Fig 2: COX activities normalized to the protein concentration (A) or COX2 subunit content (B). COX activities in Tween-20 extracts of isolated mitochondria measured as oxygen consumption using Oxygraph-2k (Oroboros) at increasing concentrations of cytochrome c (0–30 µM) are plotted as the mean ± S.D. value of three (HEK293, blue) and nine (three replicates of three clones each) COX4i1 (green) and COX4i2 KI (magenta) cells each. Hyperbolic fit of the Michaelis-Menten function is shown as full lines in respective colors.

Fig 3: The COX content and respiration of COX4i2 cysteine substitution mutant KI cell lines. (A) The COX content calculated as the COX2 subunit quantified in the WB immunodetection experiment, normalized to citrate synthase, and expressed in % relative to the COX2 content in parental HEK293 cells, is plotted as the mean ± S.D. value of two measurements in each COX4i2 cysteine-substitution mutant KI cell line as indicated below the graph compared to wild-type COX4i2 KI (magenta, n = 9). ** p < 0.01, and *** p < 0.001 mark significant differences between individual mutant cysteine KIs and wild-type COX4i2 KI. Respiratory rates (B) and p50 (C) in OXPHOS and ETC states are plotted as the mean ± S.D. value of two measurements in each COX4i2 cysteine-substitution mutant KI cell line as indicated below the graph compared to wild-type COX4i2 KI (magenta, n = 12). * p < 0.05, ** p < 0.02, *** p < 0.001 mark significant differences between individual mutant cysteine KIs and wild-type COX4i2 KI in (B) or between COX4i1 KI and wild-type or mutant variants of COX4i2 KI cells (C).

Fig 4: Model of COX functional alterations mediated by COX4 isoform exchange: Rates of oxygen consumption of COX4i1- (left) or COX4i2-containing COX (right) are depicted by variable arrow thickness in normoxia (blue arrow) and hypoxia (red arrow). P, serine58 phosphorylation site.

Fig 5: Decreased COX4I1 expression, COX activity and mitochondrial respiration with elevated glycolysis trend in COX4-1-deficient cells. mRNA expression levels of COX4 isoforms were measured by RT-qPCR in the knockdown vs. control cell lines (COX4-1 knockdown was obtained using a stable expression of COX4I1-targeting or nonmammalian-targeting shRNAs, respectively) (A) and in patient’s and healthy control fibroblasts (B). The results reveal decreased levels of COX4I1 mRNA expression and reciprocally elevated levels of COX4I2 mRNA expression in both COX4-1-deficient cells. Enzymatic activity of COX determined by spectrophotometry; shows decreased activity (C,D) in both shCOX4I1 and patient’s cells, relative to corresponding controls. Oxygen consumption rate (OCR) and extracellular acidification rate; extracellular acidification rates (ECARs) were measured in shCOX4I1 (E,G,I) and patient cells (F,H,J) and in corresponding controls, with subsequent addition of oligomycin, FCCP and antimycin/rotenone. Basal, ATP-linked and maximal OCR were calculated (G,H), and OCR was plotted against ECAR to construct energy maps (I,J). All values were normalized to cell content measured by methylene blue (A620). OCR was reduced, relative to the corresponding controls. Both COX4-1-deficient cell lines showed a tendency towards more glycolysis than the corresponding controls. Values are presented as normalized mean ± SEM; * p < 0.05 and ** p < 0.01 compared to corresponding controls.