{"doi":"10.1101/2025.01.21.634024","title":"Molecular sorting of nitrogenase catalytic cofactors","abstract":"Abstract The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (FeV-cofactor), or Fe (FeFe-cofactor). Completion of FeMo-cofactor and FeV-cofactor formation occurs on molecular scaffolds prior to delivery to their catalytic partners. In contrast, completion of FeFe-cofactor assembly occurs directly within its cognate catalytic partner. Because hybrid nitrogenase species that contain the incorrect cofactor type cannot reduce N 2 to support diazotrophic growth there must be a way to prevent misincorporation of an incorrect cofactor when different nitrogenase isozyme systems are produced at the same time. Here, we show that fidelity of the Fe-only nitrogenase is preserved by blocking the misincorporation of either FeMo-cofactor or FeV-cofactor during its maturation. This protection is accomplished by a two-domain protein, designated AnfO. It is shown that the N-terminal domain of AnfO binds to an immature form of the Fe-only nitrogenase and the C-terminal domain, tethered to the N-terminal domain by a flexible linker, has the capacity to capture FeMo- and FeV-cofactor. AnfO does not prevent the normal activation of Fe-only nitrogenase because completion of FeFe-cofactor assembly occurs within its catalytic partner and, therefore, is never available for capture by AnfO. These results support a post-translational mechanism involving the molecular sorting of structurally similar metallocofactors that involve both protein-protein interactions and metallocofactor binding while exploiting differential pathways for nitrogenase associated catalytic cofactor assembly.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":560717,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9454,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1463118,"name":"Josh Lian","orcid":null,"position":1,"is_corresponding":false},{"id":985276,"name":"Gil Namkoong","orcid":"0000-0002-0669-4889","position":2,"is_corresponding":false},{"id":890263,"name":"Daniel L. M. Suess","orcid":"0000-0002-0916-1973","position":3,"is_corresponding":false},{"id":780277,"name":"Luis M. Rubio","orcid":"0000-0003-1596-2475","position":4,"is_corresponding":false},{"id":716610,"name":"Dennis R. Dean","orcid":"0000-0001-8960-6196","position":5,"is_corresponding":false},{"id":716608,"name":"Ana Pérez‐González","orcid":"0000-0002-7724-9985","position":6,"is_corresponding":false},{"id":1462819,"name":"Alvaro Salinero‐Lanzarote","orcid":"0000-0001-5980-460X","position":0,"is_corresponding":true}],"reference_count":58,"raw_metadata":null,"created_at":"2026-07-19T02:55:46.921885Z","pmid":"39896531","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}