{"doi":"10.1016/j.jbc.2025.108444","title":"Spectroscopy and crystallography define carotenoid oxygenases as a new subclass of mononuclear non-heme FeII enzymes","abstract":"Carotenoid cleavage dioxygenases (CCDs) are non-heme Fe II enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of dioxygen (O 2 ) with their alkene substrates is unclear. Here, we apply spectroscopy in conjunction with X-ray crystallography to define the iron coordination geometry of a model CCD, CAO1 ( Neurospora crassa carotenoid oxygenase 1), in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal Fe II center that undergoes steric distortion toward a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O 2 -analog, nitric oxide, show a >100-fold increase in iron–nitric oxide affinity upon substrate binding, defining a crucial role for the substrate in activating the Fe II site for O 2 reactivity. The importance of the 5C Fe II structure for reactivity was probed through mutagenesis of the second-sphere Thr151 residue of CAO1, which occludes ligand binding at the sixth coordination position. A T151G substitution resulted in the conversion of the iron center to a six-coordinate state and a 135-fold reduction in apparent catalytic efficiency toward piceatannol compared with the wildtype enzyme. Substrate complexation resulted in partial six-coordinate to 5C conversion, indicating solvent dissociation from the iron center. Additional substitutions at this site demonstrated a general functional importance of the occluding residue within the CCD superfamily. Taken together, these data suggest an ordered mechanism of CCD catalysis occurring via substrate-promoted solvent replacement by O 2 . CCDs thus represent a new class of mononuclear non-heme Fe II enzymes.","journal":"Journal of Biological Chemistry","year":2025,"id":522958,"datarank":0.28673684867095767,"base_score":1.6094379124341003,"endowment":1.6094379124341003,"self_citation_contribution":0.24141568686511508,"citation_network_contribution":0.04532116180584257,"self_endowment_contribution":0.24141568686511508,"citer_contribution":0.04532116180584257,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":4,"citers_with_citation_signal":1,"citers_with_endowment":1,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9601,"is_data_producer":true,"deposit_databanks":{"PDB":["5U8X","8FU5","7T8P","8SRL","7T8Q","8FU2"]},"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":1234760,"name":"Michael P. Everett","orcid":null,"position":1,"is_corresponding":false},{"id":299425,"name":"Jeffrey T. Babicz","orcid":"0000-0002-1476-8390","position":2,"is_corresponding":false},{"id":310549,"name":"Anahita Daruwalla","orcid":"0000-0002-4947-9759","position":3,"is_corresponding":false},{"id":271050,"name":"Edward I. Solomon","orcid":"0000-0003-0291-3199","position":4,"is_corresponding":false},{"id":254242,"name":"Philip D. Kiser","orcid":"0000-0003-1184-9539","position":5,"is_corresponding":false},{"id":665717,"name":"Dory E. DeWeese","orcid":null,"position":0,"is_corresponding":true}],"reference_count":50,"raw_metadata":null,"created_at":"2026-07-19T02:49:58.707747Z","pmid":"40147775","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":[]}