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A key aspect of EET is the organization of electron relays, i.e., multi-heme c-type cytochromes (MHCs), within the periplasmic space of microbial cells. In this study, we investigated the mobility of periplasmic electron relays in Shewanella oneidensis MR-1, a model strain capable of EET, using in vivo protein crosslinking to the MHCs. First, we established that crosslinking efficiency correlates with the spatial proximity and diffusion coefficient of protein molecules through in vitro tests. Based on these findings, we identified distinct molecular behaviors of periplasmic MHCs, showing that the tetraheme flavocytochrome FccA, which also serves as a periplasmic fumarate reductase, forms protein complexes with limited motility, while the small tetraheme c-type cytochrome CctA remains discrete and mobile. Both MHCs contribute to EET for bioelectrochemical nitrate and nitrite reduction. These findings reveal dual mechanisms for organizing periplasmic electron relays in EET, advancing our understanding of microbial extracellular respiration.<\/jats:p>","DOI":"10.3390\/microorganisms13051144","type":"journal-article","created":{"date-parts":[[2025,5,16]],"date-time":"2025-05-16T06:57:28Z","timestamp":1747378648000},"page":"1144","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Periplasmic Protein Mobility for Extracellular Electron Transport in Shewanella oneidensis"],"prefix":"10.3390","volume":"13","author":[{"given":"Daobo","family":"Li","sequence":"first","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China"},{"name":"Guangdong Provincial Key Laboratory of Environmental Protection Microbiology and Regional Ecological Security, Guangzhou 510070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaodan","family":"Zheng","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China"},{"name":"Guangdong Provincial Key Laboratory of Environmental Protection Microbiology and Regional Ecological Security, Guangzhou 510070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yonggang","family":"Yang","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China"},{"name":"Guangdong Provincial Key Laboratory of Environmental Protection Microbiology and Regional Ecological Security, Guangzhou 510070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7276-4219","authenticated-orcid":false,"given":"Meiying","family":"Xu","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, State Key Laboratory of Applied Microbiology Southern China, Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou 510070, China"},{"name":"Guangdong Provincial Key Laboratory of Environmental Protection Microbiology and Regional Ecological Security, Guangzhou 510070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"913","DOI":"10.1128\/AEM.06803-11","article-title":"Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration","volume":"78","author":"Richter","year":"2012","journal-title":"Appl. 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