biostudies-literatureTanifuji KKyoto UniversityTakeda Science FoundationMinistry of Education, Culture, Sports, Science and TechnologyBasic Energy SciencesTatematsu FoundationNIGMS NIH HHSNational Science Foundatione202200384https://www.ebi.ac.uk/biostudies/studies/S-EPMC9547968biostudies-literatureUnknown23(19)Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe₇ S₉ C]), to reduce atmospheric N₂ to bioaccessible NH₃ . Previously, we have shown that the cofactor-free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe-S cluster [Fe₆ S₉ (SEt)₂ ]^4- . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe₅ S₉ (SH)]^3- as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold. The resultant semi-artificial enzyme catalytically reduces C₂ H₂ to C₂ H₄ , and CN<supChembiochem : a European journal of chemical biologyIncorporation of an Asymmetric Mo-Fe-S Cluster as an Artificial Cofactor into Nitrogenase.PMC954796819H0273321K205572300000720K2120721H00021P30 GM133894CHE-1904131R01 GM067626DE-AC02-76SF00515Ribbe MWHu YTanifuji KSolomon JBNagasawa TJasniewski AJLee CCHedman BOhki YTatsumi KHodgson KOfalseIncorporation of an Asymmetric Mo-Fe-S Cluster as an Artificial Cofactor into Nitrogenase.Nitrogenase employs a sophisticated electron transfer system and a Mo-Fe-S-C cofactor, designated the M-cluster [(cit)MoFe₇ S₉ C]), to reduce atmospheric N₂ to bioaccessible NH₃ . Previously, we have shown that the cofactor-free form of nitrogenase can be repurposed as a protein scaffold for the incorporation of a synthetic Fe-S cluster [Fe₆ S₉ (SEt)₂ ]^4- . Here, we demonstrate the utility of an asymmetric Mo-Fe-S cluster [Cp*MoFe₅ S₉ (SH)]^3- as an alternative artificial cofactor upon incorporation into the cofactor-free nitrogenase scaffold. The resultant semi-artificial enzyme catalytically reduces C₂ H₂ to C₂ H₄ , and CN<sup2022-01-01T00:00:00Z2022 Oct2026-05-28T01:35:38.111Z2025-04-06T10:18:57.185ZS-EPMC95479683592584310.1002/cbic.202200384