{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T08:27:43Z","timestamp":1778660863814,"version":"3.51.4"},"reference-count":45,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2024,7,28]],"date-time":"2024-07-28T00:00:00Z","timestamp":1722124800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["22-24-00985"],"award-info":[{"award-number":["22-24-00985"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["22-14-00104"],"award-info":[{"award-number":["22-14-00104"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Russian state program","award":["22-24-00985"],"award-info":[{"award-number":["22-24-00985"]}]},{"name":"Russian state program","award":["22-14-00104"],"award-info":[{"award-number":["22-14-00104"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys-&gt;Glu substitutions in the UBS and some compensating Glu-&gt;Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4\u20136 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of C\u03b1 atoms of individual residues of mutant CytC compared to WT, especially in the \u03a9-loop (70\u201385), which can cause destabilization of the Fe\u2026S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the \u03a9-loop (70\u201385) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 \u00b5M) concentrations of H2O2 and avoid complications of radical formation during the reaction.<\/jats:p>","DOI":"10.3390\/ijms25158237","type":"journal-article","created":{"date-parts":[[2024,7,30]],"date-time":"2024-07-30T09:37:25Z","timestamp":1722332245000},"page":"8237","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands"],"prefix":"10.3390","volume":"25","author":[{"given":"Rita V.","family":"Chertkova","sequence":"first","affiliation":[{"name":"Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ilya P.","family":"Oleynikov","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Bld. 40, 119992 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7757-6452","authenticated-orcid":false,"given":"Alexey A.","family":"Pakhomov","sequence":"additional","affiliation":[{"name":"Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roman V.","family":"Sudakov","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Bld. 40, 119992 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5156-942X","authenticated-orcid":false,"given":"Marina A.","family":"Semenova","sequence":"additional","affiliation":[{"name":"Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexander M.","family":"Arutyunyan","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Bld. 40, 119992 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1268-4414","authenticated-orcid":false,"given":"Vasily V.","family":"Ptushenko","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Bld. 40, 119992 Moscow, Russia"},{"name":"N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mikhail P.","family":"Kirpichnikov","sequence":"additional","affiliation":[{"name":"Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"},{"name":"Biology Department, M.V. Lomonosov Moscow State University, 119899 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9880-7202","authenticated-orcid":false,"given":"Dmitry A.","family":"Dolgikh","sequence":"additional","affiliation":[{"name":"Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"},{"name":"Biology Department, M.V. Lomonosov Moscow State University, 119899 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6373-3071","authenticated-orcid":false,"given":"Tatiana V.","family":"Vygodina","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory 1, Bld. 40, 119992 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,7,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.chemphyslip.2013.11.002","article-title":"Structural transformations of cytochrome c upon interaction with cardiolipin","volume":"179","author":"Muenzner","year":"2014","journal-title":"Chem. Phys. 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