{"gene":"RH88_085470","gene_symbol":"","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2000,"finding":"Cloning and sequencing of the T. gondii cytochrome b gene revealed mutations M129L and I254L in atovaquone-resistant parasites, both located within the ubiquinol-binding pocket (Qo domain), establishing that atovaquone interferes with electron transport at the cytochrome bc1 complex by targeting this site.","method":"Gene cloning, sequencing of wild-type and drug-resistant strains, structural modeling of Qo domain","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct sequencing of resistant mutants combined with structural modeling; cytochrome b identified as first complete mitochondrial gene of T. gondii; replicated conceptually by 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, implicating the cytochrome bc1 complex as the target of decoquinate.","method":"Drug-resistant mutant selection and cytochrome b gene sequencing","journal":"The Journal of eukaryotic microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single study, gene sequencing of resistant mutant, no full-text details available from abstract","pmids":["10519259"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"Atovaquone at nanomolar concentration totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, and was shown to inhibit the respiratory chain between cytochrome b and c1, analogous to antimycin A1 action.","method":"In situ respiration assay with digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, inhibitor pharmacology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biochemical reconstitution in permeabilized parasites with multiple inhibitors and membrane potential readout; first direct evidence of functional respiratory chain in apicomplexan","pmids":["9813002"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Using yeast bc1 complex as a surrogate, atovaquone was shown to bind competitively to the ubiquinol oxidation (Qo) pocket of cytochrome bc1, interacting with the Rieske iron-sulfur protein; a L275F mutation in yeast cytochrome b (equivalent to phenylalanine in bovine) decreased atovaquone sensitivity, confirming that residue 275 (Qo site) governs differential species sensitivity.","method":"Enzymatic inhibition kinetics (Ki determination), EPR spectroscopy of Rieske center, site-directed mutagenesis of cytochrome b, 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; directly informs T. gondii cytochrome b Qo mechanism","pmids":["12791689"],"is_preprint":false,"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2017,"finding":"Selection of T. gondii resistant to ELQ-316 yielded a Thr222Pro substitution in cytochrome b that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor, establishing that ELQ-316 acts at the Qi site of cytochrome bc1 in T. gondii.","method":"Drug-pressure selection of resistant clones, cytochrome b gene sequencing, cross-resistance analysis with known Qi site inhibitor antimycin","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via drug-resistant mutant with specific mutation, cross-resistance to mechanistically defined Qi inhibitor confirms site of action","pmids":["27919897"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Novel 4-(1H)-quinolone scaffolds target the cytochrome bc1 complex Qi site in T. gondii; mutant yeast and co-crystallographic studies demonstrated binding to the bc1 Qi site, confirming the mechanism of action against T. gondii cysts and tachyzoites.","method":"Mutant yeast surrogate system, co-crystallography of compound bound to bc1 Qi site, in vitro inhibition assays against tachyzoites and cysts","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — co-crystal structure plus mutant yeast functional validation, two orthogonal methods confirming Qi site binding","pmids":["27412848"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2018,"finding":"Structure-activity analysis of 4(1H)-quinolones against wild-type and resistant T. gondii strains demonstrated that Qo site inhibitors are the most potent, and that amino acid differences in cytochrome b between T. gondii and P. falciparum (including residues naturally equivalent to P. falciparum resistance mutations) influence differential drug susceptibility.","method":"Inhibition assays against wild-type and drug-resistant parasite strains, comparative cytochrome b sequence analysis, structure-activity relationship studies","journal":"ACS infectious diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple resistant strains tested, comparative sequence analysis, but no direct structural confirmation in T. gondii cytochrome b","pmids":["30117728"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"Artemisinin-resistant T. gondii lines evolved in vitro showed dramatic amplification of the mitochondrial genome including cytochrome b and cytochrome c oxidase I genes; high concentrations of artemisinin decreased mitochondrial membrane potential in wild-type parasites, a phenotype stably altered in resistant lines, implicating the mitochondrion (and by extension cytochrome b-containing complex III) as a target.","method":"In vitro resistance selection, whole-genome sequencing, CRISPR/Cas9 genome editing, 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 — WGS plus CRISPR validation and functional membrane potential assay; cytochrome b amplification is correlative but membrane potential data links mitochondrion functionally","pmids":["31806760"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Knockout of TgMSH1 (mitochondrial MutS homolog) led to accumulation of missense mutations in cytochrome b and reduced mitochondrial DNA content; although none of the cytochrome b mutations affected known atovaquone-sensitivity determinants, atovaquone still inhibited electron transport in these mutants. Subsequent culture under atovaquone pressure produced parasites with complete shutdown of respiration, revealing a novel atovaquone-resistance mechanism independent of cytochrome b Qo mutations.","method":"Genetic knockout of TgMSH1, cytochrome b sequencing, electron transport assay, atovaquone resistance selection","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined phenotype, sequencing of cytochrome b, and functional electron transport assay; single lab","pmids":["29141004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"Cryo-EM structures of T. gondii cytochrome bc1 complex III2 and the III2-IV supercomplex were resolved; atovaquone binding to cytochrome b revealed interactions underpinning species specificity, and ELQ-300 was shown to bind the Qi site of cytochrome b in an orientation flipped relative to its pose in the mammalian enzyme. Knockout of supercomplex assembly showed III2-IV is critical for parasite fitness but challenged the proposed role of supercomplexes in electron transfer efficiency.","method":"Cryo-electron microscopy structure determination, genetic knockout of supercomplex assembly factor, inhibitor co-structure with atovaquone and ELQ-300, parasite fitness assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structures with bound inhibitors, complemented by genetic KO with defined fitness phenotype; multiple orthogonal methods in single rigorous study","pmids":["40389671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"Combined treatment of T. gondii with HDQ (an inhibitor of the alternative NADH dehydrogenase ubiquinol binding site) and atovaquone (which blocks the ubiquinol oxidation site of cytochrome b in complex III) resulted in synergistic inhibition (fractional inhibitory concentration 0.16 nM), demonstrating that cytochrome b in complex III and the alternative NADH dehydrogenase operate in the same ubiquinone/ubiquinol cycle.","method":"Drug combination synergy assay in T. gondii-infected cells, fractional inhibitory concentration calculation","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological synergy assay places cytochrome b functionally in ubiquinone cycle with NADH dehydrogenase; single lab but mechanistically informative","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) that catalyzes ubiquinol oxidation at the Qo site and ubiquinone reduction at the Qi site; atovaquone binds competitively to the Qo pocket (as revealed by cryo-EM and mutant analyses), while endochin-like quinolones can inhibit either the Qo or Qi site (the latter confirmed by a Thr222Pro resistance mutation cross-reactive with antimycin), and mutations at these sites or adaptive mitochondrial genome amplification confer drug resistance; the complex is essential for parasite fitness and participates in a ubiquinone/ubiquinol electron-relay cycle that supports oxidative phosphorylation critical for T. gondii survival."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii cytochrome b is the mitochondrially encoded catalytic subunit of the cytochrome bc1 complex (complex III), where it drives respiratory electron transport between cytochromes b and c1 and maintains mitochondrial membrane potential [PMID:9813002, PMID:10802314]. It functions through the canonical two-site ubiquinone/ubiquinol cycle: a ubiquinol oxidation (Qo) pocket that cooperates with the Rieske iron-sulfur protein, and a ubiquinone reduction (Qi) pocket [PMID:12791689, PMID:27919897]. Cytochrome b operates in the same ubiquinone/ubiquinol redox cycle as the alternative NADH dehydrogenase, as revealed by synergistic inhibition when both entry points are blocked [PMID:17242151]. Cryo-EM of the T. gondii complex III2 and III2-IV supercomplex resolved atovaquone bound in the Qo pocket and ELQ-300 bound at the Qi site in an orientation flipped relative to the mammalian enzyme, defining the structural basis of species-selective inhibition, and knockout of supercomplex assembly established that the complex is critical for parasite fitness [PMID:40389671]. The Qo site is the target of atovaquone, whose resistance mutations (M129L, I254L) and the species-specificity residue 275 map to this pocket [PMID:10802314, PMID:12791689], whereas ELQ-class quinolones can act at either the Qo or the Qi site, the latter confirmed by a Thr222Pro mutation cross-resistant to the Qi inhibitor antimycin [PMID:27919897, PMID:27412848, PMID:30117728].","teleology":[{"year":1998,"claim":"Establishing whether apicomplexans run a functional respiratory chain and where atovaquone acts placed cytochrome b at the heart of parasite mitochondrial respiration.","evidence":"In situ respiration and membrane-potential assays in digitonin-permeabilized tachyzoites with inhibitor pharmacology","pmids":["9813002"],"confidence":"High","gaps":["Localized inhibition between cytochrome b and c1 pharmacologically but did not resolve the specific binding site","No structural detail of the target pocket"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"Sequencing a decoquinate-resistant mutant identified cytochrome b/complex III as the drug target, extending the target range beyond atovaquone.","evidence":"Drug-resistant mutant selection and cytochrome b gene sequencing","pmids":["10519259"],"confidence":"Medium","gaps":["Single study without full mechanistic detail","Binding site of decoquinate not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Cloning the gene and mapping atovaquone-resistance mutations (M129L, I254L) to the Qo pocket defined the ubiquinol oxidation site as the atovaquone-binding target.","evidence":"Gene cloning, sequencing of wild-type and resistant strains, structural modeling of the Qo domain","pmids":["10802314"],"confidence":"High","gaps":["Resistance mapping is correlative; no in vitro binding measurement in T. gondii","Qi site not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Using the yeast bc1 complex as a surrogate resolved that atovaquone binds competitively at the Qo pocket via the Rieske center and that residue 275 governs species-differential sensitivity.","evidence":"Enzyme inhibition kinetics, EPR of the Rieske center, site-directed mutagenesis, structural modeling in isolated yeast bc1","pmids":["12791689"],"confidence":"High","gaps":["Mechanism inferred in yeast, not T. gondii enzyme directly","Does not address Qi-site pharmacology"],"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2007,"claim":"Synergy between an alternative NADH dehydrogenase inhibitor and atovaquone placed cytochrome b in a shared ubiquinone/ubiquinol electron-relay cycle.","evidence":"Drug combination synergy assay with fractional inhibitory concentration calculation in infected cells","pmids":["17242151"],"confidence":"Medium","gaps":["Pharmacological inference, not direct biochemical measurement of electron flux","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Co-crystallography and mutant yeast showed 4-(1H)-quinolones bind the Qi site, establishing a second druggable pocket on cytochrome b active against both tachyzoites and cysts.","evidence":"Mutant yeast surrogate, co-crystal structure at the bc1 Qi site, in vitro inhibition of tachyzoites and cysts","pmids":["27412848"],"confidence":"High","gaps":["Structure obtained in surrogate enzyme, not T. gondii cytochrome b","In vivo efficacy not established here"],"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2017,"claim":"A Thr222Pro mutation conferring cross-resistance to the Qi inhibitor antimycin genetically confirmed that ELQ-316 acts at the Qi site in T. gondii.","evidence":"Drug-pressure selection, cytochrome b sequencing, cross-resistance analysis with antimycin","pmids":["27919897"],"confidence":"High","gaps":["Site assignment inferred from cross-resistance, not direct structural binding in T. gondii","Single resistance mutation"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Knockout of the mitochondrial MutS homolog TgMSH1 revealed cytochrome b mutation accumulation and, under atovaquone pressure, a resistance mechanism operating through complete respiration shutdown independent of Qo mutations.","evidence":"Genetic knockout of TgMSH1, cytochrome b sequencing, electron transport assay, atovaquone resistance selection","pmids":["29141004"],"confidence":"Medium","gaps":["Molecular basis of the Qo-independent resistance mechanism not defined","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Structure-activity profiling across wild-type and resistant strains established Qo inhibitors as the most potent and linked T. gondii/P. falciparum cytochrome b sequence differences to differential drug susceptibility.","evidence":"Inhibition assays against wild-type and resistant strains, comparative cytochrome b sequence analysis, SAR studies","pmids":["30117728"],"confidence":"Medium","gaps":["No direct structural confirmation in T. gondii cytochrome b","Sequence-susceptibility link is correlative"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"Artemisinin-resistant lines showing mitochondrial genome amplification of cytochrome b and altered membrane potential implicated complex III as part of the artemisinin-affected mitochondrial target.","evidence":"In vitro resistance selection, whole-genome sequencing, CRISPR/Cas9 editing, membrane potential measurement","pmids":["31806760"],"confidence":"Medium","gaps":["Cytochrome b amplification is correlative rather than a direct target demonstration","Whether cytochrome b itself mediates artemisinin action is unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Cryo-EM of the T. gondii complex III2 and III2-IV supercomplex directly visualized atovaquone in the Qo pocket and ELQ-300 in a flipped Qi orientation, defining the structural basis of species-selective inhibition and establishing the supercomplex as critical for parasite fitness.","evidence":"Cryo-EM structure determination with bound inhibitors, genetic knockout of a supercomplex assembly factor, fitness assays","pmids":["40389671"],"confidence":"High","gaps":["Challenges but does not resolve the role of supercomplexes in electron transfer efficiency","Catalytic turnover kinetics of the T. gondii enzyme not measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The molecular basis of cytochrome b-independent atovaquone resistance and the functional consequence of mitochondrial genome amplification remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Mechanism of respiration-shutdown resistance in TgMSH1 mutants unknown","Whether cytochrome b amplification directly modulates drug sensitivity untested","Electron-transfer role of the III2-IV supercomplex undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Cytochrome bc1 complex (complex III / III2)","III2-IV respiratory supercomplex"],"partners":["RIESKE IRON-SULFUR PROTEIN","CYTOCHROME C1","ALTERNATIVE NADH DEHYDROGENASE (FUNCTIONAL COUPLING VIA UBIQUINONE/UBIQUINOL POOL)"],"other_free_text":["Qo (ubiquinol oxidation) pocket is the atovaquone-binding site; resistance mutations M129L, I254L and species-specificity residue 275 map here.","Qi (ubiquinone reduction) pocket binds ELQ-class quinolones; Thr222Pro confers cross-resistance to the Qi inhibitor antimycin.","ELQ-300 binds the T. gondii Qi site in an orientation flipped relative to the mammalian enzyme, underpinning species-selective inhibition.","A cytochrome b-independent atovaquone resistance mechanism (respiration shutdown) emerges in TgMSH1 knockouts.","Mitochondrial genome amplification including cytochrome b accompanies in vitro artemisinin resistance."]},"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":3161,"usd":0.045687,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9217,"output_tokens":4465,"usd":0.078855,"stage2_stop_reason":"end_turn"},"total_usd":0.124542,"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\": \"Cloning and sequencing of the T. gondii cytochrome b gene revealed mutations M129L and I254L in atovaquone-resistant parasites, both located within the ubiquinol-binding pocket (Qo domain), establishing that atovaquone interferes with electron transport at the cytochrome bc1 complex by targeting this site.\",\n      \"method\": \"Gene cloning, sequencing of wild-type and drug-resistant strains, structural modeling of Qo domain\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct sequencing of resistant mutants combined with structural modeling; cytochrome b identified as first complete mitochondrial gene of T. gondii; replicated conceptually by 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, implicating the cytochrome bc1 complex as the target of decoquinate.\",\n      \"method\": \"Drug-resistant mutant selection and cytochrome b gene sequencing\",\n      \"journal\": \"The Journal of eukaryotic microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single study, gene sequencing of resistant mutant, no full-text details available from abstract\",\n      \"pmids\": [\"10519259\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Atovaquone at nanomolar concentration totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, and was shown to inhibit the respiratory chain between cytochrome b and c1, analogous to antimycin A1 action.\",\n      \"method\": \"In situ respiration assay with digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, inhibitor pharmacology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical reconstitution in permeabilized parasites with multiple inhibitors and membrane potential readout; first direct evidence of functional respiratory chain in apicomplexan\",\n      \"pmids\": [\"9813002\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Using yeast bc1 complex as a surrogate, atovaquone was shown to bind competitively to the ubiquinol oxidation (Qo) pocket of cytochrome bc1, interacting with the Rieske iron-sulfur protein; a L275F mutation in yeast cytochrome b (equivalent to phenylalanine in bovine) decreased atovaquone sensitivity, confirming that residue 275 (Qo site) governs differential species sensitivity.\",\n      \"method\": \"Enzymatic inhibition kinetics (Ki determination), EPR spectroscopy of Rieske center, site-directed mutagenesis of cytochrome b, 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; directly informs T. gondii cytochrome b Qo mechanism\",\n      \"pmids\": [\"12791689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Selection of T. gondii resistant to ELQ-316 yielded a Thr222Pro substitution in cytochrome b that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor, establishing that ELQ-316 acts at the Qi site of cytochrome bc1 in T. gondii.\",\n      \"method\": \"Drug-pressure selection of resistant clones, cytochrome b gene sequencing, cross-resistance analysis with known Qi site inhibitor antimycin\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via drug-resistant mutant with specific mutation, cross-resistance to mechanistically defined Qi inhibitor confirms site of action\",\n      \"pmids\": [\"27919897\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Novel 4-(1H)-quinolone scaffolds target the cytochrome bc1 complex Qi site in T. gondii; mutant yeast and co-crystallographic studies demonstrated binding to the bc1 Qi site, confirming the mechanism of action against T. gondii cysts and tachyzoites.\",\n      \"method\": \"Mutant yeast surrogate system, co-crystallography of compound bound to bc1 Qi site, in vitro inhibition assays against tachyzoites and cysts\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — co-crystal structure plus mutant yeast functional validation, two orthogonal methods confirming Qi site binding\",\n      \"pmids\": [\"27412848\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Structure-activity analysis of 4(1H)-quinolones against wild-type and resistant T. gondii strains demonstrated that Qo site inhibitors are the most potent, and that amino acid differences in cytochrome b between T. gondii and P. falciparum (including residues naturally equivalent to P. falciparum resistance mutations) influence differential drug susceptibility.\",\n      \"method\": \"Inhibition assays against wild-type and drug-resistant parasite strains, comparative cytochrome b sequence analysis, structure-activity relationship studies\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple resistant strains tested, comparative sequence analysis, but no direct structural confirmation in T. gondii cytochrome b\",\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 evolved in vitro showed dramatic amplification of the mitochondrial genome including cytochrome b and cytochrome c oxidase I genes; high concentrations of artemisinin decreased mitochondrial membrane potential in wild-type parasites, a phenotype stably altered in resistant lines, implicating the mitochondrion (and by extension cytochrome b-containing complex III) as a target.\",\n      \"method\": \"In vitro resistance selection, whole-genome sequencing, CRISPR/Cas9 genome editing, 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 — WGS plus CRISPR validation and functional membrane potential assay; cytochrome b amplification is correlative but membrane potential data links mitochondrion functionally\",\n      \"pmids\": [\"31806760\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Knockout of TgMSH1 (mitochondrial MutS homolog) led to accumulation of missense mutations in cytochrome b and reduced mitochondrial DNA content; although none of the cytochrome b mutations affected known atovaquone-sensitivity determinants, atovaquone still inhibited electron transport in these mutants. Subsequent culture under atovaquone pressure produced parasites with complete shutdown of respiration, revealing a novel atovaquone-resistance mechanism independent of cytochrome b Qo mutations.\",\n      \"method\": \"Genetic knockout of TgMSH1, cytochrome b sequencing, electron transport assay, atovaquone resistance selection\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined phenotype, sequencing of cytochrome b, and functional electron transport assay; single lab\",\n      \"pmids\": [\"29141004\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of T. gondii cytochrome bc1 complex III2 and the III2-IV supercomplex were resolved; atovaquone binding to cytochrome b revealed interactions underpinning species specificity, and ELQ-300 was shown to bind the Qi site of cytochrome b in an orientation flipped relative to its pose in the mammalian enzyme. Knockout of supercomplex assembly showed III2-IV is critical for parasite fitness but challenged the proposed role of supercomplexes in electron transfer efficiency.\",\n      \"method\": \"Cryo-electron microscopy structure determination, genetic knockout of supercomplex assembly factor, inhibitor co-structure with atovaquone and ELQ-300, parasite fitness assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structures with bound inhibitors, complemented by genetic KO with defined fitness phenotype; multiple orthogonal methods in 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 (an inhibitor of the alternative NADH dehydrogenase ubiquinol binding site) and atovaquone (which blocks the ubiquinol oxidation site of cytochrome b in complex III) resulted in synergistic inhibition (fractional inhibitory concentration 0.16 nM), demonstrating that cytochrome b in complex III and the alternative NADH dehydrogenase operate in the same ubiquinone/ubiquinol cycle.\",\n      \"method\": \"Drug combination synergy assay in T. gondii-infected cells, fractional inhibitory concentration calculation\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological synergy assay places cytochrome b functionally in ubiquinone cycle with NADH dehydrogenase; single lab but mechanistically informative\",\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) that catalyzes ubiquinol oxidation at the Qo site and ubiquinone reduction at the Qi site; atovaquone binds competitively to the Qo pocket (as revealed by cryo-EM and mutant analyses), while endochin-like quinolones can inhibit either the Qo or Qi site (the latter confirmed by a Thr222Pro resistance mutation cross-reactive with antimycin), and mutations at these sites or adaptive mitochondrial genome amplification confer drug resistance; the complex is essential for parasite fitness and participates in a ubiquinone/ubiquinol electron-relay cycle that supports oxidative phosphorylation critical for T. gondii survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Toxoplasma gondii cytochrome b is the mitochondrially encoded catalytic subunit of the cytochrome bc1 complex (complex III), where it drives respiratory electron transport between cytochromes b and c1 and maintains mitochondrial membrane potential [#2, #0]. It functions through the canonical two-site ubiquinone/ubiquinol cycle: a ubiquinol oxidation (Qo) pocket that cooperates with the Rieske iron-sulfur protein, and a ubiquinone reduction (Qi) pocket [#3, #4]. Cytochrome b operates in the same ubiquinone/ubiquinol redox cycle as the alternative NADH dehydrogenase, as revealed by synergistic inhibition when both entry points are blocked [#10]. Cryo-EM of the T. gondii complex III2 and III2-IV supercomplex resolved atovaquone bound in the Qo pocket and ELQ-300 bound at the Qi site in an orientation flipped relative to the mammalian enzyme, defining the structural basis of species-selective inhibition, and knockout of supercomplex assembly established that the complex is critical for parasite fitness [#9]. The Qo site is the target of atovaquone, whose resistance mutations (M129L, I254L) and the species-specificity residue 275 map to this pocket [#0, #3], whereas ELQ-class quinolones can act at either the Qo or the Qi site, the latter confirmed by a Thr222Pro mutation cross-resistant to the Qi inhibitor antimycin [#4, #5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing whether apicomplexans run a functional respiratory chain and where atovaquone acts placed cytochrome b at the heart of parasite mitochondrial respiration.\",\n      \"evidence\": \"In situ respiration and membrane-potential assays in digitonin-permeabilized tachyzoites with inhibitor pharmacology\",\n      \"pmids\": [\"9813002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Localized inhibition between cytochrome b and c1 pharmacologically but did not resolve the specific binding site\", \"No structural detail of the target pocket\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Sequencing a decoquinate-resistant mutant identified cytochrome b/complex III as the drug target, extending the target range beyond atovaquone.\",\n      \"evidence\": \"Drug-resistant mutant selection and cytochrome b gene sequencing\",\n      \"pmids\": [\"10519259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study without full mechanistic detail\", \"Binding site of decoquinate not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Cloning the gene and mapping atovaquone-resistance mutations (M129L, I254L) to the Qo pocket defined the ubiquinol oxidation site as the atovaquone-binding target.\",\n      \"evidence\": \"Gene cloning, sequencing of wild-type and resistant strains, structural modeling of the Qo domain\",\n      \"pmids\": [\"10802314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Resistance mapping is correlative; no in vitro binding measurement in T. gondii\", \"Qi site not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Using the yeast bc1 complex as a surrogate resolved that atovaquone binds competitively at the Qo pocket via the Rieske center and that residue 275 governs species-differential sensitivity.\",\n      \"evidence\": \"Enzyme inhibition kinetics, EPR of the Rieske center, site-directed mutagenesis, structural modeling in isolated yeast bc1\",\n      \"pmids\": [\"12791689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism inferred in yeast, not T. gondii enzyme directly\", \"Does not address Qi-site pharmacology\"],\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Synergy between an alternative NADH dehydrogenase inhibitor and atovaquone placed cytochrome b in a shared ubiquinone/ubiquinol electron-relay cycle.\",\n      \"evidence\": \"Drug combination synergy assay with fractional inhibitory concentration calculation in infected cells\",\n      \"pmids\": [\"17242151\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pharmacological inference, not direct biochemical measurement of electron flux\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Co-crystallography and mutant yeast showed 4-(1H)-quinolones bind the Qi site, establishing a second druggable pocket on cytochrome b active against both tachyzoites and cysts.\",\n      \"evidence\": \"Mutant yeast surrogate, co-crystal structure at the bc1 Qi site, in vitro inhibition of tachyzoites and cysts\",\n      \"pmids\": [\"27412848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure obtained in surrogate enzyme, not T. gondii cytochrome b\", \"In vivo efficacy not established here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A Thr222Pro mutation conferring cross-resistance to the Qi inhibitor antimycin genetically confirmed that ELQ-316 acts at the Qi site in T. gondii.\",\n      \"evidence\": \"Drug-pressure selection, cytochrome b sequencing, cross-resistance analysis with antimycin\",\n      \"pmids\": [\"27919897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Site assignment inferred from cross-resistance, not direct structural binding in T. gondii\", \"Single resistance mutation\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Knockout of the mitochondrial MutS homolog TgMSH1 revealed cytochrome b mutation accumulation and, under atovaquone pressure, a resistance mechanism operating through complete respiration shutdown independent of Qo mutations.\",\n      \"evidence\": \"Genetic knockout of TgMSH1, cytochrome b sequencing, electron transport assay, atovaquone resistance selection\",\n      \"pmids\": [\"29141004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the Qo-independent resistance mechanism not defined\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Structure-activity profiling across wild-type and resistant strains established Qo inhibitors as the most potent and linked T. gondii/P. falciparum cytochrome b sequence differences to differential drug susceptibility.\",\n      \"evidence\": \"Inhibition assays against wild-type and resistant strains, comparative cytochrome b sequence analysis, SAR studies\",\n      \"pmids\": [\"30117728\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct structural confirmation in T. gondii cytochrome b\", \"Sequence-susceptibility link is correlative\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Artemisinin-resistant lines showing mitochondrial genome amplification of cytochrome b and altered membrane potential implicated complex III as part of the artemisinin-affected mitochondrial target.\",\n      \"evidence\": \"In vitro resistance selection, whole-genome sequencing, CRISPR/Cas9 editing, membrane potential measurement\",\n      \"pmids\": [\"31806760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytochrome b amplification is correlative rather than a direct target demonstration\", \"Whether cytochrome b itself mediates artemisinin action is unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM of the T. gondii complex III2 and III2-IV supercomplex directly visualized atovaquone in the Qo pocket and ELQ-300 in a flipped Qi orientation, defining the structural basis of species-selective inhibition and establishing the supercomplex as critical for parasite fitness.\",\n      \"evidence\": \"Cryo-EM structure determination with bound inhibitors, genetic knockout of a supercomplex assembly factor, fitness assays\",\n      \"pmids\": [\"40389671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Challenges but does not resolve the role of supercomplexes in electron transfer efficiency\", \"Catalytic turnover kinetics of the T. gondii enzyme not measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular basis of cytochrome b-independent atovaquone resistance and the functional consequence of mitochondrial genome amplification remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of respiration-shutdown resistance in TgMSH1 mutants unknown\", \"Whether cytochrome b amplification directly modulates drug sensitivity untested\", \"Electron-transfer role of the III2-IV supercomplex undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008121\", \"supporting_discovery_ids\": [2, 3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006122\", \"supporting_discovery_ids\": [2, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005750\", \"supporting_discovery_ids\": [9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [2, 7], \"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, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Cytochrome bc1 complex (complex III / III2)\",\n      \"III2-IV respiratory supercomplex\"\n    ],\n    \"partners\": [\n      \"Rieske iron-sulfur protein\",\n      \"cytochrome c1\",\n      \"alternative NADH dehydrogenase (functional coupling via ubiquinone/ubiquinol pool)\"\n    ],\n    \"other_free_text\": [\n      \"Qo (ubiquinol oxidation) pocket is the atovaquone-binding site; resistance mutations M129L, I254L and species-specificity residue 275 map here.\",\n      \"Qi (ubiquinone reduction) pocket binds ELQ-class quinolones; Thr222Pro confers cross-resistance to the Qi inhibitor antimycin.\",\n      \"ELQ-300 binds the T. gondii Qi site in an orientation flipped relative to the mammalian enzyme, underpinning species-selective inhibition.\",\n      \"A cytochrome b-independent atovaquone resistance mechanism (respiration shutdown) emerges in TgMSH1 knockouts.\",\n      \"Mitochondrial genome amplification including cytochrome b accompanies in vitro artemisinin resistance.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"RH88","gene_id":"TGRH88_085470","name":null,"product":"cytochrome b","uniprot_acc":null,"chromosome":"JAAUHK010000041","start_pos":11797}]}