{"gene":"RH88_085460","gene_symbol":"","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2000,"finding":"Cytochrome b gene of T. gondii was cloned and sequenced; atovaquone-resistant mutants harbored mutations M129L and I254L in two different regions of the ubiquinol-binding pocket (Qo domain) of cytochrome b, establishing that atovaquone interferes with electron transport at the cytochrome bc1 complex by targeting the Qo site of cytochrome b.","method":"Gene cloning and sequencing of wild-type and atovaquone-resistant T. gondii strains; structural modeling of the Qo domain","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct sequencing of resistant mutants with functional drug resistance phenotype, structural modeling; foundational paper replicated conceptually across multiple subsequent studies","pmids":["10802314"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"A mutation in the cytochrome b gene was identified in a decoquinate-resistant mutant of T. gondii, indicating that cytochrome b is the target of decoquinate and that resistance arises through mutations in this mitochondrial gene.","method":"Selection of drug-resistant mutants; gene sequencing","journal":"The Journal of eukaryotic microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, direct sequencing of resistant mutant; abstract only available","pmids":["10519259"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"Atovaquone at nanomolar concentrations (0.03 µM) totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, with the site of inhibition localized between cytochrome b and c1 (consistent with bc1 complex inhibition), establishing cytochrome b as part of a functional respiratory chain in this parasite.","method":"In situ respiration assay using digitonin-permeabilized tachyzoites; safranin O mitochondrial membrane potential measurement; antimycin A and cyanide sensitivity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical assay in permeabilized parasites with multiple inhibitors and membrane potential measurement in a single rigorous study","pmids":["9813002"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Atovaquone binds competitively to the ubiquinol oxidation (Qo) pocket of the cytochrome bc1 complex, interacting with the Rieske iron-sulfur protein; a L275F mutation in cytochrome b (yeast surrogate) decreased atovaquone sensitivity, establishing that residue 275 (phenylalanine in bovine, leucine in yeast) governs species-differential inhibition.","method":"Enzymatic inhibition assays (Ki determination); EPR spectroscopy of Rieske iron-sulfur protein; site-directed mutagenesis of yeast cytochrome b (L275F); energy-minimized structural modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with isolated bc1 complex, mutagenesis, EPR, and structural modeling; mechanistically informative for T. gondii cytochrome b drug target","pmids":["12791689"],"is_preprint":false,"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2017,"finding":"A Thr222Pro substitution in cytochrome b of T. gondii confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin A (a well-characterized Qi site inhibitor), providing genetic evidence that ELQ-316 inhibits the cytochrome bc1 complex Qi site in T. gondii.","method":"Drug-pressure selection of resistant T. gondii clones; gene sequencing; pharmacological cross-resistance assays","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via resistance mutation with defined amino acid change, cross-resistance phenotype with known Qi inhibitor; single lab but two orthogonal lines of evidence","pmids":["27919897"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"Structure-activity relationship studies using wild-type and drug-resistant T. gondii and P. falciparum strains demonstrated that 4(1H)-quinolones can target either the Qo or Qi site of cytochrome bc1; T. gondii cytochrome b sequence differences relative to P. falciparum at positions associated with drug resistance influence drug susceptibility between species.","method":"Proliferation assays with wild-type and resistant parasite strains; cytochrome b sequence comparison; structure-activity relationship analysis of quinolone series","journal":"ACS infectious diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological dissection with resistant strains and sequence analysis, single lab; no direct mutagenesis of T. gondii cytochrome b in this study","pmids":["30117728"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"Novel 4-(1H)-quinolone scaffolds bind to the cytochrome bc1 complex Qi site, as demonstrated by mutant yeast and co-crystallographic studies; cytochrome b is highly expressed in T. gondii cysts (bradyzoites).","method":"Mutant yeast bc1 assay; co-crystallography; in vitro and in vivo activity assays against T. gondii tachyzoites and cysts","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — co-crystallography and mutant yeast for Qi site binding, but T. gondii cytochrome b itself not structurally resolved; bradyzoite expression is observational","pmids":["27412848"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2019,"finding":"Artemisinin-resistant T. gondii lines selected in vitro showed dramatic amplification of the mitochondrial genome, including the cytochrome b gene; treatment of wild-type parasites with high drug concentrations decreased mitochondrial membrane potential, and this phenotype was stably altered in resistant lines, implicating mitochondrial function (including cytochrome b) as an artemisinin target.","method":"Whole-genome sequencing of resistant lines; CRISPR/Cas9 mutagenesis; mitochondrial membrane potential measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genomic evidence for mitochondrial gene amplification plus functional membrane potential readout; cytochrome b not directly manipulated but implicated through copy number change","pmids":["31806760"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Parasites lacking TgMSH1 accumulate single nucleotide variations in mitochondrial DNA including cytochrome b, yet atovaquone can still inhibit electron transport in these mutants despite missense mutations in cytochrome b that do not affect known atovaquone-sensitivity determinants; under atovaquone pressure, Tgmsh1 mutants develop enhanced resistance via complete shutdown of respiration rather than via cytochrome b Qo site mutations.","method":"Tgmsh1 knockout generation; mitochondrial DNA sequencing; electron transport inhibition assays; atovaquone resistance selection","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with functional electron transport assay; novel mechanistic finding that atovaquone resistance can arise independently of cytochrome b Qo mutations","pmids":["29141004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"Cryo-EM structures of the T. gondii cytochrome bc1 complex (complex III2) and the III2-IV supercomplex were resolved; atovaquone binds to the cytochrome b Qo site in a manner revealing species-specific interactions; ELQ-300 binds the Qi site of T. gondii cytochrome b in a flipped orientation relative to its binding in the mammalian enzyme; supercomplex knockout causes disassembly and reduces parasite fitness; structure-guided design yielded more potent ELQs with subnanomolar activity against T. gondii.","method":"Cryo-electron microscopy structure determination; knockout analysis; inhibitor co-structure with atovaquone and ELQ-300; structure-guided compound design and proliferation assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures with drug-bound complexes, knockout phenotype, and structure-guided medicinal chemistry in a single rigorous study","pmids":["40389671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2011,"finding":"Atovaquone blocks mitochondrial oxidation of ubiquinol in T. gondii by binding to the ubiquinol oxidation (Qo) site of the cytochrome bc1 complex; mutations in cytochrome b associated with atovaquone resistance in Plasmodium, Pneumocystis, and Toxoplasma were characterized in isolated yeast bc1 complexes harboring equivalent mutations, establishing residue-level requirements for Qo site inhibition.","method":"Enzymatic assays with isolated yeast bc1 complexes bearing cytochrome b mutations equivalent to those in drug-resistant pathogens; synthesis and testing of novel hydroxy-naphthoquinones","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with mutagenized surrogate complexes; T. gondii resistance mutations characterized functionally in yeast system","pmids":["21251932"],"is_preprint":false,"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2007,"finding":"HDQ (1-hydroxy-2-dodecyl-4(1H)quinolone), an inhibitor of alternative (type II) NADH dehydrogenase, acts synergistically with atovaquone (which blocks the ubiquinol oxidation site of cytochrome b in complex III) against T. gondii, with a fractional inhibitory concentration of 0.16 nM, demonstrating that simultaneous interference at two sites of the ubiquinone/ubiquinol cycle is highly effective.","method":"Synergy assay (FIC index calculation) combining HDQ and atovaquone against T. gondii in cell culture","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological combination study demonstrating functional interaction at two mitochondrial sites; mechanistically informative about cytochrome b Qo site but indirect","pmids":["17242151"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) that mediates ubiquinol oxidation at the Qo site and ubiquinone reduction at the Qi site; atovaquone inhibits electron transport by binding the Qo domain (with species-specificity governed by residues lining the ubiquinol pocket), while endochin-like quinolones can inhibit either site, and drug resistance arises primarily through point mutations in the Qo or Qi domains of cytochrome b, as confirmed by cryo-EM structures, in vitro enzymatic assays, mutagenesis, and genetic selection of resistant parasites."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) that functions in the parasite's respiratory electron transport chain, where inhibition disrupts respiration and collapses mitochondrial membrane potential [PMID:9813002, PMID:40389671]. Cryo-EM structures of the T. gondii complex III2 and the III2-IV supercomplex show that cytochrome b provides two catalytically distinct drug-binding sites: the ubiquinol-oxidation Qo site, where atovaquone binds through species-specific interactions, and the Qi site, where endochin-like quinolones such as ELQ-300 bind in an orientation distinct from that in the mammalian enzyme [PMID:40389671]. Atovaquone acts as a competitive inhibitor at the Qo pocket in association with the Rieske iron-sulfur protein, and drug resistance arises through point mutations in the Qo domain (e.g. M129L, I254L), residues lining the ubiquinol pocket governing species-differential inhibition [PMID:10802314, PMID:12791689, PMID:21251932]. Resistance to Qi-site inhibitors similarly maps to cytochrome b, with a Thr222Pro substitution conferring cross-resistance to ELQ-316 and the classical Qi inhibitor antimycin A [PMID:27919897]. Cytochrome b is also the target of decoquinate [PMID:10519259], and structure-guided design against these sites has produced quinolones with subnanomolar activity [PMID:40389671]. Beyond drug-target characterization, the corpus does not detail the protein's biogenesis, assembly, or regulation independent of complex III.","teleology":[{"year":1998,"claim":"Establishing that T. gondii possesses a functional respiratory chain answered whether the parasite's mitochondrion sustains electron transport that could be drug-targeted, localizing the inhibitory site to the bc1 complex.","evidence":"In situ respiration and safranin O membrane-potential assays in digitonin-permeabilized tachyzoites with atovaquone, antimycin A, and cyanide","pmids":["9813002"],"confidence":"High","gaps":["Did not resolve which cytochrome b residues bind atovaquone","Did not distinguish Qo from Qi contributions"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"Identifying a cytochrome b mutation in a decoquinate-resistant mutant showed that cytochrome b is a drug target beyond atovaquone.","evidence":"Selection of decoquinate-resistant T. gondii mutants and gene sequencing","pmids":["10519259"],"confidence":"Medium","gaps":["Abstract-only report","Binding site of decoquinate on cytochrome b not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Cloning cytochrome b and mapping atovaquone-resistance mutations to the ubiquinol pocket answered where atovaquone acts, establishing the Qo site of cytochrome b as its molecular target.","evidence":"Gene cloning/sequencing of wild-type and atovaquone-resistant strains with Qo domain structural modeling","pmids":["10802314"],"confidence":"High","gaps":["No experimentally resolved structure","Mechanism of species-differential susceptibility not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Reconstitution in a yeast surrogate defined atovaquone as a competitive Qo-site inhibitor coupled to the Rieske protein and pinpointed residue 275 as a determinant of species-differential inhibition.","evidence":"Enzymatic Ki assays, EPR of Rieske iron-sulfur protein, and L275F mutagenesis of yeast cytochrome b","pmids":["12791689"],"confidence":"High","gaps":["Performed in yeast, not T. gondii enzyme","Does not address Qi-site inhibitors"],"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2011,"claim":"Functional testing of pathogen-equivalent mutations in isolated yeast complexes established residue-level requirements for Qo-site inhibition across Plasmodium, Pneumocystis, and Toxoplasma.","evidence":"Enzymatic assays with isolated yeast bc1 complexes bearing equivalent cytochrome b mutations plus novel hydroxy-naphthoquinones","pmids":["21251932"],"confidence":"Medium","gaps":["Surrogate yeast system rather than native T. gondii complex","Qi site not addressed"],"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2016,"claim":"Co-crystallographic and mutant-yeast work established that 4-(1H)-quinolone scaffolds engage the Qi site, extending the druggable geometry of cytochrome b beyond the Qo pocket.","evidence":"Mutant yeast bc1 assay, co-crystallography, and activity assays against T. gondii tachyzoites and cysts","pmids":["27412848"],"confidence":"Medium","gaps":["T. gondii cytochrome b not structurally resolved here","Bradyzoite expression finding is observational"],"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2017,"claim":"A Thr222Pro cytochrome b substitution conferring cross-resistance to ELQ-316 and antimycin A provided genetic evidence that ELQ-316 acts at the Qi site in T. gondii.","evidence":"Drug-pressure selection of resistant clones, sequencing, and cross-resistance profiling","pmids":["27919897"],"confidence":"High","gaps":["No structural confirmation of ELQ-316 binding pose","Fitness cost of Thr222Pro not assessed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Analysis of Tgmsh1-null parasites revealed an atovaquone-resistance route independent of cytochrome b Qo mutations, via complete respiratory shutdown.","evidence":"Tgmsh1 knockout, mitochondrial DNA sequencing, and electron transport inhibition/resistance assays","pmids":["29141004"],"confidence":"Medium","gaps":["Molecular basis of respiratory shutdown unresolved","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Structure-activity analysis with resistant strains showed quinolones can target either the Qo or Qi site and that species-specific cytochrome b sequence differences shape susceptibility.","evidence":"Proliferation assays with wild-type and resistant T. gondii and P. falciparum plus cytochrome b sequence comparison","pmids":["30117728"],"confidence":"Medium","gaps":["No direct mutagenesis of T. gondii cytochrome b","Binding sites inferred pharmacologically"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"Amplification of the mitochondrial genome including cytochrome b in artemisinin-resistant lines, alongside altered membrane potential, implicated mitochondrial function as an artemisinin target.","evidence":"Whole-genome sequencing of resistant lines, CRISPR/Cas9 mutagenesis, and membrane potential measurement","pmids":["31806760"],"confidence":"Medium","gaps":["Cytochrome b not directly manipulated; implicated only via copy-number change","Direct target of artemisinin not proven"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Cryo-EM structures of the native T. gondii complex III2 and III2-IV supercomplex resolved atovaquone at the Qo site and ELQ-300 at the Qi site, revealing species-specific and flipped binding poses and enabling structure-guided design of subnanomolar inhibitors.","evidence":"Cryo-EM of drug-bound complexes, supercomplex knockout, and structure-guided compound synthesis with proliferation assays","pmids":["40389671"],"confidence":"High","gaps":["Catalytic turnover kinetics of the native enzyme not detailed","Regulation and assembly pathway of cytochrome b not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How cytochrome b is transcribed, translated, and assembled into complex III, and how this is regulated across tachyzoite and bradyzoite stages, remains uncharacterized in the corpus.","evidence":"","pmids":[],"confidence":"Low","gaps":["No biogenesis or assembly mechanism described","Stage-specific regulation beyond observational bradyzoite expression unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Cytochrome bc1 complex (complex III2)","III2-IV respiratory supercomplex"],"partners":["RIESKE IRON-SULFUR PROTEIN (QO-SITE CATALYTIC PARTNER)","CYTOCHROME C1"],"other_free_text":["Two distinct catalytic/drug-binding sites: Qo (ubiquinol oxidation) and Qi (ubiquinone reduction).","Atovaquone binds the Qo site; resistance mutations M129L, I254L (Qo domain); residue 275 governs species-differential inhibition (shown in yeast surrogate).","ELQ-300/ELQ-316 and 4-(1H)-quinolones bind the Qi site; Thr222Pro confers ELQ-316 and antimycin A cross-resistance.","Also the target of decoquinate; implicated in artemisinin action via mitochondrial genome amplification.","Mitochondrially encoded gene; highly expressed in bradyzoite cysts.","Atovaquone resistance can also arise independently of Qo mutations via respiratory shutdown in Tgmsh1-null parasites."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"10802314","id":"PMC_10802314","title":"Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance.","date":"2000","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/10802314","citation_count":124,"is_preprint":false},{"pmid":"12791689","id":"PMC_12791689","title":"Molecular basis for atovaquone binding to the cytochrome bc1 complex.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12791689","citation_count":113,"is_preprint":false},{"pmid":"18212105","id":"PMC_18212105","title":"In vitro susceptibility of various genotypic strains of Toxoplasma gondii to pyrimethamine, sulfadiazine, and atovaquone.","date":"2008","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/18212105","citation_count":106,"is_preprint":false},{"pmid":"9813002","id":"PMC_9813002","title":"Respiration and oxidative phosphorylation in the apicomplexan parasite Toxoplasma gondii.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9813002","citation_count":91,"is_preprint":false},{"pmid":"17242151","id":"PMC_17242151","title":"Growth inhibition of Toxoplasma gondii and Plasmodium falciparum by nanomolar concentrations of 1-hydroxy-2-dodecyl-4(1H)quinolone, a high-affinity inhibitor of alternative (type II) NADH dehydrogenases.","date":"2007","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/17242151","citation_count":66,"is_preprint":false},{"pmid":"25512210","id":"PMC_25512210","title":"Molecular characterisation of Sarcocystis lutrae n. sp. and Toxoplasma gondii from the musculature of two Eurasian otters (Lutra lutra) in Norway.","date":"2014","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/25512210","citation_count":60,"is_preprint":false},{"pmid":"17826334","id":"PMC_17826334","title":"Modeling the molecular basis of atovaquone resistance in parasites and pathogenic fungi.","date":"2007","source":"Trends in parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/17826334","citation_count":52,"is_preprint":false},{"pmid":"27919897","id":"PMC_27919897","title":"Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-Quinolone-3-Diarylethers and Antimycin in Toxoplasma gondii.","date":"2017","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/27919897","citation_count":49,"is_preprint":false},{"pmid":"30117728","id":"PMC_30117728","title":"Targeted Structure-Activity Analysis of Endochin-like Quinolones Reveals Potent Qi and Qo Site Inhibitors of Toxoplasma gondii and Plasmodium falciparum Cytochrome bc1 and Identifies ELQ-400 as a Remarkably Effective Compound against Acute Experimental Toxoplasmosis.","date":"2018","source":"ACS infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/30117728","citation_count":40,"is_preprint":false},{"pmid":"27412848","id":"PMC_27412848","title":"New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27412848","citation_count":37,"is_preprint":false},{"pmid":"31806760","id":"PMC_31806760","title":"Evolution of resistance in vitro reveals mechanisms of artemisinin activity in Toxoplasma gondii.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/31806760","citation_count":35,"is_preprint":false},{"pmid":"21251932","id":"PMC_21251932","title":"Design of anti-parasitic and anti-fungal hydroxy-naphthoquinones that are less susceptible to drug resistance.","date":"2011","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/21251932","citation_count":31,"is_preprint":false},{"pmid":"23358734","id":"PMC_23358734","title":"Characterisation of full-length mitochondrial copies and partial nuclear copies (numts) of the cytochrome b and cytochrome c oxidase subunit I genes of Toxoplasma gondii, Neospora caninum, Hammondia heydorni and Hammondia triffittae (Apicomplexa: Sarcocystidae).","date":"2013","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/23358734","citation_count":30,"is_preprint":false},{"pmid":"32626661","id":"PMC_32626661","title":"Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites.","date":"2020","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32626661","citation_count":26,"is_preprint":false},{"pmid":"32540978","id":"PMC_32540978","title":"Orally Bioavailable Endochin-Like Quinolone Carbonate Ester Prodrug Reduces Toxoplasma gondii Brain Cysts.","date":"2020","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/32540978","citation_count":26,"is_preprint":false},{"pmid":"32161765","id":"PMC_32161765","title":"Activities of Endochin-Like Quinolones Against in vitro Cultured Besnoitia besnoiti Tachyzoites.","date":"2020","source":"Frontiers in veterinary science","url":"https://pubmed.ncbi.nlm.nih.gov/32161765","citation_count":16,"is_preprint":false},{"pmid":"10519259","id":"PMC_10519259","title":"Cytochrome b mutation identified in a decoquinate-resistant mutant of Toxoplasma gondii.","date":"1999","source":"The Journal of eukaryotic microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/10519259","citation_count":12,"is_preprint":false},{"pmid":"40389671","id":"PMC_40389671","title":"Structure, assembly and inhibition of the Toxoplasma gondii respiratory chain supercomplex.","date":"2025","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/40389671","citation_count":10,"is_preprint":false},{"pmid":"38820542","id":"PMC_38820542","title":"Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38820542","citation_count":10,"is_preprint":false},{"pmid":"29141004","id":"PMC_29141004","title":"Lack of mitochondrial MutS homolog 1 in Toxoplasma gondii disrupts maintenance and fidelity of mitochondrial DNA and reveals metabolic plasticity.","date":"2017","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29141004","citation_count":9,"is_preprint":false},{"pmid":"11991686","id":"PMC_11991686","title":"Are cytochrome b gene mutations the only cause of atovaquone resistance in Pneumocystis?","date":"2001","source":"Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/11991686","citation_count":7,"is_preprint":false},{"pmid":"27007245","id":"PMC_27007245","title":"Molecular phylogeny of Toxoplasmatinae: comparison between inferences based on mitochondrial and apicoplast genetic sequences.","date":"2016","source":"Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria","url":"https://pubmed.ncbi.nlm.nih.gov/27007245","citation_count":6,"is_preprint":false},{"pmid":"42058244","id":"PMC_42058244","title":"Phylogenetic study of the protozoan Toxoplasma gondii using cytochrome b and GRA20 genes.","date":"2025","source":"Archives of Razi Institute","url":"https://pubmed.ncbi.nlm.nih.gov/42058244","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14653,"output_tokens":3476,"usd":0.04805,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9659,"output_tokens":4466,"usd":0.079973,"stage2_stop_reason":"end_turn"},"total_usd":0.128023,"stage1_batch_id":"msgbatch_018xTxyBPcQFK9jdgubtxgsj","stage2_batch_id":"msgbatch_01UiG5BTaJP7BvKKL6RLeyv2","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"Cytochrome b gene of T. gondii was cloned and sequenced; atovaquone-resistant mutants harbored mutations M129L and I254L in two different regions of the ubiquinol-binding pocket (Qo domain) of cytochrome b, establishing that atovaquone interferes with electron transport at the cytochrome bc1 complex by targeting the Qo site of cytochrome b.\",\n      \"method\": \"Gene cloning and sequencing of wild-type and atovaquone-resistant T. gondii strains; structural modeling of the Qo domain\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct sequencing of resistant mutants with functional drug resistance phenotype, structural modeling; foundational paper replicated conceptually across multiple subsequent studies\",\n      \"pmids\": [\"10802314\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"A mutation in the cytochrome b gene was identified in a decoquinate-resistant mutant of T. gondii, indicating that cytochrome b is the target of decoquinate and that resistance arises through mutations in this mitochondrial gene.\",\n      \"method\": \"Selection of drug-resistant mutants; gene sequencing\",\n      \"journal\": \"The Journal of eukaryotic microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, direct sequencing of resistant mutant; abstract only available\",\n      \"pmids\": [\"10519259\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Atovaquone at nanomolar concentrations (0.03 µM) totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, with the site of inhibition localized between cytochrome b and c1 (consistent with bc1 complex inhibition), establishing cytochrome b as part of a functional respiratory chain in this parasite.\",\n      \"method\": \"In situ respiration assay using digitonin-permeabilized tachyzoites; safranin O mitochondrial membrane potential measurement; antimycin A and cyanide sensitivity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical assay in permeabilized parasites with multiple inhibitors and membrane potential measurement in a single rigorous study\",\n      \"pmids\": [\"9813002\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Atovaquone binds competitively to the ubiquinol oxidation (Qo) pocket of the cytochrome bc1 complex, interacting with the Rieske iron-sulfur protein; a L275F mutation in cytochrome b (yeast surrogate) decreased atovaquone sensitivity, establishing that residue 275 (phenylalanine in bovine, leucine in yeast) governs species-differential inhibition.\",\n      \"method\": \"Enzymatic inhibition assays (Ki determination); EPR spectroscopy of Rieske iron-sulfur protein; site-directed mutagenesis of yeast cytochrome b (L275F); energy-minimized structural modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with isolated bc1 complex, mutagenesis, EPR, and structural modeling; mechanistically informative for T. gondii cytochrome b drug target\",\n      \"pmids\": [\"12791689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A Thr222Pro substitution in cytochrome b of T. gondii confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin A (a well-characterized Qi site inhibitor), providing genetic evidence that ELQ-316 inhibits the cytochrome bc1 complex Qi site in T. gondii.\",\n      \"method\": \"Drug-pressure selection of resistant T. gondii clones; gene sequencing; pharmacological cross-resistance assays\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via resistance mutation with defined amino acid change, cross-resistance phenotype with known Qi inhibitor; single lab but two orthogonal lines of evidence\",\n      \"pmids\": [\"27919897\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Structure-activity relationship studies using wild-type and drug-resistant T. gondii and P. falciparum strains demonstrated that 4(1H)-quinolones can target either the Qo or Qi site of cytochrome bc1; T. gondii cytochrome b sequence differences relative to P. falciparum at positions associated with drug resistance influence drug susceptibility between species.\",\n      \"method\": \"Proliferation assays with wild-type and resistant parasite strains; cytochrome b sequence comparison; structure-activity relationship analysis of quinolone series\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological dissection with resistant strains and sequence analysis, single lab; no direct mutagenesis of T. gondii cytochrome b in this study\",\n      \"pmids\": [\"30117728\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Novel 4-(1H)-quinolone scaffolds bind to the cytochrome bc1 complex Qi site, as demonstrated by mutant yeast and co-crystallographic studies; cytochrome b is highly expressed in T. gondii cysts (bradyzoites).\",\n      \"method\": \"Mutant yeast bc1 assay; co-crystallography; in vitro and in vivo activity assays against T. gondii tachyzoites and cysts\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — co-crystallography and mutant yeast for Qi site binding, but T. gondii cytochrome b itself not structurally resolved; bradyzoite expression is observational\",\n      \"pmids\": [\"27412848\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Artemisinin-resistant T. gondii lines selected in vitro showed dramatic amplification of the mitochondrial genome, including the cytochrome b gene; treatment of wild-type parasites with high drug concentrations decreased mitochondrial membrane potential, and this phenotype was stably altered in resistant lines, implicating mitochondrial function (including cytochrome b) as an artemisinin target.\",\n      \"method\": \"Whole-genome sequencing of resistant lines; CRISPR/Cas9 mutagenesis; mitochondrial membrane potential measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genomic evidence for mitochondrial gene amplification plus functional membrane potential readout; cytochrome b not directly manipulated but implicated through copy number change\",\n      \"pmids\": [\"31806760\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Parasites lacking TgMSH1 accumulate single nucleotide variations in mitochondrial DNA including cytochrome b, yet atovaquone can still inhibit electron transport in these mutants despite missense mutations in cytochrome b that do not affect known atovaquone-sensitivity determinants; under atovaquone pressure, Tgmsh1 mutants develop enhanced resistance via complete shutdown of respiration rather than via cytochrome b Qo site mutations.\",\n      \"method\": \"Tgmsh1 knockout generation; mitochondrial DNA sequencing; electron transport inhibition assays; atovaquone resistance selection\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with functional electron transport assay; novel mechanistic finding that atovaquone resistance can arise independently of cytochrome b Qo mutations\",\n      \"pmids\": [\"29141004\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of the T. gondii cytochrome bc1 complex (complex III2) and the III2-IV supercomplex were resolved; atovaquone binds to the cytochrome b Qo site in a manner revealing species-specific interactions; ELQ-300 binds the Qi site of T. gondii cytochrome b in a flipped orientation relative to its binding in the mammalian enzyme; supercomplex knockout causes disassembly and reduces parasite fitness; structure-guided design yielded more potent ELQs with subnanomolar activity against T. gondii.\",\n      \"method\": \"Cryo-electron microscopy structure determination; knockout analysis; inhibitor co-structure with atovaquone and ELQ-300; structure-guided compound design and proliferation assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures with drug-bound complexes, knockout phenotype, and structure-guided medicinal chemistry in a single rigorous study\",\n      \"pmids\": [\"40389671\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Atovaquone blocks mitochondrial oxidation of ubiquinol in T. gondii by binding to the ubiquinol oxidation (Qo) site of the cytochrome bc1 complex; mutations in cytochrome b associated with atovaquone resistance in Plasmodium, Pneumocystis, and Toxoplasma were characterized in isolated yeast bc1 complexes harboring equivalent mutations, establishing residue-level requirements for Qo site inhibition.\",\n      \"method\": \"Enzymatic assays with isolated yeast bc1 complexes bearing cytochrome b mutations equivalent to those in drug-resistant pathogens; synthesis and testing of novel hydroxy-naphthoquinones\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with mutagenized surrogate complexes; T. gondii resistance mutations characterized functionally in yeast system\",\n      \"pmids\": [\"21251932\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HDQ (1-hydroxy-2-dodecyl-4(1H)quinolone), an inhibitor of alternative (type II) NADH dehydrogenase, acts synergistically with atovaquone (which blocks the ubiquinol oxidation site of cytochrome b in complex III) against T. gondii, with a fractional inhibitory concentration of 0.16 nM, demonstrating that simultaneous interference at two sites of the ubiquinone/ubiquinol cycle is highly effective.\",\n      \"method\": \"Synergy assay (FIC index calculation) combining HDQ and atovaquone against T. gondii in cell culture\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological combination study demonstrating functional interaction at two mitochondrial sites; mechanistically informative about cytochrome b Qo site but indirect\",\n      \"pmids\": [\"17242151\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) that mediates ubiquinol oxidation at the Qo site and ubiquinone reduction at the Qi site; atovaquone inhibits electron transport by binding the Qo domain (with species-specificity governed by residues lining the ubiquinol pocket), while endochin-like quinolones can inhibit either site, and drug resistance arises primarily through point mutations in the Qo or Qi domains of cytochrome b, as confirmed by cryo-EM structures, in vitro enzymatic assays, mutagenesis, and genetic selection of resistant parasites.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) that functions in the parasite's respiratory electron transport chain, where inhibition disrupts respiration and collapses mitochondrial membrane potential [#2, #9]. Cryo-EM structures of the T. gondii complex III2 and the III2-IV supercomplex show that cytochrome b provides two catalytically distinct drug-binding sites: the ubiquinol-oxidation Qo site, where atovaquone binds through species-specific interactions, and the Qi site, where endochin-like quinolones such as ELQ-300 bind in an orientation distinct from that in the mammalian enzyme [#9]. Atovaquone acts as a competitive inhibitor at the Qo pocket in association with the Rieske iron-sulfur protein, and drug resistance arises through point mutations in the Qo domain (e.g. M129L, I254L), residues lining the ubiquinol pocket governing species-differential inhibition [#0, #3, #10]. Resistance to Qi-site inhibitors similarly maps to cytochrome b, with a Thr222Pro substitution conferring cross-resistance to ELQ-316 and the classical Qi inhibitor antimycin A [#4]. Cytochrome b is also the target of decoquinate [#1], and structure-guided design against these sites has produced quinolones with subnanomolar activity [#9]. Beyond drug-target characterization, the corpus does not detail the protein's biogenesis, assembly, or regulation independent of complex III.\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that T. gondii possesses a functional respiratory chain answered whether the parasite's mitochondrion sustains electron transport that could be drug-targeted, localizing the inhibitory site to the bc1 complex.\",\n      \"evidence\": \"In situ respiration and safranin O membrane-potential assays in digitonin-permeabilized tachyzoites with atovaquone, antimycin A, and cyanide\",\n      \"pmids\": [\"9813002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which cytochrome b residues bind atovaquone\", \"Did not distinguish Qo from Qi contributions\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identifying a cytochrome b mutation in a decoquinate-resistant mutant showed that cytochrome b is a drug target beyond atovaquone.\",\n      \"evidence\": \"Selection of decoquinate-resistant T. gondii mutants and gene sequencing\",\n      \"pmids\": [\"10519259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Abstract-only report\", \"Binding site of decoquinate on cytochrome b not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Cloning cytochrome b and mapping atovaquone-resistance mutations to the ubiquinol pocket answered where atovaquone acts, establishing the Qo site of cytochrome b as its molecular target.\",\n      \"evidence\": \"Gene cloning/sequencing of wild-type and atovaquone-resistant strains with Qo domain structural modeling\",\n      \"pmids\": [\"10802314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimentally resolved structure\", \"Mechanism of species-differential susceptibility not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Reconstitution in a yeast surrogate defined atovaquone as a competitive Qo-site inhibitor coupled to the Rieske protein and pinpointed residue 275 as a determinant of species-differential inhibition.\",\n      \"evidence\": \"Enzymatic Ki assays, EPR of Rieske iron-sulfur protein, and L275F mutagenesis of yeast cytochrome b\",\n      \"pmids\": [\"12791689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in yeast, not T. gondii enzyme\", \"Does not address Qi-site inhibitors\"],\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Functional testing of pathogen-equivalent mutations in isolated yeast complexes established residue-level requirements for Qo-site inhibition across Plasmodium, Pneumocystis, and Toxoplasma.\",\n      \"evidence\": \"Enzymatic assays with isolated yeast bc1 complexes bearing equivalent cytochrome b mutations plus novel hydroxy-naphthoquinones\",\n      \"pmids\": [\"21251932\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Surrogate yeast system rather than native T. gondii complex\", \"Qi site not addressed\"],\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Co-crystallographic and mutant-yeast work established that 4-(1H)-quinolone scaffolds engage the Qi site, extending the druggable geometry of cytochrome b beyond the Qo pocket.\",\n      \"evidence\": \"Mutant yeast bc1 assay, co-crystallography, and activity assays against T. gondii tachyzoites and cysts\",\n      \"pmids\": [\"27412848\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"T. gondii cytochrome b not structurally resolved here\", \"Bradyzoite expression finding is observational\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A Thr222Pro cytochrome b substitution conferring cross-resistance to ELQ-316 and antimycin A provided genetic evidence that ELQ-316 acts at the Qi site in T. gondii.\",\n      \"evidence\": \"Drug-pressure selection of resistant clones, sequencing, and cross-resistance profiling\",\n      \"pmids\": [\"27919897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural confirmation of ELQ-316 binding pose\", \"Fitness cost of Thr222Pro not assessed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Analysis of Tgmsh1-null parasites revealed an atovaquone-resistance route independent of cytochrome b Qo mutations, via complete respiratory shutdown.\",\n      \"evidence\": \"Tgmsh1 knockout, mitochondrial DNA sequencing, and electron transport inhibition/resistance assays\",\n      \"pmids\": [\"29141004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of respiratory shutdown unresolved\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Structure-activity analysis with resistant strains showed quinolones can target either the Qo or Qi site and that species-specific cytochrome b sequence differences shape susceptibility.\",\n      \"evidence\": \"Proliferation assays with wild-type and resistant T. gondii and P. falciparum plus cytochrome b sequence comparison\",\n      \"pmids\": [\"30117728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct mutagenesis of T. gondii cytochrome b\", \"Binding sites inferred pharmacologically\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Amplification of the mitochondrial genome including cytochrome b in artemisinin-resistant lines, alongside altered membrane potential, implicated mitochondrial function as an artemisinin target.\",\n      \"evidence\": \"Whole-genome sequencing of resistant lines, CRISPR/Cas9 mutagenesis, and membrane potential measurement\",\n      \"pmids\": [\"31806760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytochrome b not directly manipulated; implicated only via copy-number change\", \"Direct target of artemisinin not proven\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM structures of the native T. gondii complex III2 and III2-IV supercomplex resolved atovaquone at the Qo site and ELQ-300 at the Qi site, revealing species-specific and flipped binding poses and enabling structure-guided design of subnanomolar inhibitors.\",\n      \"evidence\": \"Cryo-EM of drug-bound complexes, supercomplex knockout, and structure-guided compound synthesis with proliferation assays\",\n      \"pmids\": [\"40389671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic turnover kinetics of the native enzyme not detailed\", \"Regulation and assembly pathway of cytochrome b not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How cytochrome b is transcribed, translated, and assembled into complex III, and how this is regulated across tachyzoite and bradyzoite stages, remains uncharacterized in the corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No biogenesis or assembly mechanism described\", \"Stage-specific regulation beyond observational bradyzoite expression unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008121\", \"supporting_discovery_ids\": [2, 3, 9, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006122\", \"supporting_discovery_ids\": [2, 3, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [2, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005750\", \"supporting_discovery_ids\": [9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0022904\", \"supporting_discovery_ids\": [2, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0042775\", \"supporting_discovery_ids\": [2, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Cytochrome bc1 complex (complex III2)\",\n      \"III2-IV respiratory supercomplex\"\n    ],\n    \"partners\": [\n      \"Rieske iron-sulfur protein (Qo-site catalytic partner)\",\n      \"Cytochrome c1\"\n    ],\n    \"other_free_text\": [\n      \"Two distinct catalytic/drug-binding sites: Qo (ubiquinol oxidation) and Qi (ubiquinone reduction).\",\n      \"Atovaquone binds the Qo site; resistance mutations M129L, I254L (Qo domain); residue 275 governs species-differential inhibition (shown in yeast surrogate).\",\n      \"ELQ-300/ELQ-316 and 4-(1H)-quinolones bind the Qi site; Thr222Pro confers ELQ-316 and antimycin A cross-resistance.\",\n      \"Also the target of decoquinate; implicated in artemisinin action via mitochondrial genome amplification.\",\n      \"Mitochondrially encoded gene; highly expressed in bradyzoite cysts.\",\n      \"Atovaquone resistance can also arise independently of Qo mutations via respiratory shutdown in Tgmsh1-null parasites.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"RH88","gene_id":"TGRH88_085460","name":null,"product":"cytochrome b","uniprot_acc":null,"chromosome":"JAAUHK010000041","start_pos":8748}]}