{"gene":"NDUFS7","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":1999,"finding":"PSST (NDUFS7) subunit of complex I is the primary site of photoaffinity labeling by the potent inhibitor [3H]TDP (trifluoromethyldiazirinyl-pyridaben), establishing that PSST functionally couples electron transfer from iron-sulfur cluster N2 to ubiquinone; all structurally diverse potent complex I inhibitors (rotenone, piericidin A, bullatacin, pyridaben) compete for this same binding site on PSST, and the equivalent bacterial subunit NQO6 shares this conserved function.","method":"Photoaffinity labeling with [3H]TDP, protein sequencing, immunoprecipitation, competitive binding assays, enzyme inhibition assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro biochemical reconstitution with photoaffinity labeling, protein sequencing, immunoprecipitation, and competitive inhibition, replicated in bacterial homologs","pmids":["10097178"],"is_preprint":false},{"year":2001,"finding":"PSST (NDUFS7) and ND1 subunits of complex I are functionally coupled at the quinone-binding region: NADH increases PSST labeling while decreasing ND1 labeling; MPP+ and stigmatellin show opposite effects (increased ND1, decreased PSST labeling), indicating dynamic interaction between these two subunits at a semiquinone binding site.","method":"Photoaffinity labeling with [3H]TDP, competitive binding, preincubation with NADH/inhibitors","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 — in vitro photoaffinity labeling with multiple orthogonal inhibitors demonstrating reciprocal labeling changes between PSST and ND1","pmids":["11418099"],"is_preprint":false},{"year":2000,"finding":"Conserved acidic residues D136 and E140 in the PSST homologue (Yarrowia lipolytica) play a central role in proton translocation and ubiquinone interaction; E89 (proposed fourth ligand of iron-sulfur center N2) is not a ligand of N2 as EPR spectra show unchanged N2 amount but shifted spectrum upon mutation to glutamine, alanine, or cysteine.","method":"Site-directed mutagenesis, EPR spectroscopy, enzymatic activity assays in Yarrowia lipolytica","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — site-directed mutagenesis combined with EPR spectroscopy and activity assays in a tractable eukaryotic model","pmids":["10811805"],"is_preprint":false},{"year":2003,"finding":"Two aspartic acid residues D99 and D115 in the PSST homologue (NUKM, Yarrowia lipolytica) are essential for complex I catalytic activity; their mutation to Asn, Glu, or Gly reduces activity to 5–8% of wild-type and reduces the EPR N2 signal by ~50%, while complex I remains stably assembled. No conserved acidic residue in PSST serves as the fourth ligand of iron-sulfur cluster N2.","method":"Site-directed mutagenesis of all 8 conserved acidic residues, EPR spectroscopy, enzyme activity assays, Blue Native PAGE","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — comprehensive mutagenesis of all conserved acidic residues combined with EPR and activity assays","pmids":["12930834"],"is_preprint":false},{"year":2012,"finding":"Fenpyroximate binds at the interface between the PSST and 49 kDa subunits of complex I, not at the distal membrane domain (ND5); the pharmacophoric pyrazole ring orients toward PSST (labeled in region Ser43–Arg66) while the side chain orients toward the 49 kDa subunit, within the quinone-binding pocket formed at the PSST/49 kDa/ND1 interface.","method":"Photoaffinity labeling with two 125I-labeled fenpyroximate derivatives, limited proteolysis, doubled SDS-PAGE, mass spectrometry","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — two orthogonal photoaffinity probes with fragment mapping by proteolysis pinpointing specific regions","pmids":["22353032"],"is_preprint":false},{"year":2016,"finding":"NDUFAF5, an S-adenosylmethionine-dependent assembly factor, hydroxylates Arg-73 of NDUFS7 early in the complex I assembly pathway, before formation of the junction between the peripheral and membrane arms.","method":"Mass spectrometry identification of hydroxylation, functional assembly analysis by Blue Native PAGE, biochemical characterization of NDUFAF5 activity","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — post-translational modification identified by MS with assembly-stage determination by native PAGE","pmids":["27226634"],"is_preprint":false},{"year":2019,"finding":"Conserved residues in the loop of PSST (NDUFS7) facing the ~30 Å quinone-binding tunnel are critical for ubiquinone reductase activity; mutation of conserved arginine residues drastically reduces Q reductase activity despite full complex I assembly, and molecular dynamics simulations show these residues dynamically control ubiquinone diffusion and retention near the terminal electron donor N2.","method":"Site-directed mutagenesis, enzymatic activity assays, Blue Native PAGE assembly analysis, molecular dynamics simulations (microsecond scale)","journal":"Biochimica et biophysica acta. Bioenergetics","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis with activity assays and multi-microsecond MD simulations providing mechanistic insight","pmids":["31226318"],"is_preprint":false},{"year":2007,"finding":"NDUFS7 is essential for biogenesis of fully assembled complex I; an intronic mutation (c.17-1167 C>G) creates a cryptic exon producing a truncated 41-amino-acid protein, resulting in marked decrease in fully assembled complex I on Blue Native PAGE while other respiratory chain complexes are unaffected.","method":"Blue Native PAGE assembly analysis, patient-derived cell studies, RT-PCR demonstration of cryptic exon","journal":"Molecular genetics and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 — patient-derived cells with defined molecular mechanism (cryptic exon) and assembly assay","pmids":["17604671"],"is_preprint":false},{"year":2023,"finding":"NDUFS7 contains a direct drug-binding site: exome sequencing of six independently selected DX2-201-resistant clones all revealed a p.V91M mutation in NDUFS7, demonstrating that this residue is within the compound's binding site and that NDUFS7 inhibition suppresses oxidative phosphorylation.","method":"Exome sequencing of resistant clones, cell viability assays, in vivo syngeneic tumor model","journal":"ACS pharmacology & translational science","confidence":"Medium","confidence_rationale":"Tier 2 — convergent resistance mutations in 6/6 independent clones pinpointing drug-binding site","pmids":["37588763"],"is_preprint":false},{"year":2024,"finding":"NDUFS7 deficiency in HEK293T cells causes reduced cell proliferation, elevated cell death, and increased ROS; upregulation of SLC7A11 compensates by increasing cystine import and glutathione biosynthesis to mitigate apoptosis.","method":"NDUFS7 knockout in HEK293T cells, ROS measurement, SLC7A11 knockdown/overexpression, glutathione assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function with defined cellular phenotype and pathway placement via SLC7A11/GSH axis","pmids":["38823363"],"is_preprint":false},{"year":2024,"finding":"The canine NDUFS7 p.Val179Met missense variant fails to rescue lethality upon knockdown of the Drosophila ortholog ND-20 (whereas wildtype NDUFS7 partially rescues), establishing this residue as functionally critical for NDUFS7 activity in vivo.","method":"Drosophila in vivo complementation assay with ubiquitous knockdown of ND-20 and overexpression of wildtype vs. mutant canine NDUFS7","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo genetic complementation in Drosophila ortholog model","pmids":["38316835"],"is_preprint":false},{"year":2026,"finding":"NRF2 directly regulates NDUFS7 transcription (ChIP-seq confirmed binding), and NDUFS7 is required for mitochondrial complex I integrity in trabecular meshwork cells; restoration of NDUFS7 in NRF2-deficient cells or mice rescues mitochondrial impairment.","method":"ChIP-seq, NRF2 knockout mice, NRF2 knockdown/overexpression, NDUFS7 rescue experiments, mitochondrial function assays","journal":"Research (Washington, D.C.)","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-seq plus genetic rescue experiments with functional readout","pmids":["41869290"],"is_preprint":false}],"current_model":"NDUFS7 (PSST) is a core subunit of mitochondrial complex I that occupies the junction between the hydrophilic and membrane arms, harboring the high-affinity binding site for ubiquinone and diverse complex I inhibitors (rotenone, piericidin A, fenpyroximate, pyridaben) within a ~30 Å quinone-binding tunnel formed at the PSST/49 kDa/ND1 interface; it functionally couples electron transfer from iron-sulfur cluster N2 to ubiquinone through conserved loop residues and is post-translationally hydroxylated at Arg-73 by the assembly factor NDUFAF5 early in complex I biogenesis, and its loss disrupts fully assembled complex I and elevates reactive oxygen species."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of NDUFS7 as the subunit harboring the inhibitor/ubiquinone-binding site resolved where electron transfer from cluster N2 converges with quinone reduction and where all major complex I inhibitors act.","evidence":"Photoaffinity labeling with [³H]TDP, competitive binding with rotenone/piericidin A/bullatacin, protein sequencing in bovine heart and bacterial complex I","pmids":["10097178"],"confidence":"High","gaps":["Atomic-resolution binding pose of inhibitors not determined","Relative contribution of NDUFS7 vs. adjacent subunits to binding affinity not quantified"]},{"year":2000,"claim":"Systematic mutagenesis of conserved acidic residues in the PSST homolog established which residues are essential for catalysis versus cluster N2 ligation, ruling out a conserved acidic fourth ligand for N2.","evidence":"Site-directed mutagenesis of D136, E140, E89 and others in Yarrowia lipolytica with EPR spectroscopy and activity assays","pmids":["10811805","12930834"],"confidence":"High","gaps":["Identity of the true fourth N2 ligand remained unresolved at this stage","Proton translocation mechanism at D136/E140 not fully delineated"]},{"year":2001,"claim":"Demonstrating reciprocal redox-dependent labeling of NDUFS7 and ND1 established that these two subunits dynamically interact at the semiquinone-binding site, coupling electron input to quinone chemistry.","evidence":"Photoaffinity labeling with [³H]TDP modulated by NADH preincubation and competitive inhibitors MPP+/stigmatellin","pmids":["11418099"],"confidence":"High","gaps":["Structural basis of dynamic PSST–ND1 interaction not resolved","Whether semiquinone is a catalytic intermediate or side product not settled"]},{"year":2007,"claim":"A disease-causing intronic mutation producing truncated NDUFS7 demonstrated that this subunit is indispensable for biogenesis of fully assembled complex I, linking NDUFS7 deficiency to isolated complex I deficiency.","evidence":"Patient-derived cells with c.17-1167 C>G mutation, Blue Native PAGE showing selective complex I loss, RT-PCR identifying cryptic exon","pmids":["17604671"],"confidence":"Medium","gaps":["No complementation/rescue experiment performed in patient cells","Residual complex I activity not quantified"]},{"year":2012,"claim":"Mapping fenpyroximate binding to the PSST/49 kDa interface pinpointed the pharmacophore orientation within the quinone-binding pocket, refining the topology of the shared inhibitor-binding site.","evidence":"Two orthogonal ¹²⁵I-fenpyroximate photoaffinity probes with limited proteolysis and mass spectrometry fragment mapping in bovine complex I","pmids":["22353032"],"confidence":"High","gaps":["No cryo-EM or crystal structure with bound inhibitor at this point","Contribution of ND1 interface residues not mapped with equivalent resolution"]},{"year":2016,"claim":"Discovery that NDUFAF5 hydroxylates NDUFS7 Arg-73 early in assembly revealed the first known post-translational modification of this subunit and placed it within the complex I assembly pathway before junction formation.","evidence":"Mass spectrometry identification of Arg-73 hydroxylation, Blue Native PAGE assembly intermediates, NDUFAF5 functional characterization","pmids":["27226634"],"confidence":"High","gaps":["Functional consequence of Arg-73 hydroxylation on catalytic activity not directly tested","Whether hydroxylation is reversible or regulatory is unknown"]},{"year":2019,"claim":"Mutagenesis of conserved loop arginines facing the quinone tunnel, combined with microsecond molecular dynamics, established that NDUFS7 loop residues actively control ubiquinone diffusion and retention near N2 rather than serving a purely structural role.","evidence":"Site-directed mutagenesis with activity assays and Blue Native PAGE in Yarrowia lipolytica, multi-microsecond MD simulations","pmids":["31226318"],"confidence":"High","gaps":["Experimental validation of MD-predicted quinone poses lacking","Whether loop dynamics change with proton-motive force not tested"]},{"year":2023,"claim":"Convergent p.V91M resistance mutations across six independent clones confirmed NDUFS7 as a direct drug target for the complex I inhibitor DX2-201, validating pharmacological targeting of NDUFS7 in cancer cells.","evidence":"Exome sequencing of six independently selected DX2-201-resistant clones, cell viability assays, syngeneic tumor model","pmids":["37588763"],"confidence":"Medium","gaps":["Binding affinity and structural basis of V91M resistance not determined","In vivo efficacy not tested with NDUFS7-mutant tumors"]},{"year":2024,"claim":"NDUFS7 knockout in human cells revealed that its loss elevates ROS and triggers compensatory SLC7A11-mediated cystine import and glutathione biosynthesis, placing NDUFS7 upstream of a defined redox-homeostasis pathway.","evidence":"NDUFS7 knockout in HEK293T cells with ROS measurement, SLC7A11 knockdown/overexpression, glutathione assays","pmids":["38823363"],"confidence":"Medium","gaps":["Whether SLC7A11 compensation occurs in primary or differentiated cell types unknown","Source of ROS (complex I flavin site vs. quinone site) not discriminated"]},{"year":2024,"claim":"Cross-species complementation in Drosophila validated that the canine p.Val179Met variant is functionally deleterious, establishing an in vivo system to assess NDUFS7 variant pathogenicity.","evidence":"Drosophila ND-20 knockdown with canine wildtype vs. V179M NDUFS7 overexpression rescue assay","pmids":["38316835"],"confidence":"Medium","gaps":["Biochemical mechanism of Val179Met dysfunction not characterized","Partial rescue by wildtype suggests dosage or species-compatibility limitations"]},{"year":2026,"claim":"Identification of NRF2 as a direct transcriptional regulator of NDUFS7 linked oxidative stress signaling to complex I biogenesis, with NDUFS7 restoration rescuing mitochondrial defects from NRF2 loss.","evidence":"ChIP-seq for NRF2 binding at NDUFS7 locus, NRF2 knockout mice, NDUFS7 rescue in NRF2-deficient trabecular meshwork cells","pmids":["41869290"],"confidence":"Medium","gaps":["Whether NRF2 regulation of NDUFS7 operates in all tissues or is cell-type restricted","Mechanism by which NDUFS7 alone rescues complex I in NRF2-deficient background not explained"]},{"year":null,"claim":"The structural basis for how NDUFS7 Arg-73 hydroxylation influences complex I assembly competence, the precise atomic mechanism by which loop dynamics couple electron transfer to proton translocation, and whether pharmacological targeting of the NDUFS7 quinone site can be therapeutically exploited remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of NDUFS7 with bound inhibitor in a catalytically defined state","Functional role of Arg-73 hydroxylation untested by non-hydroxylatable mutant in mammalian system","Therapeutic window for NDUFS7-targeted complex I inhibitors in cancer not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1,2,3,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,6]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,5,7,9]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,2,3,6,8]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[9,11]}],"complexes":["Mitochondrial complex I (NADH:ubiquinone oxidoreductase)"],"partners":["NDUFS2","ND1","NDUFAF5","SLC7A11","NRF2"],"other_free_text":[]},"mechanistic_narrative":"NDUFS7 (PSST) is a core catalytic subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase) that couples electron transfer from iron-sulfur cluster N2 to ubiquinone reduction within a ~30 Å quinone-binding tunnel formed at the NDUFS7/NDUFS2(49 kDa)/ND1 interface. Conserved loop residues, including critical arginine and aspartate residues, control ubiquinone diffusion, retention, and proton translocation, while the subunit harbors the shared high-affinity binding site for structurally diverse complex I inhibitors including rotenone, piericidin A, pyridaben, and fenpyroximate [PMID:10097178, PMID:12930834, PMID:31226318, PMID:22353032]. NDUFS7 is post-translationally hydroxylated at Arg-73 by the assembly factor NDUFAF5 early in complex I biogenesis, and its loss abolishes fully assembled complex I, elevates reactive oxygen species, and triggers compensatory upregulation of the SLC7A11/glutathione axis [PMID:27226634, PMID:17604671, PMID:38823363]. NRF2 directly regulates NDUFS7 transcription, and biallelic NDUFS7 mutations cause isolated complex I deficiency [PMID:41869290, PMID:17604671]."},"prefetch_data":{"uniprot":{"accession":"O75251","full_name":"NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial","aliases":["Complex I-20kD","CI-20kD","NADH-ubiquinone oxidoreductase 20 kDa subunit","PSST subunit"],"length_aa":213,"mass_kda":23.6,"function":"Core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) which catalyzes electron transfer from NADH through the respiratory chain, using ubiquinone as an electron acceptor (PubMed:17275378). Essential for the catalytic activity of complex I (PubMed:17275378)","subcellular_location":"Mitochondrion inner membrane","url":"https://www.uniprot.org/uniprotkb/O75251/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NDUFS7","classification":"Not Classified","n_dependent_lines":217,"n_total_lines":1208,"dependency_fraction":0.17963576158940397},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NDUFS7","total_profiled":1310},"omim":[{"mim_id":"618224","title":"MITOCHONDRIAL COMPLEX I DEFICIENCY, NUCLEAR TYPE 3; MC1DN3","url":"https://www.omim.org/entry/618224"},{"mim_id":"603846","title":"NADH-UBIQUINONE OXIDOREDUCTASE Fe-S PROTEIN 3; NDUFS3","url":"https://www.omim.org/entry/603846"},{"mim_id":"601825","title":"NADH-UBIQUINONE OXIDOREDUCTASE Fe-S PROTEIN 7; NDUFS7","url":"https://www.omim.org/entry/601825"},{"mim_id":"256000","title":"LEIGH SYNDROME, NUCLEAR; NULS","url":"https://www.omim.org/entry/256000"},{"mim_id":"252010","title":"MITOCHONDRIAL COMPLEX I DEFICIENCY, NUCLEAR TYPE 1; MC1DN1","url":"https://www.omim.org/entry/252010"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":623.2}],"url":"https://www.proteinatlas.org/search/NDUFS7"},"hgnc":{"alias_symbol":["PSST","FLJ46880","FLJ45860","CI-20"],"prev_symbol":[]},"alphafold":{"accession":"O75251","domains":[{"cath_id":"3.40.50.12280","chopping":"81-204","consensus_level":"high","plddt":96.0886,"start":81,"end":204}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75251","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75251-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75251-F1-predicted_aligned_error_v6.png","plddt_mean":81.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NDUFS7","jax_strain_url":"https://www.jax.org/strain/search?query=NDUFS7"},"sequence":{"accession":"O75251","fasta_url":"https://rest.uniprot.org/uniprotkb/O75251.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75251/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75251"}},"corpus_meta":[{"pmid":"10360771","id":"PMC_10360771","title":"Leigh syndrome associated with a mutation in the NDUFS7 (PSST) nuclear encoded subunit of complex I.","date":"1999","source":"Annals of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/10360771","citation_count":157,"is_preprint":false},{"pmid":"10097178","id":"PMC_10097178","title":"NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone.","date":"1999","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10097178","citation_count":146,"is_preprint":false},{"pmid":"11418099","id":"PMC_11418099","title":"Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling.","date":"2001","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11418099","citation_count":88,"is_preprint":false},{"pmid":"27919778","id":"PMC_27919778","title":"A mutation in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Tetranychus urticae is associated with resistance to METI acaricides.","date":"2016","source":"Insect biochemistry and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27919778","citation_count":66,"is_preprint":false},{"pmid":"10811805","id":"PMC_10811805","title":"Function of conserved acidic residues in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10811805","citation_count":64,"is_preprint":false},{"pmid":"11695186","id":"PMC_11695186","title":"The insecticide target in the PSST subunit of complex I.","date":"2001","source":"Pest management science","url":"https://pubmed.ncbi.nlm.nih.gov/11695186","citation_count":52,"is_preprint":false},{"pmid":"22353032","id":"PMC_22353032","title":"Fenpyroximate binds to the interface between PSST and 49 kDa subunits in mitochondrial NADH-ubiquinone oxidoreductase.","date":"2012","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22353032","citation_count":47,"is_preprint":false},{"pmid":"27226634","id":"PMC_27226634","title":"NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27226634","citation_count":43,"is_preprint":false},{"pmid":"12930834","id":"PMC_12930834","title":"Two aspartic acid residues in the PSST-homologous NUKM subunit of complex I from Yarrowia lipolytica are essential for catalytic activity.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12930834","citation_count":40,"is_preprint":false},{"pmid":"17275378","id":"PMC_17275378","title":"A novel mutation in the human complex I NDUFS7 subunit associated with Leigh syndrome.","date":"2007","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17275378","citation_count":38,"is_preprint":false},{"pmid":"17604671","id":"PMC_17604671","title":"A novel mutation of the NDUFS7 gene leads to activation of a cryptic exon and impaired assembly of mitochondrial complex I in a patient with Leigh syndrome.","date":"2007","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17604671","citation_count":38,"is_preprint":false},{"pmid":"31226318","id":"PMC_31226318","title":"Mutations in a conserved loop in the PSST subunit of respiratory complex I affect ubiquinone binding and dynamics.","date":"2019","source":"Biochimica et biophysica acta. 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research","url":"https://pubmed.ncbi.nlm.nih.gov/41631271","citation_count":0,"is_preprint":false},{"pmid":"12151107","id":"PMC_12151107","title":"The gene encoding the PSST subunit of respiratory chain complex I is present in more than one copy in yellow lupine.","date":"2002","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/12151107","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.08.663799","title":"NDUFS2 (NADH:Ubiquinone oxidoreductase core subunit S2) in Mitochondrial Electron Transport Chain Complex I is Critical to Oxygen Responsiveness of Human Ductus Arteriosus Smooth Muscle Cells","date":"2025-07-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.08.663799","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14492,"output_tokens":2978,"usd":0.044073},"stage2":{"model":"claude-opus-4-6","input_tokens":6335,"output_tokens":2846,"usd":0.154237},"total_usd":0.19831,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"PSST (NDUFS7) subunit of complex I is the primary site of photoaffinity labeling by the potent inhibitor [3H]TDP (trifluoromethyldiazirinyl-pyridaben), establishing that PSST functionally couples electron transfer from iron-sulfur cluster N2 to ubiquinone; all structurally diverse potent complex I inhibitors (rotenone, piericidin A, bullatacin, pyridaben) compete for this same binding site on PSST, and the equivalent bacterial subunit NQO6 shares this conserved function.\",\n      \"method\": \"Photoaffinity labeling with [3H]TDP, protein sequencing, immunoprecipitation, competitive binding assays, enzyme inhibition assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical reconstitution with photoaffinity labeling, protein sequencing, immunoprecipitation, and competitive inhibition, replicated in bacterial homologs\",\n      \"pmids\": [\"10097178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PSST (NDUFS7) and ND1 subunits of complex I are functionally coupled at the quinone-binding region: NADH increases PSST labeling while decreasing ND1 labeling; MPP+ and stigmatellin show opposite effects (increased ND1, decreased PSST labeling), indicating dynamic interaction between these two subunits at a semiquinone binding site.\",\n      \"method\": \"Photoaffinity labeling with [3H]TDP, competitive binding, preincubation with NADH/inhibitors\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro photoaffinity labeling with multiple orthogonal inhibitors demonstrating reciprocal labeling changes between PSST and ND1\",\n      \"pmids\": [\"11418099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Conserved acidic residues D136 and E140 in the PSST homologue (Yarrowia lipolytica) play a central role in proton translocation and ubiquinone interaction; E89 (proposed fourth ligand of iron-sulfur center N2) is not a ligand of N2 as EPR spectra show unchanged N2 amount but shifted spectrum upon mutation to glutamine, alanine, or cysteine.\",\n      \"method\": \"Site-directed mutagenesis, EPR spectroscopy, enzymatic activity assays in Yarrowia lipolytica\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site-directed mutagenesis combined with EPR spectroscopy and activity assays in a tractable eukaryotic model\",\n      \"pmids\": [\"10811805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Two aspartic acid residues D99 and D115 in the PSST homologue (NUKM, Yarrowia lipolytica) are essential for complex I catalytic activity; their mutation to Asn, Glu, or Gly reduces activity to 5–8% of wild-type and reduces the EPR N2 signal by ~50%, while complex I remains stably assembled. No conserved acidic residue in PSST serves as the fourth ligand of iron-sulfur cluster N2.\",\n      \"method\": \"Site-directed mutagenesis of all 8 conserved acidic residues, EPR spectroscopy, enzyme activity assays, Blue Native PAGE\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — comprehensive mutagenesis of all conserved acidic residues combined with EPR and activity assays\",\n      \"pmids\": [\"12930834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Fenpyroximate binds at the interface between the PSST and 49 kDa subunits of complex I, not at the distal membrane domain (ND5); the pharmacophoric pyrazole ring orients toward PSST (labeled in region Ser43–Arg66) while the side chain orients toward the 49 kDa subunit, within the quinone-binding pocket formed at the PSST/49 kDa/ND1 interface.\",\n      \"method\": \"Photoaffinity labeling with two 125I-labeled fenpyroximate derivatives, limited proteolysis, doubled SDS-PAGE, mass spectrometry\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — two orthogonal photoaffinity probes with fragment mapping by proteolysis pinpointing specific regions\",\n      \"pmids\": [\"22353032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NDUFAF5, an S-adenosylmethionine-dependent assembly factor, hydroxylates Arg-73 of NDUFS7 early in the complex I assembly pathway, before formation of the junction between the peripheral and membrane arms.\",\n      \"method\": \"Mass spectrometry identification of hydroxylation, functional assembly analysis by Blue Native PAGE, biochemical characterization of NDUFAF5 activity\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — post-translational modification identified by MS with assembly-stage determination by native PAGE\",\n      \"pmids\": [\"27226634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Conserved residues in the loop of PSST (NDUFS7) facing the ~30 Å quinone-binding tunnel are critical for ubiquinone reductase activity; mutation of conserved arginine residues drastically reduces Q reductase activity despite full complex I assembly, and molecular dynamics simulations show these residues dynamically control ubiquinone diffusion and retention near the terminal electron donor N2.\",\n      \"method\": \"Site-directed mutagenesis, enzymatic activity assays, Blue Native PAGE assembly analysis, molecular dynamics simulations (microsecond scale)\",\n      \"journal\": \"Biochimica et biophysica acta. Bioenergetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis with activity assays and multi-microsecond MD simulations providing mechanistic insight\",\n      \"pmids\": [\"31226318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NDUFS7 is essential for biogenesis of fully assembled complex I; an intronic mutation (c.17-1167 C>G) creates a cryptic exon producing a truncated 41-amino-acid protein, resulting in marked decrease in fully assembled complex I on Blue Native PAGE while other respiratory chain complexes are unaffected.\",\n      \"method\": \"Blue Native PAGE assembly analysis, patient-derived cell studies, RT-PCR demonstration of cryptic exon\",\n      \"journal\": \"Molecular genetics and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — patient-derived cells with defined molecular mechanism (cryptic exon) and assembly assay\",\n      \"pmids\": [\"17604671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NDUFS7 contains a direct drug-binding site: exome sequencing of six independently selected DX2-201-resistant clones all revealed a p.V91M mutation in NDUFS7, demonstrating that this residue is within the compound's binding site and that NDUFS7 inhibition suppresses oxidative phosphorylation.\",\n      \"method\": \"Exome sequencing of resistant clones, cell viability assays, in vivo syngeneic tumor model\",\n      \"journal\": \"ACS pharmacology & translational science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — convergent resistance mutations in 6/6 independent clones pinpointing drug-binding site\",\n      \"pmids\": [\"37588763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NDUFS7 deficiency in HEK293T cells causes reduced cell proliferation, elevated cell death, and increased ROS; upregulation of SLC7A11 compensates by increasing cystine import and glutathione biosynthesis to mitigate apoptosis.\",\n      \"method\": \"NDUFS7 knockout in HEK293T cells, ROS measurement, SLC7A11 knockdown/overexpression, glutathione assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined cellular phenotype and pathway placement via SLC7A11/GSH axis\",\n      \"pmids\": [\"38823363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The canine NDUFS7 p.Val179Met missense variant fails to rescue lethality upon knockdown of the Drosophila ortholog ND-20 (whereas wildtype NDUFS7 partially rescues), establishing this residue as functionally critical for NDUFS7 activity in vivo.\",\n      \"method\": \"Drosophila in vivo complementation assay with ubiquitous knockdown of ND-20 and overexpression of wildtype vs. mutant canine NDUFS7\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic complementation in Drosophila ortholog model\",\n      \"pmids\": [\"38316835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NRF2 directly regulates NDUFS7 transcription (ChIP-seq confirmed binding), and NDUFS7 is required for mitochondrial complex I integrity in trabecular meshwork cells; restoration of NDUFS7 in NRF2-deficient cells or mice rescues mitochondrial impairment.\",\n      \"method\": \"ChIP-seq, NRF2 knockout mice, NRF2 knockdown/overexpression, NDUFS7 rescue experiments, mitochondrial function assays\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-seq plus genetic rescue experiments with functional readout\",\n      \"pmids\": [\"41869290\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NDUFS7 (PSST) is a core subunit of mitochondrial complex I that occupies the junction between the hydrophilic and membrane arms, harboring the high-affinity binding site for ubiquinone and diverse complex I inhibitors (rotenone, piericidin A, fenpyroximate, pyridaben) within a ~30 Å quinone-binding tunnel formed at the PSST/49 kDa/ND1 interface; it functionally couples electron transfer from iron-sulfur cluster N2 to ubiquinone through conserved loop residues and is post-translationally hydroxylated at Arg-73 by the assembly factor NDUFAF5 early in complex I biogenesis, and its loss disrupts fully assembled complex I and elevates reactive oxygen species.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NDUFS7 (PSST) is a core catalytic subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase) that couples electron transfer from iron-sulfur cluster N2 to ubiquinone reduction within a ~30 Å quinone-binding tunnel formed at the NDUFS7/NDUFS2(49 kDa)/ND1 interface. Conserved loop residues, including critical arginine and aspartate residues, control ubiquinone diffusion, retention, and proton translocation, while the subunit harbors the shared high-affinity binding site for structurally diverse complex I inhibitors including rotenone, piericidin A, pyridaben, and fenpyroximate [PMID:10097178, PMID:12930834, PMID:31226318, PMID:22353032]. NDUFS7 is post-translationally hydroxylated at Arg-73 by the assembly factor NDUFAF5 early in complex I biogenesis, and its loss abolishes fully assembled complex I, elevates reactive oxygen species, and triggers compensatory upregulation of the SLC7A11/glutathione axis [PMID:27226634, PMID:17604671, PMID:38823363]. NRF2 directly regulates NDUFS7 transcription, and biallelic NDUFS7 mutations cause isolated complex I deficiency [PMID:41869290, PMID:17604671].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of NDUFS7 as the subunit harboring the inhibitor/ubiquinone-binding site resolved where electron transfer from cluster N2 converges with quinone reduction and where all major complex I inhibitors act.\",\n      \"evidence\": \"Photoaffinity labeling with [³H]TDP, competitive binding with rotenone/piericidin A/bullatacin, protein sequencing in bovine heart and bacterial complex I\",\n      \"pmids\": [\"10097178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution binding pose of inhibitors not determined\", \"Relative contribution of NDUFS7 vs. adjacent subunits to binding affinity not quantified\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Systematic mutagenesis of conserved acidic residues in the PSST homolog established which residues are essential for catalysis versus cluster N2 ligation, ruling out a conserved acidic fourth ligand for N2.\",\n      \"evidence\": \"Site-directed mutagenesis of D136, E140, E89 and others in Yarrowia lipolytica with EPR spectroscopy and activity assays\",\n      \"pmids\": [\"10811805\", \"12930834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the true fourth N2 ligand remained unresolved at this stage\", \"Proton translocation mechanism at D136/E140 not fully delineated\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating reciprocal redox-dependent labeling of NDUFS7 and ND1 established that these two subunits dynamically interact at the semiquinone-binding site, coupling electron input to quinone chemistry.\",\n      \"evidence\": \"Photoaffinity labeling with [³H]TDP modulated by NADH preincubation and competitive inhibitors MPP+/stigmatellin\",\n      \"pmids\": [\"11418099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of dynamic PSST–ND1 interaction not resolved\", \"Whether semiquinone is a catalytic intermediate or side product not settled\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"A disease-causing intronic mutation producing truncated NDUFS7 demonstrated that this subunit is indispensable for biogenesis of fully assembled complex I, linking NDUFS7 deficiency to isolated complex I deficiency.\",\n      \"evidence\": \"Patient-derived cells with c.17-1167 C>G mutation, Blue Native PAGE showing selective complex I loss, RT-PCR identifying cryptic exon\",\n      \"pmids\": [\"17604671\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No complementation/rescue experiment performed in patient cells\", \"Residual complex I activity not quantified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapping fenpyroximate binding to the PSST/49 kDa interface pinpointed the pharmacophore orientation within the quinone-binding pocket, refining the topology of the shared inhibitor-binding site.\",\n      \"evidence\": \"Two orthogonal ¹²⁵I-fenpyroximate photoaffinity probes with limited proteolysis and mass spectrometry fragment mapping in bovine complex I\",\n      \"pmids\": [\"22353032\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No cryo-EM or crystal structure with bound inhibitor at this point\", \"Contribution of ND1 interface residues not mapped with equivalent resolution\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery that NDUFAF5 hydroxylates NDUFS7 Arg-73 early in assembly revealed the first known post-translational modification of this subunit and placed it within the complex I assembly pathway before junction formation.\",\n      \"evidence\": \"Mass spectrometry identification of Arg-73 hydroxylation, Blue Native PAGE assembly intermediates, NDUFAF5 functional characterization\",\n      \"pmids\": [\"27226634\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Arg-73 hydroxylation on catalytic activity not directly tested\", \"Whether hydroxylation is reversible or regulatory is unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mutagenesis of conserved loop arginines facing the quinone tunnel, combined with microsecond molecular dynamics, established that NDUFS7 loop residues actively control ubiquinone diffusion and retention near N2 rather than serving a purely structural role.\",\n      \"evidence\": \"Site-directed mutagenesis with activity assays and Blue Native PAGE in Yarrowia lipolytica, multi-microsecond MD simulations\",\n      \"pmids\": [\"31226318\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Experimental validation of MD-predicted quinone poses lacking\", \"Whether loop dynamics change with proton-motive force not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Convergent p.V91M resistance mutations across six independent clones confirmed NDUFS7 as a direct drug target for the complex I inhibitor DX2-201, validating pharmacological targeting of NDUFS7 in cancer cells.\",\n      \"evidence\": \"Exome sequencing of six independently selected DX2-201-resistant clones, cell viability assays, syngeneic tumor model\",\n      \"pmids\": [\"37588763\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding affinity and structural basis of V91M resistance not determined\", \"In vivo efficacy not tested with NDUFS7-mutant tumors\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"NDUFS7 knockout in human cells revealed that its loss elevates ROS and triggers compensatory SLC7A11-mediated cystine import and glutathione biosynthesis, placing NDUFS7 upstream of a defined redox-homeostasis pathway.\",\n      \"evidence\": \"NDUFS7 knockout in HEK293T cells with ROS measurement, SLC7A11 knockdown/overexpression, glutathione assays\",\n      \"pmids\": [\"38823363\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SLC7A11 compensation occurs in primary or differentiated cell types unknown\", \"Source of ROS (complex I flavin site vs. quinone site) not discriminated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Cross-species complementation in Drosophila validated that the canine p.Val179Met variant is functionally deleterious, establishing an in vivo system to assess NDUFS7 variant pathogenicity.\",\n      \"evidence\": \"Drosophila ND-20 knockdown with canine wildtype vs. V179M NDUFS7 overexpression rescue assay\",\n      \"pmids\": [\"38316835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical mechanism of Val179Met dysfunction not characterized\", \"Partial rescue by wildtype suggests dosage or species-compatibility limitations\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of NRF2 as a direct transcriptional regulator of NDUFS7 linked oxidative stress signaling to complex I biogenesis, with NDUFS7 restoration rescuing mitochondrial defects from NRF2 loss.\",\n      \"evidence\": \"ChIP-seq for NRF2 binding at NDUFS7 locus, NRF2 knockout mice, NDUFS7 rescue in NRF2-deficient trabecular meshwork cells\",\n      \"pmids\": [\"41869290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NRF2 regulation of NDUFS7 operates in all tissues or is cell-type restricted\", \"Mechanism by which NDUFS7 alone rescues complex I in NRF2-deficient background not explained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis for how NDUFS7 Arg-73 hydroxylation influences complex I assembly competence, the precise atomic mechanism by which loop dynamics couple electron transfer to proton translocation, and whether pharmacological targeting of the NDUFS7 quinone site can be therapeutically exploited remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of NDUFS7 with bound inhibitor in a catalytically defined state\", \"Functional role of Arg-73 hydroxylation untested by non-hydroxylatable mutant in mammalian system\", \"Therapeutic window for NDUFS7-targeted complex I inhibitors in cancer not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 5, 7, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 2, 3, 6, 8]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [9, 11]}\n    ],\n    \"complexes\": [\n      \"Mitochondrial complex I (NADH:ubiquinone oxidoreductase)\"\n    ],\n    \"partners\": [\n      \"NDUFS2\",\n      \"ND1\",\n      \"NDUFAF5\",\n      \"SLC7A11\",\n      \"NRF2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}