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Here, we reveal an unexpected alternative mediator interaction site in FDH1 of\n                    <jats:italic>Methylorubrum extorquens<\/jats:italic>\n                    AM1. Removing the native flavin mononucleotide cofactor abolishes NAD\n                    <jats:sup>+<\/jats:sup>\n                    \u2010dependent activity but preserves EV\u2010driven catalysis. Through mutagenesis, kinetic analysis, and molecular docking, we identified a cooperative aromatic network\u2014comprising residues F232, F471, and Y329\u2014that stabilizes EV via stacking interactions near the proximal B1 iron\u2013sulfur cluster. Disrupting these residues impairs EV\u2010mediated electron transfer without destabilizing global structure. These findings reveal a dual interaction strategy for artificial mediators in tungsten FDHs, offering a structural framework to rationally engineer biocatalysts for CO\n                    <jats:sub>2<\/jats:sub>\n                    conversion.\n                  <\/jats:p>","DOI":"10.1002\/1873-3468.70373","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T12:05:23Z","timestamp":1779969923000},"page":"2094-2107","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An unexpected alternative viologen electron mediator site in tungsten\u2010containing formate dehydrogenase"],"prefix":"10.1002","volume":"600","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9672-0114","authenticated-orcid":false,"given":"Eleni G.","family":"Poloniataki","sequence":"first","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST)  Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4729-693X","authenticated-orcid":false,"given":"Yong Hwan","family":"Kim","sequence":"additional","affiliation":[{"name":"School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST)  Republic of Korea"},{"name":"Graduate School of Carbon Neutrality, Ulsan National Institute of Science and Technology (UNIST)  Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,28]]},"reference":[{"key":"e_1_2_9_2_1","unstructured":"Masson\u2010DelmotteV ZhaiP PiraniA ConnorsSL P\u00e9anC BergerS CaudN ChenY GoldfarbL GomisMIet\u00a0al. 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