Coletta, Genna2025-07-072025-07-072025-07-072025-06-25https://hdl.handle.net/10012/21973Cytochrome 𝘤 oxidase (COX) is the terminal enzyme of the electron transport chain and plays a crucial role in cellular respiration. As a multisubunit enzyme, COX consists of catalytic core subunits encoded by the mitochondrial genome and requires the coordinated action of more than 30 nuclear-encoded assembly factors for proper biogenesis and function. Human COX deficiencies have been associated with mutations in both nuclear and mitochondrial genes and are thus characterized by immense genetic heterogeneity and a vast spectrum of clinical phenotypes. 𝘊𝘖𝘟11 is a nuclear-encoded copper chaperone that is required for COX assembly. Beyond this well-characterized role, COX11 has an additional, uncharacterized role in cellular redox homeostasis. Caron-Godon et al. reported a patient with compound heterozygous mutations in 𝘊𝘖𝘟11, whose homologous mutations were studied in haploid yeast. When grown on non-fermentable carbon sources, one of the mutant alleles, P238T, demonstrated robust growth, indicative of respiratory competence, in contrast to the truncation mutants, Y250* and R254*, which exhibited a complete and partial respiratory deficiency, respectively. Given that COX deficiencies are inherited in an autosomal recessive manner, this finding adds an unexpected complexity to the patient’s phenotype, which may suggest the possibility of a hypomorphic or dominant negative allele. To better recapitulate the patient’s genotype, I employed a pseudodiploid system in 𝘚𝘢𝘤𝘤𝘩𝘢𝘳𝘰𝘮𝘺𝘤𝘦𝘴 𝘤𝘦𝘳𝘦𝘷𝘪𝘴𝘪𝘢𝘦, which involves the stable co-expression of two mutant 𝘊𝘖𝘟11 alleles in a single haploid background. Functional assays, including growth on non-fermentable carbon and COX enzymatic activity, as well as immunoblotting of core COX subunits, demonstrated that the pseudodiploid double mutants, representative of the patient, supported robust respiration and maintained COX assembly at levels comparable to those of wild-type. In contrast, evaluation of oxidative stress markers revealed defects in cellular redox balance. Double mutant strains, particularly P238T/R254*, exhibited significantly elevated superoxide dismutase activity, a pronounced decrease in mitochondrial aconitase activity, and increased sensitivity to hydrogen peroxide. These data indicate that the redox equilibrium is compromised even when cytochrome 𝘤 oxidase function is preserved.enMaster Thesis