{"doi":"10.1101/2024.01.10.575067","title":"Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO","abstract":"Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":493252,"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":0.9606,"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":1241738,"name":"Joseph Palazzo","orcid":"0000-0001-5688-8588","position":1,"is_corresponding":false},{"id":370533,"name":"Simon Schlanger","orcid":null,"position":2,"is_corresponding":false},{"id":492274,"name":"Dhanya T. Jayaram","orcid":"0000-0002-3139-7157","position":3,"is_corresponding":false},{"id":1079785,"name":"Sidra Islam","orcid":"0000-0003-0617-1698","position":4,"is_corresponding":false},{"id":353430,"name":"Richard C. Page","orcid":"0000-0002-3006-3171","position":5,"is_corresponding":false},{"id":360859,"name":"Dennis J. Stuehr","orcid":"0000-0002-0960-6848","position":6,"is_corresponding":false},{"id":884837,"name":"Pranjal Biswas","orcid":"0000-0003-4173-4214","position":0,"is_corresponding":true}],"reference_count":44,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:00.148120Z","pmid":"38260356","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":[]}