{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,2]],"date-time":"2025-11-02T06:59:39Z","timestamp":1762066779994,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2022,9,20]],"date-time":"2022-09-20T00:00:00Z","timestamp":1663632000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Deputyship for Research &amp; Innovation, Ministry of Education in Saudi Arabia","award":["149\/442"],"award-info":[{"award-number":["149\/442"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>A cytochrome c552 mutant from Thermus thermophilus HB8 (rC552 C14A) was reported, where the polypeptide with replaced Cys14 by alanine, overexpressed in the cytosol of E. coli. The apo-form of the C14A mutant (apo-C14A) without the original prosthetic group was obtained by simple chemical treatments that retained compact conformation amenable to reconstitution with heme b and zinc(II)-protoporphyrin(IX), gradually followed by spontaneous formation of a covalent bond between the polypeptide and porphyrin ring in the reconstituted apo-C14A. Further analysis suggested that the residual Cys11 and vinyl group of the porphyrin ring linked through the thiol-ene reaction promoted by light under ambient conditions. In this study, we describe the kinetic improvement of the covalent bond formation in accordance with the mechanism of the photoinduced thiol-ene reaction, which involves a thiyl radical as a reaction intermediate. Adding a radical generator to the reconstituted C14A mutant with either heme-b or zinc(II) porphyrin accelerated the bond-forming reaction, which supported the involvement of a radical species in the reaction. Partial observation of the reconstituted C14A in a dimer form and detection of sulfuryl radical by EPR spectroscopy indicated a thiyl radical on Cys11, a unique cysteinyl residue in rC552 C14A. The covalent bond forming mediated by the radical generator was also adaptable to the reconstituted apo-C14A with manganese(II)-protoporphyrin(IX), which also exhibits light-mediated covalent linkage formation. Therefore, the radical generator extends the versatility of producing c-type-like cytochrome starting from a metallo-protoporphyrin(IX) and the apo-C14A instantaneously.<\/jats:p>","DOI":"10.3390\/biom12101329","type":"journal-article","created":{"date-parts":[[2022,9,20]],"date-time":"2022-09-20T21:12:53Z","timestamp":1663708373000},"page":"1329","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Radical Mediated Rapid In Vitro Formation of c-Type Cytochrome"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3995-8940","authenticated-orcid":false,"given":"Sheikh Muhammad","family":"Ibrahim","sequence":"first","affiliation":[{"name":"Chemistry Department, Faculty of Science, Islamic University of Madinah, Al-Madinah Al-Munawarah 42351, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9026-4779","authenticated-orcid":false,"given":"Sami","family":"Ben Aoun","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah P.O. Box 30002, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0079-1274","authenticated-orcid":false,"given":"Hiroshi","family":"Nakajima","sequence":"additional","affiliation":[{"name":"Graduate School of Science, Osaka City University, Osaka 558-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fethi","family":"Kooli","sequence":"additional","affiliation":[{"name":"Chemistry Department, Faculty of Science, Islamic University of Madinah, Al-Madinah Al-Munawarah 42351, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yoshihito","family":"Watanabe","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan"},{"name":"Research Center of Materials Science, Nagoya University, Nagoya 464-8602, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1098\/rstl.1886.0007","article-title":"VI. 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