{"doi":"10.1101/2020.06.23.165407","title":"<sup>13</sup> C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding ‘Alcove’","abstract":"Abstract EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Ni p ) that bridges a [4Fe-4S] cluster and distal Ni d . An alcove seen in the ACS crystal-structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO and is suggested to ‘cradle’ this CO. Previously, we only knew WT ACS bound a single CO in the A red -CO intermediate, here seen as forming Ni p (I)-CO with CO on-axis of the d z 2 odd-electron orbital (g ⊥ &gt;g ‖ ∼2). The two-dimensional field-frequency pattern of 2K-35 GHz 13 C-ENDOR spectra collected across the A red -CO EPR envelope now reveals a second CO bound in the d z 2 orbital’s equatorial plane. This WT A-cluster conformer dominates the nearly-conservative F229W variant, but 13 C-ENDOR reveals a minority “A” conformation with (g ‖ &gt;g ⊥ ∼2) characteristic of a ‘cloverleaf’ (eg. d x 2 - y 2 ) odd-electron orbital, and with Ni p binding two, apparently ‘in-plane’ CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) ∼tenfold and introduces extensive cluster flexibility. 13 C-ENDOR shows the F229A cluster is mostly (60%) in the “A” conformation, but with ∼20% each of the WT conformer and an “O” state in which d z 2 Ni p (I) (g ⊥ &gt;g ‖ ∼2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO 2 fixation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":130203,"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":0.9557,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":382711,"name":"Seth Wiley","orcid":"0000-0002-2447-5153","position":1,"is_corresponding":false},{"id":328499,"name":"Stephen W. Ragsdale","orcid":"0000-0003-3938-8906","position":2,"is_corresponding":false},{"id":382712,"name":"Brian M. Hoffman","orcid":"0000-0002-3100-0746","position":3,"is_corresponding":false},{"id":382710,"name":"Christopher D. James","orcid":"0000-0002-6207-6380","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-18T23:15:53.196774Z","pmid":null,"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":[]}