemical Society},
year = {2026},
month = sep,
doi = {10.1021/jacs.6c13605},
url = {https://hal.science/hal-05764317v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Direct Visualization of a Hydroperoxy Intermediate in Fluorescent Protein Chromophore Maturation in PAmCherry1
AU - Hanifa Bibi
AU - Leonie De Vrieze
AU - Elke De Zitter
AU - Nicolas Coquelle
AU - Janko Civic
AU - Eduard Fron
AU - Kenichi Kamata
AU - Masaya Usui
AU - Kaori Otsuki
AU - Gilles Garré
AU - Ronald Rios-Santacruz
AU - Giorgio Schirò
AU - Fangjia Luo
AU - Nipawan Nuemket
AU - Jungmin Kang
AU - Shigeki Owada
AU - Kensuke Tono
AU - Thi Yen Hang Bui
AU - Olivia Olislaegers
AU - Hidetsugu Asada
AU - Dohyun Im
AU - Luc Van Meervelt
AU - So Iwata
AU - Nobuhiko Miyasaka
AU - Jeremy N Harvey
AU - Eriko Nango
AU - Martin Weik
AU - Hideaki Mizuno
PY - 2026
DA - 2026/09/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c13605
UR - https://hal.science/hal-05764317v1
AB - 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.
SN - 0002-7863
SN - 1520-5126
ER -