Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...

Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...

The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....

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