{"doi":"10.1016/j.jbc.2024.107381","title":"An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons","abstract":"Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O 2 ) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase ( Pa DADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of Pa DADH reveals the interaction of the glutamate 246 (E 246 ) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E 246 likely ionizes to facilitate substrate binding during Pa DADH catalysis. This study aimed to investigate how replacing the E 246 residue with leucine affects Pa DADH catalysis and its ability to react with O 2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O 2 reactivity in the E 246 L variant, resulting in a reduced flavin semiquinone species and superoxide (O 2 • ˉ ) during substrate oxidation. The O 2 • ˉ reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log ( k cat / K m ) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O 2 • ˉ can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.","journal":"Journal of Biological Chemistry","year":2024,"id":497663,"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":0.9562,"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":1344526,"name":"Kendall E. Wood","orcid":null,"position":1,"is_corresponding":false},{"id":1344272,"name":"Claire Snelgrove","orcid":"0009-0001-5064-1071","position":2,"is_corresponding":false},{"id":727973,"name":"Daniel Ouedraogo","orcid":null,"position":3,"is_corresponding":false},{"id":692300,"name":"Giovanni Gadda","orcid":"0000-0002-7508-4195","position":4,"is_corresponding":false},{"id":1095182,"name":"Joanna Afokai Quaye","orcid":"0000-0002-7041-4570","position":0,"is_corresponding":true}],"reference_count":109,"raw_metadata":null,"created_at":"2026-07-19T02:09:34.764412Z","pmid":"38762175","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":[]}