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Mitochondria, 'the powerhouses of the cell', house the integral metabolism pathway of oxidative phosphorylation to produce the majority of cellular energy. Mammalian cytochrome c oxidase, also called complex IV (cIV), is indispensable for the overall oxidative phosphorylation function as the terminal oxidase, and for its regulation to sustain energetic needs. Since cIV is a multimeric enzyme composed of subunits encoded by nuclear and mitochondrial genomes, its biogenesis is a complicated process, which needs to be coordinated to complete a fully functional complex. Further, the setup of individual nuclear-encoded subunits isoforms of cIV may fine-tune cIV function based on the tissue or the environment context. Despite the physiological and pathological relevance of cIV composition, biogenesis, and the secondary deficiencies triggered by cIV defects, nuclear-encoded subunits' function remains poorly understood. At first, mammalian COX4 subunit isoforms with tissue- and oxygen-dependent expression were studied in the HEK293 cellular model with an exclusive expression of COX4I1 or COX4I2 isoform. Remarkably, the COX4I2 isozyme showed lower affinity to oxygen, which may imply regulation of cIV activity under hypoxia, and is of physiological relevance for the oxygen-sensing mechanism. Further,...

Oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes were shown to form supramolecular assemblies termed supercomplexes. It has been repeatedly shown that complexes are not linked only by their function but also by interdependence of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV, COX) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomer as well as within supercomplexes. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used HEK293 cellular model with complete knockout of COX4 subunit, which serves as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process are disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by selective decrease in cI subunits amount and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII and cIV were missing in COX4dKO due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labelling of mtDNA-encoded proteins uncovered decrease of cIV and cI subunits translation. Moreover, partial impairment of mitochondrial proteosynthesis correlated with decreased level of mitochondrial ribosomal proteins. In addition, complexome profiling approach uncovered accumulation of cI assembly intermediates indicating that cI biogenesis was affected rather than stability. We propose that impairment of mitochondrial proteosynthesis caused by cIV deficiency represents one of the mechanisms which may couple biogenesis of cI and cIV.

Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain and its main function is transfer of electrons to the terminal acceptor, oxygen. Moreover, COX contributes to the generation of proton gradient, which is indispensable for the production of vast majority of ATP molecules in the mammalian cells. The present work summarizes available data concerning the structure and function of nuclear-encoded subunits, which are specific for the eukaryotic form of COX. Particular emphasis is put on the role of subunit Cox4 and its regulatory properties within the enzyme. The study also describes other proteins associated with the enzyme, which are able to regulate the activity of COX, but are mainly involved in formation and stabilization of the respiratory supercomplexes. Based on the summarized data, three levels model of quaternary COX structure is postulated. They correspond to subunits of the catalytic center, nuclear-encoded subunits and substoichiometrically asociated proteins, which may give rise to several forms of COX with varying composition and differentially regulated function.