{"doi":"10.1021/acs.biochem.1c00619","title":"Accumulation and Pulse Electron Paramagnetic Resonance Spectroscopic Investigation of the 4-Oxidobenzyl Radical Generated in the Radical<i>S</i>-Adenosyl-<scp>l</scp>-methionine Enzyme HydG","abstract":"The radical S-adenosyl-l-methionine (SAM) enzyme HydG cleaves tyrosine to generate CO and CN– ligands of the [FeFe] hydrogenase H-cluster, accompanied by the formation of a 4-oxidobenzyl radical (4-OB•), which is the precursor to the HydG p-cresol byproduct. Native HydG only generates a small amount of 4-OB•, limiting detailed electron paramagnetic resonance (EPR) spectral characterization beyond our initial EPR lineshape study employing various tyrosine isotopologues. Here, we show that the concentration of trapped 4-OB• is significantly increased in reactions using HydG variants, in which the “dangler Fe” to which CO and CN– bind is missing or substituted by a redox-inert Zn2+ ion. This allows for the detailed characterization of 4-OB• using high-field EPR and electron nuclear double resonance spectroscopy to extract its g-values and 1H/13C hyperfine couplings. These results are compared to density functional theory-predicted values of several 4-OB• models with different sizes and protonation states, with a best fit to the deprotonated radical anion configuration of 4-OB•. Overall, our results depict a clearer electronic structure of the transient 4-OB• radical and provide new insights into the radical SAM chemistry of HydG.","journal":"Biochemistry","year":2022,"id":273563,"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":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9494,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":885103,"name":"Nanhao Chen","orcid":null,"position":1,"is_corresponding":false},{"id":683039,"name":"David A. Marchiori","orcid":"0000-0001-9738-3674","position":2,"is_corresponding":false},{"id":356693,"name":"Lee‐Ping Wang","orcid":"0000-0003-3072-9946","position":3,"is_corresponding":false},{"id":334945,"name":"R. David Britt","orcid":"0000-0003-0889-8436","position":4,"is_corresponding":false},{"id":334946,"name":"Guodong Rao","orcid":"0000-0001-8043-3436","position":0,"is_corresponding":true}],"reference_count":66,"raw_metadata":null,"created_at":"2026-07-19T00:28:03.504676Z","pmid":"34989236","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":[]}