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The conformational dynamics of human CPR in solution, which involves transitions from a \u201clocked\/closed\u201d to an \u201cunlocked\/open\u201d state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site\u2010Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non\u2010canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH<jats:sup>\u2022<\/jats:sup> and the nitroxide. The DEER data revealed a salt concentration\u2010dependent distance distribution, evidence of an \u201copen\u201d CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the \u201copen\u201d semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.<\/jats:p>","DOI":"10.1002\/chem.202304307","type":"journal-article","created":{"date-parts":[[2024,1,26]],"date-time":"2024-01-26T13:35:10Z","timestamp":1706276110000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non\u2010canonical amino acid"],"prefix":"10.1002","volume":"30","author":[{"given":"Maxime","family":"Bizet","sequence":"first","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Deborah","family":"Byrne","sequence":"additional","affiliation":[{"name":"Protein Expression Facility Aix Marseille Univ CNRS IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fr\u00e9d\u00e9ric","family":"Biaso","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guillaume","family":"Gerbaud","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emilien","family":"Etienne","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Giuseppina","family":"Briola","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruno","family":"Guigliarelli","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philippe","family":"Urban","sequence":"additional","affiliation":[{"name":"TBI Universit\u00e9 de Toulouse CNRS INRAE INSA  31077 Toulouse France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pierre","family":"Dorlet","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tamas","family":"Kalai","sequence":"additional","affiliation":[{"name":"Department of Organic and Medicinal Chemistry Faculty of Pharmacy University of P\u00e9cs  PO Box 99 Szigeti st. 12 H-7602 7624 P\u00e9cs Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gilles","family":"Truan","sequence":"additional","affiliation":[{"name":"TBI Universit\u00e9 de Toulouse CNRS INRAE INSA  31077 Toulouse France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8576-5675","authenticated-orcid":false,"given":"Marl\u00e8ne","family":"Martinho","sequence":"additional","affiliation":[{"name":"Aix Marseille Univ CNRS Bio\u00e9nerg\u00e9tique et Ing\u00e9nierie des Prot\u00e9ines IMM  13402 Marseille France"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,2,13]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00204-020-02936-7"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21186669"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms131115012"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M807868200"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/febs.13501"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2015.01.032"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201800607"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1111\/febs.14757"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/embor.2009.82"},{"key":"e_1_2_8_10_1","first-page":"1083","volume":"13","author":"Esteves F.","year":"2023","journal-title":"Biomol. 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