{"doi":"10.1021/jacs.3c09428","title":"ENDOR Spectroscopy Reveals the “Free” 5′-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe–4S]<sup>2+</sup> Cluster","abstract":"1/2 H and 13 C hyperfine coupling constants to 5′-deoxyadenosyl (5′-dAdo•) radical trapped within the active site of the radical S -adenosyl- l -methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp 2 C5′-carbon ( J. Am. Chem. Soc. 2019, 141, 12139–12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5′-dAdo• free radical is never truly “free”: tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5′-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57 Fe, 13 C, 15 N, and 2 H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe–4S] 2+ cluster cogenerated with 5′-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57 Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry–density functional theory computations. Incorporation of ENDOR-derived distances from C5′(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5′-dAdo• with a short, nonbonded C5′-Fe distance (∼3 Å). This distance involves substantial motion of 5′-dAdo• toward the unique Fe of the [4Fe–4S] 2+ cluster upon S–C(5′) bond-cleavage, plausibly an initial step toward formation of the Fe–C5′ bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.","journal":"Journal of the American Chemical Society","year":2024,"id":437755,"datarank":0.3596842909197557,"base_score":2.3978952727983707,"endowment":2.3978952727983707,"self_citation_contribution":0.3596842909197557,"citation_network_contribution":0.0,"self_endowment_contribution":0.3596842909197557,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9586,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":925661,"name":"Madeline B. Ho","orcid":"0000-0001-6351-2132","position":1,"is_corresponding":false},{"id":1052965,"name":"Maike N. Lundahl","orcid":"0000-0002-4391-7279","position":2,"is_corresponding":false},{"id":258617,"name":"Martín A. Mosquera","orcid":"0000-0003-2170-5651","position":3,"is_corresponding":false},{"id":744434,"name":"William E. Broderick","orcid":"0000-0001-5782-7322","position":4,"is_corresponding":false},{"id":744435,"name":"Joan Broderick","orcid":"0000-0001-7057-9124","position":5,"is_corresponding":false},{"id":382712,"name":"Brian M. Hoffman","orcid":"0000-0002-3100-0746","position":6,"is_corresponding":false},{"id":802682,"name":"Hao Yang","orcid":"0000-0001-7229-0957","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:00:34.490087Z","pmid":"38308759","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":[]}