{"doi":"10.1073/pnas.2517347122","title":"In vitro maturation of fully active [FeFe]-hydrogenase in a defined system including the iron carrier NfuA","abstract":"The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H 2 . A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that the Escherichia coli iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H 2 production rates comparable to that of the in vivo - matured Chlamydomonas reinhardtii [FeFe]-hydrogenase ( Cr HydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [Fe II (cys)(CN)(CO) 2 ] – synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (Mb H64L ) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited H ox -CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and Mb H64L to the fully defined maturation system thus results in an in vitro [FeFe]-hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.","journal":"Proceedings of the National Academy of Sciences","year":2025,"id":524471,"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":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9424,"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":890645,"name":"Adrien Pagnier","orcid":"0000-0002-1951-2415","position":1,"is_corresponding":false},{"id":744433,"name":"Eric M. Shepard","orcid":"0000-0002-4244-1045","position":2,"is_corresponding":false},{"id":890646,"name":"Batuhan Balci","orcid":"0000-0002-1626-2705","position":3,"is_corresponding":false},{"id":1398528,"name":"Abraham Teye","orcid":null,"position":4,"is_corresponding":false},{"id":890647,"name":"Douglas M. Warui","orcid":"0000-0001-8950-9433","position":5,"is_corresponding":false},{"id":802682,"name":"Hao Yang","orcid":"0000-0001-7229-0957","position":6,"is_corresponding":false},{"id":1268547,"name":"Alex Drena","orcid":"0000-0002-8864-6740","position":7,"is_corresponding":false},{"id":649879,"name":"Squire J. Booker","orcid":"0000-0002-7211-5937","position":8,"is_corresponding":false},{"id":382712,"name":"Brian M. Hoffman","orcid":"0000-0002-3100-0746","position":9,"is_corresponding":false},{"id":744434,"name":"William E. Broderick","orcid":"0000-0001-5782-7322","position":10,"is_corresponding":false},{"id":744435,"name":"Joan Broderick","orcid":"0000-0001-7057-9124","position":11,"is_corresponding":false},{"id":1397979,"name":"Alexander Marlott","orcid":"0009-0008-8305-2186","position":0,"is_corresponding":true}],"reference_count":93,"raw_metadata":null,"created_at":"2026-07-19T02:50:12.083054Z","pmid":"40982687","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":[]}