{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200G bus 2403, 3001Leuven, Belgium.",
    "Université Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale , 38000Grenoble, France.",
    "Quantum Chemistry and Physical Chemistry Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200F bus 2404, 3001Leuven, Belgium.",
    "Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F bus 2404, 3001Leuven, Belgium.",
    "College of Science and Technology, Nihon University, 1-5-1 Kandasurugadai, Chiyoda-ku, Tokyo101-0062, Japan.",
    "Support Unit for Bio-Material Analysis, RIKEN Center for Brain Science, 2-1 Hirosawa, Wako-city, Saitama351-0198, Japan.",
    "Japan Synchrotron Radiation Research Institute , 1-1-1 Kouto, Sayo, Hyogo679-5198, Japan.",
    "RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo679-5148, Japan.",
    "Faculty of Chemistry, Hanoi National University of Education, 136 Xuan Thuy street, Cau giay district, Hanoi123105, Vietnam.",
    "Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto606-8501, Japan.",
    "Laboratory of Biomolecular Architecture, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Celestijnenlaan 200G bus 2403, 3001Leuven, Belgium.",
    "Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai980-8577, Japan."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 28,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Hanifa Bibi"
    },
    {
      "affs": [
        0
      ],
      "name": "Leonie De Vrieze",
      "orcid": "0009-0005-5409-3251"
    },
    {
      "affs": [
        1
      ],
      "name": "Elke De Zitter",
      "orcid": "0000-0001-6325-7354"
    },
    {
      "affs": [
        1
      ],
      "name": "Nicolas Coquelle"
    },
    {
      "affs": [
        2
      ],
      "name": "Janko Civic",
      "orcid": "0009-0002-9207-835X"
    },
    {
      "affs": [
        3
      ],
      "name": "Eduard Fron",
      "orcid": "0000-0003-2260-0798"
    },
    {
      "affs": [
        4
      ],
      "name": "Kenichi Kamata",
      "orcid": "0000-0003-4344-8473"
    },
    {
      "affs": [
        5
      ],
      "name": "Masaya Usui"
    },
    {
      "affs": [
        5
      ],
      "name": "Kaori Otsuki"
    },
    {
      "affs": [
        0
      ],
      "name": "Gilles Garré"
    },
    {
      "affs": [
        1
      ],
      "name": "Ronald Rios-Santacruz"
    },
    {
      "affs": [
        1
      ],
      "name": "Giorgio Schirò"
    },
    {
      "affs": [
        6
      ],
      "name": "Fangjia Luo",
      "orcid": "0000-0003-0631-6236"
    },
    {
      "affs": [
        6,
        7
      ],
      "name": "Nipawan Nuemket"
    },
    {
      "affs": [
        7
      ],
      "name": "Jungmin Kang",
      "orcid": "0000-0002-3506-6400"
    },
    {
      "affs": [
        6,
        7
      ],
      "name": "Shigeki Owada"
    },
    {
      "affs": [
        6,
        7
      ],
      "name": "Kensuke Tono"
    },
    {
      "affs": [
        8
      ],
      "name": "Thi Yen Hang Bui"
    },
    {
      "affs": [
        0
      ],
      "name": "Olivia Olislaegers",
      "orcid": "0000-0002-0987-7808"
    },
    {
      "affs": [
        9
      ],
      "name": "Hidetsugu Asada"
    },
    {
      "affs": [
        9
      ],
      "name": "Dohyun Im"
    },
    {
      "affs": [
        10
      ],
      "name": "Luc Van Meervelt",
      "orcid": "0000-0003-2186-5209"
    },
    {
      "affs": [
        9
      ],
      "name": "So Iwata"
    },
    {
      "affs": [
        5
      ],
      "name": "Nobuhiko Miyasaka"
    },
    {
      "affs": [
        2
      ],
      "name": "Jeremy N Harvey",
      "orcid": "0000-0002-1728-1596"
    },
    {
      "affs": [
        11
      ],
      "name": "Eriko Nango"
    },
    {
      "affs": [
        1
      ],
      "name": "Martin Weik"
    },
    {
      "affs": [
        0
      ],
      "name": "Hideaki Mizuno",
      "orcid": "0000-0002-6983-5255"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/e3nq4skqgrm2y.bib",
  "cited_by": 0,
  "doi": "10.1021/jacs.6c13605",
  "fulltext_url": "https://europepmc.org/articles/PMC13632553",
  "id": "e3nq4skqgrm2y",
  "issns": [
    "0002-7863",
    "1520-5126"
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  "landing_url": "https://hal.science/hal-05764317v1",
  "language": "en",
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
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      "1304|Advanced Fluorescence Microscopy Techniques"
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  "pdf_url": "https://hal.science/hal-05764317/document",
  "pmcid": "PMC13632553",
  "pmid": "42937899",
  "published": "2026-09-01",
  "publisher": "American Chemical Society",
  "ris": "https://research.draconitic.com/paper/e3nq4skqgrm2y.ris",
  "sources": [
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      "first_seen": "2026-10-30T12:17:42Z",
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  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1304|Advanced Fluorescence Microscopy Techniques",
      "subfield": "biophysics"
    }
  ],
  "title": "Direct Visualization of a Hydroperoxy Intermediate in Fluorescent Protein Chromophore Maturation in PAmCherry1",
  "title_text": "Direct Visualization of a Hydroperoxy Intermediate in Fluorescent Protein Chromophore Maturation in PAmCherry1",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:22:24Z",
  "url": "https://research.draconitic.com/paper/e3nq4skqgrm2y",
  "venue": "Journal of the American Chemical Society",
  "versions": []
}