{"gene":"RH88_085360","gene_symbol":"","run_date":"2026-09-07T10:25:42","timeline":{"discoveries":[{"year":2000,"finding":"Cytochrome b of T. gondii was cloned and sequenced; drug-resistant mutants selected under atovaquone pressure carried mutations M129L and I254L within the Qo (ubiquinol-binding) domain of cytochrome b, indicating that atovaquone inhibits electron transport by targeting the cytochrome bc1 complex Qo site. Structural modelling suggested the protein may terminate within the mitochondrial membrane, differing from other homologues.","method":"Gene cloning and sequencing of wild-type and atovaquone-resistant strains; structural modelling of the Qo domain","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct cloning, sequencing of resistance mutants, and structural modelling; foundational paper establishing Qo-site mechanism, later independently confirmed","pmids":["10802314"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"finding":"A mutation in cytochrome b was identified in a decoquinate-resistant mutant of T. gondii, providing genetic evidence that cytochrome b is the target of decoquinate at the cytochrome bc1 complex.","method":"Selection of drug-resistant mutants and sequencing of the cytochrome b gene","journal":"The Journal of eukaryotic microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, genetic identification of resistance mutation, but abstract detail is limited","pmids":["10519259"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1998,"finding":"Atovaquone at nanomolar concentrations totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, and the site of inhibition was demonstrated biochemically to be between cytochrome b and cytochrome c1 (i.e., at the bc1 complex), analogous to the action of antimycin A1.","method":"In situ respirometry with digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, use of selective respiratory chain inhibitors and substrates","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical inhibition assay with multiple substrates and inhibitors, first direct biochemical demonstration of functional respiratory chain in apicomplexans","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 and interacts with the Rieske iron-sulfur protein; the L275F substitution in cytochrome b (equivalent position) reduces atovaquone sensitivity, establishing the molecular basis for differential inhibition. These findings from yeast/bovine surrogates inform the mechanism relevant to T. gondii cytochrome b.","method":"Inhibition kinetics of isolated bc1 complex, EPR spectroscopy of Rieske center, site-directed mutagenesis (L275F) in yeast cytochrome b, energy-minimized structural modelling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified complex, mutagenesis, EPR, and structural modelling; mechanistically informs T. gondii cytochrome b Qo site","pmids":["12791689"],"is_preprint":false,"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2017,"finding":"A Thr222Pro amino acid 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 inhibits the Qi site of the cytochrome bc1 complex in T. gondii.","method":"Selection of drug-resistant clones under ELQ-316 pressure; sequencing of cytochrome b gene; cross-resistance testing with antimycin","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic resistance mapping with cross-resistance validation to a reference Qi inhibitor, directly attributing ELQ-316 mechanism to the Qi site of T. gondii cytochrome bc1","pmids":["27919897"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Novel 4-(1H)-quinolone scaffolds targeting the cytochrome bc1 complex Qi site inhibit T. gondii active infection and cysts in vitro and in vivo; mutant yeast and co-crystallographic studies demonstrate compound binding to the bc1 complex Qi site, establishing Qi-site inhibition as a mechanism against T. gondii.","method":"Mutant yeast bc1 complex assays, co-crystallography, in vitro and in vivo T. gondii infection models","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — co-crystal structure and mutant yeast enzymatic assay in a single study, directly demonstrating Qi site binding","pmids":["27412848"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2025,"finding":"Cryo-EM structures of the T. gondii III2-IV respiratory chain supercomplex and of complex III2 (containing cytochrome b) revealed an apicomplexan-conserved III2-IV interface with a unique, kinked architecture. Atovaquone was shown to bind the Qo site of T. gondii cytochrome b with species-specific interactions. ELQ-300 binds the Qi site of apicomplexan cytochrome b in a flipped orientation relative to its binding in the mammalian enzyme. Knockout of a supercomplex subunit disrupts III2-IV assembly but atovaquone and ELQ-300 still bind complex III2, and structure-guided design produced more potent ELQs with subnanomolar activity against T. gondii.","method":"Cryo-electron microscopy structure determination; knockout analysis; inhibitor co-structure determination; compound potency testing","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures with inhibitor co-complexes, knockout validation, and structure-guided drug design, multiple orthogonal methods in one rigorous study","pmids":["40389671"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"T. gondii lines resistant to artemisinin showed dramatic amplification of the mitochondrial genome including the cytochrome b gene, and wild-type parasites treated with high concentrations of artemisinin showed decreased mitochondrial membrane potential, suggesting the mitochondrion (including cytochrome b-containing complex III) is a target of artemisinin action.","method":"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 point mutation introduction, mitochondrial membrane potential measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — mitochondrial genome amplification and membrane potential data from a single lab; cytochrome b involvement is inferred from amplification rather than direct functional assay of cytochrome b specifically","pmids":["31806760"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"Parasites lacking TgMSH1 accumulate single nucleotide variations in mitochondrial DNA including missense mutations in cytochrome b; however, none of these mutations affect amino acids known to determine atovaquone sensitivity, and atovaquone still inhibits electron transport in these mutants. Under continued atovaquone pressure, Tgmsh1 knockouts develop enhanced resistance by completely shutting down respiration (metabolic plasticity), rather than through cytochrome b Qo mutations.","method":"Tgmsh1 knockout generation, mitochondrial DNA sequencing, atovaquone sensitivity assays, respiration measurements, glucose-dependence assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout with multiple orthogonal readouts (sequencing, respiration, drug sensitivity); establishes that cytochrome b Qo mutations are not required for atovaquone resistance when oxidative phosphorylation is bypassed","pmids":["29141004"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2007,"finding":"Combined treatment of T. gondii-infected cells 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 of parasite replication (fractional inhibitory concentration of 0.16 nM), demonstrating that simultaneous interference at two distinct sites in the mitochondrial ubiquinone/ubiquinol cycle is highly effective.","method":"In vitro T. gondii growth assay, drug combination analysis (fractional inhibitory concentration calculation)","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, cell-based combination assay confirming cytochrome b Qo site as atovaquone target through synergy with upstream inhibitor","pmids":["17242151"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) whose Qo (ubiquinol oxidation) and Qi (quinone reduction) sites are the direct targets of clinically used and preclinical antiparasitic drugs: atovaquone binds the Qo site (confirmed by cryo-EM co-structure, resistance mutations M129L/I254L, and biochemical inhibition data), while endochin-like quinolones such as ELQ-316 and ELQ-300 bind the Qi site (confirmed by resistance mutation T222P, cross-resistance with antimycin, and cryo-EM co-structure showing an apicomplexan-specific flipped binding orientation), and disruption of either site collapses the mitochondrial membrane potential and blocks oxidative phosphorylation essential for parasite survival."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) that carries out ubiquinol oxidation and quinone reduction, driving the respiratory electron transport chain and maintenance of the mitochondrial membrane potential essential for parasite survival [PMID:10802314, PMID:9813002]. Biochemical respirometry localized the site of atovaquone inhibition to the segment between cytochrome b and cytochrome c1 within the bc1 complex, where nanomolar atovaquone collapses respiration and membrane potential [PMID:9813002]. Two distinct catalytic sites of cytochrome b are pharmacologically targetable: the Qo (ubiquinol oxidation) pocket, where atovaquone binds — defined by resistance mutations M129L and I254L and confirmed by cryo-EM co-structure [PMID:10802314, PMID:40389671] — and the Qi (quinone reduction) site, where endochin-like quinolones such as ELQ-316 and ELQ-300 bind, defined by the T222P resistance mutation with cross-resistance to the reference Qi inhibitor antimycin and by a cryo-EM co-structure showing an apicomplexan-specific flipped ELQ-300 orientation [PMID:27919897, PMID:40389671]. Cryo-EM further resolved cytochrome b within a III2-IV supercomplex with a uniquely kinked apicomplexan interface, and knockout of a supercomplex subunit disrupts III2-IV assembly without abolishing inhibitor binding to complex III2 [PMID:40389671]. Cytochrome b is additionally the identified target of decoquinate through a resistance mutation [PMID:10519259], and simultaneous blockade of the Qo site with atovaquone and of an upstream ubiquinol-binding site with HDQ produces strong synergy, underscoring the centrality of the ubiquinone/ubiquinol cycle [PMID:17242151].","teleology":[{"year":1998,"claim":"Establishing whether apicomplexan parasites possess a functional respiratory chain and where atovaquone acts answered the basic question of the drug's molecular target within mitochondrial electron transport.","evidence":"In situ respirometry and safranin O membrane potential measurement in digitonin-permeabilized T. gondii tachyzoites with selective inhibitors and substrates","pmids":["9813002"],"confidence":"High","gaps":["Did not identify the specific residues or binding pocket engaged by atovaquone","Did not distinguish Qo from Qi involvement structurally"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":1999,"claim":"Genetic mapping of a decoquinate-resistance mutation to cytochrome b established cytochrome b as a drug target at the bc1 complex beyond atovaquone.","evidence":"Selection of decoquinate-resistant mutants and sequencing of the cytochrome b gene in T. gondii","pmids":["10519259"],"confidence":"Medium","gaps":["Limited detail on the specific mutation and binding site","No biochemical confirmation of decoquinate-cytochrome b interaction"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2000,"claim":"Cloning the cytochrome b gene and mapping atovaquone-resistance mutations to the Qo domain pinpointed the ubiquinol oxidation site as the atovaquone binding region, moving from a functional segment to specific residues.","evidence":"Gene cloning and sequencing of wild-type and atovaquone-resistant T. gondii strains with structural modelling of the Qo domain (M129L, I254L)","pmids":["10802314"],"confidence":"High","gaps":["Modelling only — no experimental structure of the atovaquone-bound complex","Did not address the Qi site or other inhibitors"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Surrogate biochemistry defined how atovaquone occupies the Qo pocket and interacts with the Rieske iron-sulfur protein, providing molecular mechanism informing the T. gondii Qo site.","evidence":"Inhibition kinetics of isolated bc1 complex, EPR of the Rieske center, and L275F mutagenesis in yeast cytochrome b","pmids":["12791689"],"confidence":"High","gaps":["Derived from yeast/bovine surrogates, not T. gondii enzyme directly","Species-specific differences in binding not resolved"],"attribution":[{"species":"Saccharomyces cerevisiae","strain":""}]},{"year":2007,"claim":"Demonstrating synergy between atovaquone (Qo blockade) and HDQ (upstream ubiquinol-binding inhibition) confirmed the central role of the ubiquinone/ubiquinol cycle and validated combined-site targeting.","evidence":"In vitro T. gondii growth assay with fractional inhibitory concentration analysis of the atovaquone-HDQ combination","pmids":["17242151"],"confidence":"Medium","gaps":["Cell-based only, no direct enzymatic measurement of dual inhibition","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Identifying 4-(1H)-quinolone scaffolds that bind the Qi site opened a second, distinct pharmacological site on cytochrome b for anti-Toxoplasma action.","evidence":"Mutant yeast bc1 assays, co-crystallography, and in vitro/in vivo T. gondii infection models","pmids":["27412848"],"confidence":"High","gaps":["Co-crystal structure was in surrogate rather than T. gondii enzyme","Did not map T. gondii-specific Qi resistance residues"],"attribution":[{"species":"Toxoplasma gondii","strain":"EGS"}]},{"year":2017,"claim":"Mapping the T222P resistance mutation with antimycin cross-resistance provided direct genetic proof that ELQ-316 inhibits the Qi site of T. gondii cytochrome b, distinguishing it mechanistically from Qo-site atovaquone.","evidence":"Selection of ELQ-316-resistant clones, cytochrome b sequencing, and cross-resistance testing against the reference Qi inhibitor antimycin","pmids":["27919897"],"confidence":"High","gaps":["No structural depiction of the ELQ-316-Qi complex at this stage","Fitness cost of T222P not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"The TgMSH1 knockout showed that atovaquone resistance can arise via metabolic shutdown of respiration rather than cytochrome b Qo mutation, clarifying that Qo mutations are not the only resistance route.","evidence":"Tgmsh1 knockout with mtDNA sequencing, respiration measurements, glucose-dependence and atovaquone sensitivity assays","pmids":["29141004"],"confidence":"Medium","gaps":["Accumulated cytochrome b missense mutations did not alter atovaquone-relevant residues","Metabolic bypass mechanism not fully dissected"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Amplification of the cytochrome b-containing mitochondrial genome in artemisinin-resistant lines and loss of membrane potential on treatment implicated complex III in artemisinin action, extending cytochrome b's relevance beyond bc1-specific inhibitors.","evidence":"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 point mutation, and membrane potential measurement","pmids":["31806760"],"confidence":"Medium","gaps":["Cytochrome b involvement inferred from genome amplification, not a direct functional assay of cytochrome b","Precise artemisinin target within the mitochondrion unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Cryo-EM structures of the III2-IV supercomplex and complex III2 with bound inhibitors resolved atomic-level, species-specific atovaquone Qo binding and a flipped apicomplexan ELQ-300 Qi orientation, converting inferred mechanisms into direct structural models and enabling structure-guided drug design.","evidence":"Cryo-EM structure determination with inhibitor co-complexes, supercomplex-subunit knockout, and potency testing of new ELQs","pmids":["40389671"],"confidence":"High","gaps":["In vivo efficacy and resistance profiles of the new subnanomolar ELQs not yet defined","Functional consequence of the unique III2-IV kinked interface not fully explained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How cytochrome b sequence and structural divergence across apicomplexan species and strains shapes clinical resistance and selectivity of Qo/Qi inhibitors remains to be systematically resolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No comprehensive map of Qo/Qi resistance-mutation fitness landscapes","Selectivity margin versus mammalian cytochrome b not fully quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Cytochrome bc1 complex (complex III2)","III2-IV respiratory chain supercomplex"],"partners":["CYTOCHROME C1","RIESKE IRON-SULFUR PROTEIN"],"other_free_text":["Qo (ubiquinol oxidation) site is the atovaquone binding pocket; resistance mutations M129L, I254L [#0]","Qi (quinone reduction) site is the endochin-like quinolone (ELQ-316/ELQ-300) binding pocket; resistance mutation T222P with antimycin cross-resistance [#4]","ELQ-300 binds the apicomplexan Qi site in a flipped orientation relative to the mammalian enzyme [#6]","Atovaquone resistance can also arise via metabolic shutdown of respiration independent of cytochrome b Qo mutations [#8]"]},"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, 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cytochrome bc1 complex Qo site. Structural modelling suggested the protein may terminate within the mitochondrial membrane, differing from other homologues.\",\n      \"method\": \"Gene cloning and sequencing of wild-type and atovaquone-resistant strains; structural modelling of the Qo domain\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct cloning, sequencing of resistance mutants, and structural modelling; foundational paper establishing Qo-site mechanism, later independently confirmed\",\n      \"pmids\": [\"10802314\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"A mutation in cytochrome b was identified in a decoquinate-resistant mutant of T. gondii, providing genetic evidence that cytochrome b is the target of decoquinate at the cytochrome bc1 complex.\",\n      \"method\": \"Selection of drug-resistant mutants and sequencing of the cytochrome b gene\",\n      \"journal\": \"The Journal of eukaryotic microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, genetic identification of resistance mutation, but abstract detail is limited\",\n      \"pmids\": [\"10519259\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Atovaquone at nanomolar concentrations totally inhibited respiration and disrupted mitochondrial membrane potential in T. gondii tachyzoites, and the site of inhibition was demonstrated biochemically to be between cytochrome b and cytochrome c1 (i.e., at the bc1 complex), analogous to the action of antimycin A1.\",\n      \"method\": \"In situ respirometry with digitonin-permeabilized tachyzoites, mitochondrial membrane potential measurement with safranin O, use of selective respiratory chain inhibitors and substrates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical inhibition assay with multiple substrates and inhibitors, first direct biochemical demonstration of functional respiratory chain in apicomplexans\",\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 and interacts with the Rieske iron-sulfur protein; the L275F substitution in cytochrome b (equivalent position) reduces atovaquone sensitivity, establishing the molecular basis for differential inhibition. These findings from yeast/bovine surrogates inform the mechanism relevant to T. gondii cytochrome b.\",\n      \"method\": \"Inhibition kinetics of isolated bc1 complex, EPR spectroscopy of Rieske center, site-directed mutagenesis (L275F) in yeast cytochrome b, energy-minimized structural modelling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified complex, mutagenesis, EPR, and structural modelling; mechanistically informs T. gondii cytochrome b Qo site\",\n      \"pmids\": [\"12791689\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A Thr222Pro amino acid 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 inhibits the Qi site of the cytochrome bc1 complex in T. gondii.\",\n      \"method\": \"Selection of drug-resistant clones under ELQ-316 pressure; sequencing of cytochrome b gene; cross-resistance testing with antimycin\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic resistance mapping with cross-resistance validation to a reference Qi inhibitor, directly attributing ELQ-316 mechanism to the Qi site of T. gondii cytochrome bc1\",\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 targeting the cytochrome bc1 complex Qi site inhibit T. gondii active infection and cysts in vitro and in vivo; mutant yeast and co-crystallographic studies demonstrate compound binding to the bc1 complex Qi site, establishing Qi-site inhibition as a mechanism against T. gondii.\",\n      \"method\": \"Mutant yeast bc1 complex assays, co-crystallography, in vitro and in vivo T. gondii infection models\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — co-crystal structure and mutant yeast enzymatic assay in a single study, directly demonstrating Qi site binding\",\n      \"pmids\": [\"27412848\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of the T. gondii III2-IV respiratory chain supercomplex and of complex III2 (containing cytochrome b) revealed an apicomplexan-conserved III2-IV interface with a unique, kinked architecture. Atovaquone was shown to bind the Qo site of T. gondii cytochrome b with species-specific interactions. ELQ-300 binds the Qi site of apicomplexan cytochrome b in a flipped orientation relative to its binding in the mammalian enzyme. Knockout of a supercomplex subunit disrupts III2-IV assembly but atovaquone and ELQ-300 still bind complex III2, and structure-guided design produced more potent ELQs with subnanomolar activity against T. gondii.\",\n      \"method\": \"Cryo-electron microscopy structure determination; knockout analysis; inhibitor co-structure determination; compound potency testing\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures with inhibitor co-complexes, knockout validation, and structure-guided drug design, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"40389671\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"T. gondii lines resistant to artemisinin showed dramatic amplification of the mitochondrial genome including the cytochrome b gene, and wild-type parasites treated with high concentrations of artemisinin showed decreased mitochondrial membrane potential, suggesting the mitochondrion (including cytochrome b-containing complex III) is a target of artemisinin action.\",\n      \"method\": \"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 point mutation introduction, 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 / Weak — mitochondrial genome amplification and membrane potential data from a single lab; cytochrome b involvement is inferred from amplification rather than direct functional assay of cytochrome b specifically\",\n      \"pmids\": [\"31806760\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Parasites lacking TgMSH1 accumulate single nucleotide variations in mitochondrial DNA including missense mutations in cytochrome b; however, none of these mutations affect amino acids known to determine atovaquone sensitivity, and atovaquone still inhibits electron transport in these mutants. Under continued atovaquone pressure, Tgmsh1 knockouts develop enhanced resistance by completely shutting down respiration (metabolic plasticity), rather than through cytochrome b Qo mutations.\",\n      \"method\": \"Tgmsh1 knockout generation, mitochondrial DNA sequencing, atovaquone sensitivity assays, respiration measurements, glucose-dependence assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout with multiple orthogonal readouts (sequencing, respiration, drug sensitivity); establishes that cytochrome b Qo mutations are not required for atovaquone resistance when oxidative phosphorylation is bypassed\",\n      \"pmids\": [\"29141004\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Combined treatment of T. gondii-infected cells 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 of parasite replication (fractional inhibitory concentration of 0.16 nM), demonstrating that simultaneous interference at two distinct sites in the mitochondrial ubiquinone/ubiquinol cycle is highly effective.\",\n      \"method\": \"In vitro T. gondii growth assay, drug combination analysis (fractional inhibitory concentration calculation)\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, cell-based combination assay confirming cytochrome b Qo site as atovaquone target through synergy with upstream inhibitor\",\n      \"pmids\": [\"17242151\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) whose Qo (ubiquinol oxidation) and Qi (quinone reduction) sites are the direct targets of clinically used and preclinical antiparasitic drugs: atovaquone binds the Qo site (confirmed by cryo-EM co-structure, resistance mutations M129L/I254L, and biochemical inhibition data), while endochin-like quinolones such as ELQ-316 and ELQ-300 bind the Qi site (confirmed by resistance mutation T222P, cross-resistance with antimycin, and cryo-EM co-structure showing an apicomplexan-specific flipped binding orientation), and disruption of either site collapses the mitochondrial membrane potential and blocks oxidative phosphorylation essential for parasite survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Toxoplasma gondii cytochrome b is a mitochondrially encoded subunit of the cytochrome bc1 complex (complex III) that carries out ubiquinol oxidation and quinone reduction, driving the respiratory electron transport chain and maintenance of the mitochondrial membrane potential essential for parasite survival [#0, #2]. Biochemical respirometry localized the site of atovaquone inhibition to the segment between cytochrome b and cytochrome c1 within the bc1 complex, where nanomolar atovaquone collapses respiration and membrane potential [#2]. Two distinct catalytic sites of cytochrome b are pharmacologically targetable: the Qo (ubiquinol oxidation) pocket, where atovaquone binds \\u2014 defined by resistance mutations M129L and I254L and confirmed by cryo-EM co-structure [#0, #6] \\u2014 and the Qi (quinone reduction) site, where endochin-like quinolones such as ELQ-316 and ELQ-300 bind, defined by the T222P resistance mutation with cross-resistance to the reference Qi inhibitor antimycin and by a cryo-EM co-structure showing an apicomplexan-specific flipped ELQ-300 orientation [#4, #6]. Cryo-EM further resolved cytochrome b within a III2-IV supercomplex with a uniquely kinked apicomplexan interface, and knockout of a supercomplex subunit disrupts III2-IV assembly without abolishing inhibitor binding to complex III2 [#6]. Cytochrome b is additionally the identified target of decoquinate through a resistance mutation [#1], and simultaneous blockade of the Qo site with atovaquone and of an upstream ubiquinol-binding site with HDQ produces strong synergy, underscoring the centrality of the ubiquinone/ubiquinol cycle [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing whether apicomplexan parasites possess a functional respiratory chain and where atovaquone acts answered the basic question of the drug's molecular target within mitochondrial electron transport.\",\n      \"evidence\": \"In situ respirometry and safranin O membrane potential measurement in digitonin-permeabilized T. gondii tachyzoites with selective inhibitors and substrates\",\n      \"pmids\": [\"9813002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the specific residues or binding pocket engaged by atovaquone\", \"Did not distinguish Qo from Qi involvement structurally\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Genetic mapping of a decoquinate-resistance mutation to cytochrome b established cytochrome b as a drug target at the bc1 complex beyond atovaquone.\",\n      \"evidence\": \"Selection of decoquinate-resistant mutants and sequencing of the cytochrome b gene in T. gondii\",\n      \"pmids\": [\"10519259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Limited detail on the specific mutation and binding site\", \"No biochemical confirmation of decoquinate-cytochrome b interaction\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Cloning the cytochrome b gene and mapping atovaquone-resistance mutations to the Qo domain pinpointed the ubiquinol oxidation site as the atovaquone binding region, moving from a functional segment to specific residues.\",\n      \"evidence\": \"Gene cloning and sequencing of wild-type and atovaquone-resistant T. gondii strains with structural modelling of the Qo domain (M129L, I254L)\",\n      \"pmids\": [\"10802314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Modelling only \\u2014 no experimental structure of the atovaquone-bound complex\", \"Did not address the Qi site or other inhibitors\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Surrogate biochemistry defined how atovaquone occupies the Qo pocket and interacts with the Rieske iron-sulfur protein, providing molecular mechanism informing the T. gondii Qo site.\",\n      \"evidence\": \"Inhibition kinetics of isolated bc1 complex, EPR of the Rieske center, and L275F mutagenesis in yeast cytochrome b\",\n      \"pmids\": [\"12791689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Derived from yeast/bovine surrogates, not T. gondii enzyme directly\", \"Species-specific differences in binding not resolved\"],\n      \"attribution\": [{\"species\": \"Saccharomyces cerevisiae\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating synergy between atovaquone (Qo blockade) and HDQ (upstream ubiquinol-binding inhibition) confirmed the central role of the ubiquinone/ubiquinol cycle and validated combined-site targeting.\",\n      \"evidence\": \"In vitro T. gondii growth assay with fractional inhibitory concentration analysis of the atovaquone-HDQ combination\",\n      \"pmids\": [\"17242151\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-based only, no direct enzymatic measurement of dual inhibition\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identifying 4-(1H)-quinolone scaffolds that bind the Qi site opened a second, distinct pharmacological site on cytochrome b for anti-Toxoplasma action.\",\n      \"evidence\": \"Mutant yeast bc1 assays, co-crystallography, and in vitro/in vivo T. gondii infection models\",\n      \"pmids\": [\"27412848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Co-crystal structure was in surrogate rather than T. gondii enzyme\", \"Did not map T. gondii-specific Qi resistance residues\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"EGS\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapping the T222P resistance mutation with antimycin cross-resistance provided direct genetic proof that ELQ-316 inhibits the Qi site of T. gondii cytochrome b, distinguishing it mechanistically from Qo-site atovaquone.\",\n      \"evidence\": \"Selection of ELQ-316-resistant clones, cytochrome b sequencing, and cross-resistance testing against the reference Qi inhibitor antimycin\",\n      \"pmids\": [\"27919897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural depiction of the ELQ-316-Qi complex at this stage\", \"Fitness cost of T222P not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The TgMSH1 knockout showed that atovaquone resistance can arise via metabolic shutdown of respiration rather than cytochrome b Qo mutation, clarifying that Qo mutations are not the only resistance route.\",\n      \"evidence\": \"Tgmsh1 knockout with mtDNA sequencing, respiration measurements, glucose-dependence and atovaquone sensitivity assays\",\n      \"pmids\": [\"29141004\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Accumulated cytochrome b missense mutations did not alter atovaquone-relevant residues\", \"Metabolic bypass mechanism not fully dissected\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Amplification of the cytochrome b-containing mitochondrial genome in artemisinin-resistant lines and loss of membrane potential on treatment implicated complex III in artemisinin action, extending cytochrome b's relevance beyond bc1-specific inhibitors.\",\n      \"evidence\": \"In vitro resistance evolution, whole-genome sequencing, CRISPR/Cas9 point mutation, and membrane potential measurement\",\n      \"pmids\": [\"31806760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytochrome b involvement inferred from genome amplification, not a direct functional assay of cytochrome b\", \"Precise artemisinin target within the mitochondrion unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cryo-EM structures of the III2-IV supercomplex and complex III2 with bound inhibitors resolved atomic-level, species-specific atovaquone Qo binding and a flipped apicomplexan ELQ-300 Qi orientation, converting inferred mechanisms into direct structural models and enabling structure-guided drug design.\",\n      \"evidence\": \"Cryo-EM structure determination with inhibitor co-complexes, supercomplex-subunit knockout, and potency testing of new ELQs\",\n      \"pmids\": [\"40389671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy and resistance profiles of the new subnanomolar ELQs not yet defined\", \"Functional consequence of the unique III2-IV kinked interface not fully explained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How cytochrome b sequence and structural divergence across apicomplexan species and strains shapes clinical resistance and selectivity of Qo/Qi inhibitors remains to be systematically resolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No comprehensive map of Qo/Qi resistance-mutation fitness landscapes\", \"Selectivity margin versus mammalian cytochrome b not fully quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008121\", \"supporting_discovery_ids\": [2, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0045153\", \"supporting_discovery_ids\": [2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005743\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005750\", \"supporting_discovery_ids\": [6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006119\", \"supporting_discovery_ids\": [2, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0022904\", \"supporting_discovery_ids\": [2, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"Cytochrome bc1 complex (complex III2)\",\n      \"III2-IV respiratory chain supercomplex\"\n    ],\n    \"partners\": [\n      \"Cytochrome c1\",\n      \"Rieske iron-sulfur protein\"\n    ],\n    \"other_free_text\": [\n      \"Qo (ubiquinol oxidation) site is the atovaquone binding pocket; resistance mutations M129L, I254L [#0]\",\n      \"Qi (quinone reduction) site is the endochin-like quinolone (ELQ-316/ELQ-300) binding pocket; resistance mutation T222P with antimycin cross-resistance [#4]\",\n      \"ELQ-300 binds the apicomplexan Qi site in a flipped orientation relative to the mammalian enzyme [#6]\",\n      \"Atovaquone resistance can also arise via metabolic shutdown of respiration independent of cytochrome b Qo mutations [#8]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"RH88","gene_id":"TGRH88_085360","name":null,"product":"cytochrome b","uniprot_acc":null,"chromosome":"JAAUHK010000041","start_pos":22313}]}