{"gene":"RH88_085380","gene_symbol":"","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2000,"finding":"T. gondii cytochrome b was cloned and sequenced; atovaquone-resistant mutants carry mutations M129L and I254L within the ubiquinol-binding pocket (Qo domain) of cytochrome b, establishing that atovaquone inhibits electron transport by targeting the cytochrome bc1 complex Qo site in T. gondii.","method":"Gene cloning, sequencing of wild-type and atovaquone-resistant 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 site-specific mutations mapped to functional domain, first description of the gene, replicated by 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 molecular target of decoquinate in T. gondii.","method":"Sequencing of cytochrome b gene from drug-resistant mutants","journal":"The Journal of eukaryotic microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, single method (sequencing of resistant mutant), no functional reconstitution","pmids":["10519259"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"Atovaquone at nanomolar concentrations inhibits the T. gondii respiratory chain between cytochrome b and c1 (analogous to antimycin A1 inhibition), disrupts mitochondrial membrane potential, and blocks oxidative phosphorylation in permeabilized tachyzoites.","method":"In situ respiration assay with digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, inhibitor profiling (antimycin A, cyanide, atovaquone)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical assay in permeabilized parasites with multiple inhibitors and multiple readouts (respiration, membrane potential, ADP phosphorylation) in a single rigorous study","pmids":["9813002"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Atovaquone binds to the ubiquinol oxidation (Qo) pocket of the cytochrome bc1 complex, interacting with the Rieske iron-sulfur protein; the identity of residue 275 in cytochrome b (leucine in yeast vs. phenylalanine in bovine) explains differential species sensitivity, confirmed by L275F mutagenesis in yeast.","method":"Biochemical inhibition assays (Ki determination), EPR spectroscopy of Rieske center, midpoint potential measurement, site-directed mutagenesis (L275F in yeast cytochrome b), computational energy-minimized structural modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified bc1 complex, mutagenesis validation, multiple orthogonal methods (kinetics, EPR, redox potential, modeling); directly informs T. gondii cytochrome b mechanism","pmids":["12791689"],"is_preprint":false,"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2017,"finding":"A Thr222Pro substitution in T. gondii cytochrome b confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor, providing genetic evidence that ELQ-316 and related 4(1H)-quinolone-3-diarylethers inhibit the cytochrome bc1 complex Qi site in T. gondii.","method":"Drug-pressure selection of resistant clones, whole-gene sequencing of cytochrome b, quantitative resistance assays comparing wild-type and mutant parasites","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via resistance mutation mapped to Qi site confirmed by cross-resistance to antimycin; single lab but two orthogonal lines of evidence (mutation identification + cross-resistance profile)","pmids":["27919897"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Bradyzoite cysts of T. gondii highly express cytochrome b; novel 4-(1H)-quinolone scaffolds targeting the cytochrome bc1 complex Qi site inhibit both active infection (IC50 30 nM) and cysts (IC50 4 μM) in vitro; mutant yeast and co-crystallographic studies confirm binding to the bc1 complex Qi site.","method":"In vitro cyst model with EGS strain, cat oocyst production assay, inhibition assays, mutant yeast complementation, co-crystallography of compound bound to bc1 Qi site","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — co-crystallographic structure plus mutant yeast validation plus functional inhibition assays in T. gondii models in a single study","pmids":["27412848"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2018,"finding":"Comparison of T. gondii and P. falciparum cytochrome b sequences identified natural amino acid differences at positions associated with drug resistance in P. falciparum, and structure-activity studies with wild-type and drug-resistant strains showed that substituents on the quinolone ring influence selectivity between Qo and Qi site inhibition of cytochrome bc1.","method":"Cytochrome b sequence comparison, inhibition assays with wild-type and resistant T. gondii and P. falciparum strains, structure-activity relationship analysis of 4(1H)-quinolone series","journal":"ACS infectious diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cross-species cytochrome b sequence analysis combined with pharmacological profiling on resistant strains; single lab, no structural data for T. gondii specifically","pmids":["30117728"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"Artemisinin-resistant T. gondii lines selected in vitro showed dramatic amplification of the mitochondrial genome including cytochrome b and cytochrome c oxidase I genes, and wild-type parasites treated with high artemisinin concentrations showed decreased mitochondrial membrane potential—a phenotype stably altered in resistant parasites—suggesting the mitochondrion (including cytochrome b-containing complex III) is an important artemisinin target.","method":"In vitro resistance selection, whole-genome sequencing, CRISPR/Cas9 point mutation introduction, mitochondrial membrane potential measurement, competitive growth assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome amplification confirmed by sequencing, mitochondrial membrane potential assay, CRISPR validation of specific mutations; cytochrome b involvement inferred from genomic amplification rather than direct functional assay of the protein","pmids":["31806760"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Parasites lacking TgMSH1 accumulate missense mutations in cytochrome b but none at residues known to determine atovaquone sensitivity; atovaquone still inhibits electron transport in these mutants, yet the parasites survive atovaquone treatment by shutting down oxidative phosphorylation entirely—demonstrating a novel, cytochrome b mutation-independent mechanism of atovaquone resistance.","method":"TgMSH1 knockout generation, mitochondrial DNA sequencing, electron transport inhibition assays, growth assays under mitochondrial inhibitors with diverse targets, glucose deprivation experiments","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with functional electron transport assay and multiple inhibitor profiling; single lab but multiple orthogonal readouts","pmids":["29141004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"Cryo-EM structures of the T. gondii III2-IV respiratory supercomplex and isolated complex III2 (containing cytochrome b) were solved; atovaquone was shown by structure to bind the cytochrome b Qo site with parasite-specific interactions explaining species selectivity; ELQ-300 binds the cytochrome b Qi site in a flipped orientation relative to the mammalian enzyme; structure-guided design produced more potent ELQs with subnanomolar activity against T. gondii.","method":"Cryo-electron microscopy structure determination, knockout analysis of supercomplex assembly, inhibitor soaking/co-structure with atovaquone and ELQ-300, structure-guided medicinal chemistry, T. gondii proliferation assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures at high resolution with functional validation by knockout and inhibitor assays; multiple orthogonal methods in a single rigorous study","pmids":["40389671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"Combined treatment of T. gondii with HDQ (type II NADH dehydrogenase inhibitor) and atovaquone (cytochrome b Qo site inhibitor) produced synergistic inhibition of parasite replication (fractional inhibitory concentration 0.16 nM), demonstrating that simultaneous blockade of the ubiquinone/ubiquinol cycle at the NADH dehydrogenase step and at cytochrome b is highly effective.","method":"In vitro T. gondii growth inhibition assays, fractional inhibitory concentration calculation for drug combination","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological combination assay establishing functional synergy between two mitochondrial targets; mechanism inferred from known drug targets rather than direct biochemical dissection of cytochrome b itself","pmids":["17242151"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"T. gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) whose Qo and Qi ubiquinol/quinone-binding pockets are the molecular targets of atovaquone and endochin-like quinolones respectively; resistance arises from point mutations in these pockets (e.g., M129L, I254L at Qo; T222P at Qi), and cryo-EM structures have revealed parasite-specific binding geometries that underpin species-selective inhibition and guide next-generation drug design."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii cytochrome b is a mitochondrially encoded, ubiquinol-oxidizing subunit of the cytochrome bc1 complex (complex III) that carries respiratory electron transport between cytochrome b and c1; its inhibition disrupts mitochondrial membrane potential and blocks oxidative phosphorylation in tachyzoites [PMID:9813002]. It is the molecular target of atovaquone, which binds the ubiquinol-oxidation (Qo) pocket in cooperation with the Rieske iron-sulfur protein, and atovaquone-resistant parasites carry Qo-domain point mutations (M129L, I254L) [PMID:10802314, PMID:12791689]. A structurally distinct class of 4(1H)-quinolone diaryl ethers (endochin-like quinolones, ELQs) instead engages the quinone-reduction (Qi) site: a Thr222Pro substitution confers resistance to ELQ-316 with cross-resistance to the Qi inhibitor antimycin, and Qi binding was confirmed co-crystallographically [PMID:27919897, PMID:27412848]. Cryo-EM structures of the T. gondii III2-IV supercomplex and isolated complex III2 resolved atovaquone in the Qo site and ELQ-300 in the Qi site with a flipped, parasite-specific binding geometry that explains species-selective inhibition and enabled design of subnanomolar ELQs [PMID:40389671]. Cytochrome b is highly expressed in bradyzoite cysts, extending its relevance to the latent stage [PMID:27412848], and it also functions as a node for combination therapy, acting synergistically with type II NADH dehydrogenase inhibition [PMID:17242151]. Resistance is not solely genetic: parasites can survive atovaquone by shutting down oxidative phosphorylation entirely, independent of cytochrome b mutation [PMID:29141004].","teleology":[{"year":1998,"claim":"Establishing what respiratory step atovaquone blocks was needed to define its antiparasitic mechanism; direct assay placed the block within complex III and linked it to loss of membrane potential and ATP synthesis.","evidence":"In situ respiration and safranin O membrane-potential assays in digitonin-permeabilized tachyzoites with inhibitor profiling","pmids":["9813002"],"confidence":"High","gaps":["Did not identify the specific cytochrome b residues or subsite engaged","No structural resolution of the binding pocket"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"Identifying the target of decoquinate addressed whether multiple quinolone-class drugs converge on the same protein; a resistance mutation implicated cytochrome b.","evidence":"Cytochrome b gene sequencing from a decoquinate-resistant mutant","pmids":["10519259"],"confidence":"Medium","gaps":["Single method (sequencing) without functional reconstitution","Subsite (Qo vs Qi) not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Cloning the gene and mapping atovaquone-resistance mutations to a functional domain established cytochrome b as the atovaquone target and localized the drug to the Qo site.","evidence":"Cloning/sequencing of wild-type and resistant strains with Qo-domain structural modeling","pmids":["10802314"],"confidence":"High","gaps":["No experimental structure of the T. gondii Qo pocket","Mechanism of species selectivity not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Determining how atovaquone selectivity is encoded clarified species-specific inhibition; the drug binds the Qo pocket contacting the Rieske protein, with residue 275 identity governing sensitivity.","evidence":"Purified bc1 kinetics, EPR of the Rieske center, redox measurement, and L275F mutagenesis in yeast","pmids":["12791689"],"confidence":"High","gaps":["Performed in yeast, not T. gondii cytochrome b directly","Does not address Qi-site inhibitors"],"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2007,"claim":"Testing combination pharmacology showed cytochrome b inhibition can be potentiated by blocking an upstream ubiquinone step, demonstrating synergistic vulnerability of the respiratory chain.","evidence":"In vitro growth inhibition with HDQ + atovaquone and fractional inhibitory concentration analysis","pmids":["17242151"],"confidence":"Medium","gaps":["Synergy inferred from known targets, not direct biochemical dissection of cytochrome b","No structural correlate"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Defining a second druggable subsite and stage-specific expression established the Qi site as a target active against latent cysts, broadening therapeutic reach.","evidence":"In vitro cyst model (EGS strain), inhibition assays, mutant yeast complementation, and co-crystallography at the bc1 Qi site","pmids":["27412848"],"confidence":"High","gaps":["Cyst-stage IC50 remains high (4 uM)","Qi structural data from surrogate systems, not T. gondii enzyme"],"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2017,"claim":"Genetically localizing ELQ-316 action distinguished Qi- from Qo-site inhibitors; a Thr222Pro mutation conferring ELQ and antimycin cross-resistance placed ELQs at the Qi site.","evidence":"Drug-pressure selection, cytochrome b sequencing, and cross-resistance quantitation","pmids":["27919897"],"confidence":"High","gaps":["No direct structure of the T. gondii Qi pocket in this study","Fitness cost of T222P not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Uncovering a non-genetic resistance route revealed that cytochrome b inhibition can be bypassed metabolically; TgMSH1-null parasites survive atovaquone by shutting down oxidative phosphorylation rather than by Qo mutation.","evidence":"TgMSH1 knockout, mitochondrial DNA sequencing, electron transport and multi-inhibitor growth assays, glucose deprivation","pmids":["29141004"],"confidence":"Medium","gaps":["Molecular basis of the OXPHOS-shutdown state undefined","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Cross-species sequence and SAR comparison clarified how quinolone substituents partition between Qo and Qi inhibition, guiding selectivity engineering.","evidence":"T. gondii vs P. falciparum cytochrome b sequence comparison and SAR profiling on wild-type and resistant strains","pmids":["30117728"],"confidence":"Medium","gaps":["No T. gondii-specific structural data","Selectivity determinants inferred pharmacologically"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"Probing artemisinin action implicated the cytochrome b-containing mitochondrion; resistant lines amplified the mitochondrial genome and showed altered membrane potential.","evidence":"In vitro resistance selection, whole-genome sequencing, CRISPR mutation introduction, and membrane-potential assays","pmids":["31806760"],"confidence":"Medium","gaps":["Cytochrome b involvement inferred from genome amplification, not direct protein assay","No structural or biochemical link to complex III activity"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Solving the parasite respiratory supercomplex structures resolved the long-standing gap in T. gondii-specific inhibitor geometry, showing atovaquone at Qo and ELQ-300 in a flipped Qi pose that explains selectivity and enabled potency optimization.","evidence":"Cryo-EM of III2-IV supercomplex and complex III2, inhibitor co-structures, knockout assembly analysis, and structure-guided medicinal chemistry","pmids":["40389671"],"confidence":"High","gaps":["In vivo efficacy of optimized ELQs not established here","Resistance liabilities of new compounds unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How cytochrome b mutation-independent atovaquone resistance is enacted metabolically, and whether structure-guided ELQs retain activity against Qi-mutant and latent-stage parasites in vivo, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Molecular mechanism of OXPHOS-shutdown survival undefined","In vivo efficacy against cysts and resistant mutants untested","No T. gondii-specific atovaquone/ELQ cross-resistance structural map beyond the reported poses"],"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","CYTOCHROME C1","CYTOCHROME C OXIDASE (COMPLEX IV)"],"other_free_text":["Qo (ubiquinol-oxidation) site is the binding pocket for atovaquone and decoquinate; resistance mutations M129L and I254L map here","Qi (quinone-reduction) site is the binding pocket for endochin-like quinolones (ELQ-316, ELQ-300) and antimycin; T222P confers ELQ/antimycin cross-resistance","Highly expressed in bradyzoite cysts, making complex III a target relevant to the latent stage","Mitochondrial genome (including cytochrome b) is amplified in artemisinin-resistant lines","Cytochrome b mutation-independent atovaquone survival occurs via shutdown of oxidative phosphorylation"]},"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":"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":14412,"output_tokens":3219,"usd":0.045761,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9285,"output_tokens":4443,"usd":0.07875,"stage2_stop_reason":"end_turn"},"total_usd":0.124511,"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\": \"T. gondii cytochrome b was cloned and sequenced; atovaquone-resistant mutants carry mutations M129L and I254L within the ubiquinol-binding pocket (Qo domain) of cytochrome b, establishing that atovaquone inhibits electron transport by targeting the cytochrome bc1 complex Qo site in T. gondii.\",\n      \"method\": \"Gene cloning, sequencing of wild-type and atovaquone-resistant 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 site-specific mutations mapped to functional domain, first description of the gene, replicated by 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 molecular target of decoquinate in T. gondii.\",\n      \"method\": \"Sequencing of cytochrome b gene from drug-resistant mutants\",\n      \"journal\": \"The Journal of eukaryotic microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, single method (sequencing of resistant mutant), no functional reconstitution\",\n      \"pmids\": [\"10519259\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Atovaquone at nanomolar concentrations inhibits the T. gondii respiratory chain between cytochrome b and c1 (analogous to antimycin A1 inhibition), disrupts mitochondrial membrane potential, and blocks oxidative phosphorylation in permeabilized tachyzoites.\",\n      \"method\": \"In situ respiration assay with digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, inhibitor profiling (antimycin A, cyanide, atovaquone)\",\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 multiple readouts (respiration, membrane potential, ADP phosphorylation) 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 to the ubiquinol oxidation (Qo) pocket of the cytochrome bc1 complex, interacting with the Rieske iron-sulfur protein; the identity of residue 275 in cytochrome b (leucine in yeast vs. phenylalanine in bovine) explains differential species sensitivity, confirmed by L275F mutagenesis in yeast.\",\n      \"method\": \"Biochemical inhibition assays (Ki determination), EPR spectroscopy of Rieske center, midpoint potential measurement, site-directed mutagenesis (L275F in yeast cytochrome b), computational energy-minimized structural modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified bc1 complex, mutagenesis validation, multiple orthogonal methods (kinetics, EPR, redox potential, modeling); directly informs T. gondii cytochrome b mechanism\",\n      \"pmids\": [\"12791689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A Thr222Pro substitution in T. gondii cytochrome b confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor, providing genetic evidence that ELQ-316 and related 4(1H)-quinolone-3-diarylethers inhibit the cytochrome bc1 complex Qi site in T. gondii.\",\n      \"method\": \"Drug-pressure selection of resistant clones, whole-gene sequencing of cytochrome b, quantitative resistance assays comparing wild-type and mutant parasites\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via resistance mutation mapped to Qi site confirmed by cross-resistance to antimycin; single lab but two orthogonal lines of evidence (mutation identification + cross-resistance profile)\",\n      \"pmids\": [\"27919897\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Bradyzoite cysts of T. gondii highly express cytochrome b; novel 4-(1H)-quinolone scaffolds targeting the cytochrome bc1 complex Qi site inhibit both active infection (IC50 30 nM) and cysts (IC50 4 μM) in vitro; mutant yeast and co-crystallographic studies confirm binding to the bc1 complex Qi site.\",\n      \"method\": \"In vitro cyst model with EGS strain, cat oocyst production assay, inhibition assays, mutant yeast complementation, co-crystallography of compound bound to bc1 Qi site\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — co-crystallographic structure plus mutant yeast validation plus functional inhibition assays in T. gondii models in a single study\",\n      \"pmids\": [\"27412848\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Comparison of T. gondii and P. falciparum cytochrome b sequences identified natural amino acid differences at positions associated with drug resistance in P. falciparum, and structure-activity studies with wild-type and drug-resistant strains showed that substituents on the quinolone ring influence selectivity between Qo and Qi site inhibition of cytochrome bc1.\",\n      \"method\": \"Cytochrome b sequence comparison, inhibition assays with wild-type and resistant T. gondii and P. falciparum strains, structure-activity relationship analysis of 4(1H)-quinolone series\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cross-species cytochrome b sequence analysis combined with pharmacological profiling on resistant strains; single lab, no structural data for T. gondii specifically\",\n      \"pmids\": [\"30117728\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Artemisinin-resistant T. gondii lines selected in vitro showed dramatic amplification of the mitochondrial genome including cytochrome b and cytochrome c oxidase I genes, and wild-type parasites treated with high artemisinin concentrations showed decreased mitochondrial membrane potential—a phenotype stably altered in resistant parasites—suggesting the mitochondrion (including cytochrome b-containing complex III) is an important artemisinin target.\",\n      \"method\": \"In vitro resistance selection, whole-genome sequencing, CRISPR/Cas9 point mutation introduction, mitochondrial membrane potential measurement, competitive growth assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome amplification confirmed by sequencing, mitochondrial membrane potential assay, CRISPR validation of specific mutations; cytochrome b involvement inferred from genomic amplification rather than direct functional assay of the protein\",\n      \"pmids\": [\"31806760\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Parasites lacking TgMSH1 accumulate missense mutations in cytochrome b but none at residues known to determine atovaquone sensitivity; atovaquone still inhibits electron transport in these mutants, yet the parasites survive atovaquone treatment by shutting down oxidative phosphorylation entirely—demonstrating a novel, cytochrome b mutation-independent mechanism of atovaquone resistance.\",\n      \"method\": \"TgMSH1 knockout generation, mitochondrial DNA sequencing, electron transport inhibition assays, growth assays under mitochondrial inhibitors with diverse targets, glucose deprivation experiments\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with functional electron transport assay and multiple inhibitor profiling; single lab but multiple orthogonal readouts\",\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 III2-IV respiratory supercomplex and isolated complex III2 (containing cytochrome b) were solved; atovaquone was shown by structure to bind the cytochrome b Qo site with parasite-specific interactions explaining species selectivity; ELQ-300 binds the cytochrome b Qi site in a flipped orientation relative to the mammalian enzyme; structure-guided design produced more potent ELQs with subnanomolar activity against T. gondii.\",\n      \"method\": \"Cryo-electron microscopy structure determination, knockout analysis of supercomplex assembly, inhibitor soaking/co-structure with atovaquone and ELQ-300, structure-guided medicinal chemistry, T. gondii proliferation assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures at high resolution with functional validation by knockout and inhibitor assays; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"40389671\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Combined treatment of T. gondii with HDQ (type II NADH dehydrogenase inhibitor) and atovaquone (cytochrome b Qo site inhibitor) produced synergistic inhibition of parasite replication (fractional inhibitory concentration 0.16 nM), demonstrating that simultaneous blockade of the ubiquinone/ubiquinol cycle at the NADH dehydrogenase step and at cytochrome b is highly effective.\",\n      \"method\": \"In vitro T. gondii growth inhibition assays, fractional inhibitory concentration calculation for drug combination\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological combination assay establishing functional synergy between two mitochondrial targets; mechanism inferred from known drug targets rather than direct biochemical dissection of cytochrome b itself\",\n      \"pmids\": [\"17242151\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"T. gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) whose Qo and Qi ubiquinol/quinone-binding pockets are the molecular targets of atovaquone and endochin-like quinolones respectively; resistance arises from point mutations in these pockets (e.g., M129L, I254L at Qo; T222P at Qi), and cryo-EM structures have revealed parasite-specific binding geometries that underpin species-selective inhibition and guide next-generation drug design.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Toxoplasma gondii cytochrome b is a mitochondrially encoded, ubiquinol-oxidizing subunit of the cytochrome bc1 complex (complex III) that carries respiratory electron transport between cytochrome b and c1; its inhibition disrupts mitochondrial membrane potential and blocks oxidative phosphorylation in tachyzoites [#2]. It is the molecular target of atovaquone, which binds the ubiquinol-oxidation (Qo) pocket in cooperation with the Rieske iron-sulfur protein, and atovaquone-resistant parasites carry Qo-domain point mutations (M129L, I254L) [#0, #3]. A structurally distinct class of 4(1H)-quinolone diaryl ethers (endochin-like quinolones, ELQs) instead engages the quinone-reduction (Qi) site: a Thr222Pro substitution confers resistance to ELQ-316 with cross-resistance to the Qi inhibitor antimycin, and Qi binding was confirmed co-crystallographically [#4, #5]. Cryo-EM structures of the T. gondii III2-IV supercomplex and isolated complex III2 resolved atovaquone in the Qo site and ELQ-300 in the Qi site with a flipped, parasite-specific binding geometry that explains species-selective inhibition and enabled design of subnanomolar ELQs [#9]. Cytochrome b is highly expressed in bradyzoite cysts, extending its relevance to the latent stage [#5], and it also functions as a node for combination therapy, acting synergistically with type II NADH dehydrogenase inhibition [#10]. Resistance is not solely genetic: parasites can survive atovaquone by shutting down oxidative phosphorylation entirely, independent of cytochrome b mutation [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing what respiratory step atovaquone blocks was needed to define its antiparasitic mechanism; direct assay placed the block within complex III and linked it to loss of membrane potential and ATP synthesis.\",\n      \"evidence\": \"In situ respiration and safranin O membrane-potential assays in digitonin-permeabilized tachyzoites with inhibitor profiling\",\n      \"pmids\": [\"9813002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the specific cytochrome b residues or subsite engaged\", \"No structural resolution of the binding pocket\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identifying the target of decoquinate addressed whether multiple quinolone-class drugs converge on the same protein; a resistance mutation implicated cytochrome b.\",\n      \"evidence\": \"Cytochrome b gene sequencing from a decoquinate-resistant mutant\",\n      \"pmids\": [\"10519259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method (sequencing) without functional reconstitution\", \"Subsite (Qo vs Qi) not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Cloning the gene and mapping atovaquone-resistance mutations to a functional domain established cytochrome b as the atovaquone target and localized the drug to the Qo site.\",\n      \"evidence\": \"Cloning/sequencing of wild-type and resistant strains with Qo-domain structural modeling\",\n      \"pmids\": [\"10802314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental structure of the T. gondii Qo pocket\", \"Mechanism of species selectivity not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Determining how atovaquone selectivity is encoded clarified species-specific inhibition; the drug binds the Qo pocket contacting the Rieske protein, with residue 275 identity governing sensitivity.\",\n      \"evidence\": \"Purified bc1 kinetics, EPR of the Rieske center, redox measurement, and L275F mutagenesis in yeast\",\n      \"pmids\": [\"12791689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in yeast, not T. gondii cytochrome b directly\", \"Does not address Qi-site inhibitors\"],\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Testing combination pharmacology showed cytochrome b inhibition can be potentiated by blocking an upstream ubiquinone step, demonstrating synergistic vulnerability of the respiratory chain.\",\n      \"evidence\": \"In vitro growth inhibition with HDQ + atovaquone and fractional inhibitory concentration analysis\",\n      \"pmids\": [\"17242151\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Synergy inferred from known targets, not direct biochemical dissection of cytochrome b\", \"No structural correlate\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defining a second druggable subsite and stage-specific expression established the Qi site as a target active against latent cysts, broadening therapeutic reach.\",\n      \"evidence\": \"In vitro cyst model (EGS strain), inhibition assays, mutant yeast complementation, and co-crystallography at the bc1 Qi site\",\n      \"pmids\": [\"27412848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cyst-stage IC50 remains high (4 uM)\", \"Qi structural data from surrogate systems, not T. gondii enzyme\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Genetically localizing ELQ-316 action distinguished Qi- from Qo-site inhibitors; a Thr222Pro mutation conferring ELQ and antimycin cross-resistance placed ELQs at the Qi site.\",\n      \"evidence\": \"Drug-pressure selection, cytochrome b sequencing, and cross-resistance quantitation\",\n      \"pmids\": [\"27919897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No direct structure of the T. gondii Qi pocket in this study\", \"Fitness cost of T222P not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Uncovering a non-genetic resistance route revealed that cytochrome b inhibition can be bypassed metabolically; TgMSH1-null parasites survive atovaquone by shutting down oxidative phosphorylation rather than by Qo mutation.\",\n      \"evidence\": \"TgMSH1 knockout, mitochondrial DNA sequencing, electron transport and multi-inhibitor growth assays, glucose deprivation\",\n      \"pmids\": [\"29141004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the OXPHOS-shutdown state undefined\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Cross-species sequence and SAR comparison clarified how quinolone substituents partition between Qo and Qi inhibition, guiding selectivity engineering.\",\n      \"evidence\": \"T. gondii vs P. falciparum cytochrome b sequence comparison and SAR profiling on wild-type and resistant strains\",\n      \"pmids\": [\"30117728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No T. gondii-specific structural data\", \"Selectivity determinants inferred pharmacologically\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Probing artemisinin action implicated the cytochrome b-containing mitochondrion; resistant lines amplified the mitochondrial genome and showed altered membrane potential.\",\n      \"evidence\": \"In vitro resistance selection, whole-genome sequencing, CRISPR mutation introduction, and membrane-potential assays\",\n      \"pmids\": [\"31806760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytochrome b involvement inferred from genome amplification, not direct protein assay\", \"No structural or biochemical link to complex III activity\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Solving the parasite respiratory supercomplex structures resolved the long-standing gap in T. gondii-specific inhibitor geometry, showing atovaquone at Qo and ELQ-300 in a flipped Qi pose that explains selectivity and enabled potency optimization.\",\n      \"evidence\": \"Cryo-EM of III2-IV supercomplex and complex III2, inhibitor co-structures, knockout assembly analysis, and structure-guided medicinal chemistry\",\n      \"pmids\": [\"40389671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy of optimized ELQs not established here\", \"Resistance liabilities of new compounds unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How cytochrome b mutation-independent atovaquone resistance is enacted metabolically, and whether structure-guided ELQs retain activity against Qi-mutant and latent-stage parasites in vivo, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of OXPHOS-shutdown survival undefined\", \"In vivo efficacy against cysts and resistant mutants untested\", \"No T. gondii-specific atovaquone/ELQ cross-resistance structural map beyond the reported poses\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008121\", \"supporting_discovery_ids\": [2, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006122\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"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\": [0, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0022904\", \"supporting_discovery_ids\": [2, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006119\", \"supporting_discovery_ids\": [2, 8], \"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\",\n      \"cytochrome c1\",\n      \"cytochrome c oxidase (complex IV)\"\n    ],\n    \"other_free_text\": [\n      \"Qo (ubiquinol-oxidation) site is the binding pocket for atovaquone and decoquinate; resistance mutations M129L and I254L map here\",\n      \"Qi (quinone-reduction) site is the binding pocket for endochin-like quinolones (ELQ-316, ELQ-300) and antimycin; T222P confers ELQ/antimycin cross-resistance\",\n      \"Highly expressed in bradyzoite cysts, making complex III a target relevant to the latent stage\",\n      \"Mitochondrial genome (including cytochrome b) is amplified in artemisinin-resistant lines\",\n      \"Cytochrome b mutation-independent atovaquone survival occurs via shutdown of oxidative phosphorylation\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"RH88","gene_id":"TGRH88_085380","name":null,"product":"cytochrome b","uniprot_acc":null,"chromosome":"JAAUHK010000041","start_pos":31525}]}