Cunatova, K.; Vrbacky, M.; Knezu, M.; Pecinova, A.; Alan, L.; Houstek, J.; Fernandez-Vizarra, E.; Mracek, T.; Pecina, P.

COX6B is one of the eleven nuclear-encoded subunits of the human mitochondrial cytochrome c oxidase (cIV). Within the cIV structure, COX6B is located in the intermembrane space-facing region. In mammals, there are two COX6B isoforms: the ubiquitous COX6B1, expressed in all cell types and tissues, and COX6B2 present only in testes and certain types of cancer. Rare COX6B1 missense pathogenic variants have been identified as the cause of childhood-onset mitochondrial encephalopathy associated with cIV deficiency. Despite the relevance of COX6B1 in mitochondrial physiopathology, its molecular role in cIV biogenesis and/or maintenance had not been thoroughly characterized. In spite of the assigned role for COX6B1 as a late incorporation subunit into an almost complete cIV, a COX6B1 human cell line knock-out (KO) exhibited a total loss of cIV. To get a deeper insight into the mechanisms that drive the lack of assembly or destabilization of cIV in the absence of COX6B1, we also used the COX6B1 KO cell background to express an alternative oxidase (AOX) and COX6B1 carrying the R20C and R20H pathogenic variants. These analyses have led us to conclude that the COX6B1 subunit does not only contribute to the stabilization of cIV in the late assembly stages, but is also indispensable for redox-sensitive early cIV assembly steps. In addition, this study has evidenced the incorporation of partially assembled cIV modules directly into supercomplex structures, supporting the \"cooperative assembly\" model for respiratory chain biogenesis.