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. Author manuscript; available in PMC: 2016 Jul 18.
Published in final edited form as: Clin Sci (Lond). 2016 Mar;130(6):393–407. doi: 10.1042/CS20150707

Figure 1. Cytochrome oxidase location in the respiratory chain and activity assay in human skin fibroblasts.

Figure 1

A. Schematic representation of the respiratory chain in the inner mitochondrial membrane showing the interaction of cytochrome oxidase (complex IV) with complexes I and III in a super-complex (respirasome). The site of action of specific inhibitors is indicated in red. The green arrow shows the alternative oxidase (AOX) by-pass, which when expressed in COX-defective human mitochondria or flies rescues their various phenotypes. The assay of COX with externally added cytochrome c requires the permeabilization of the outer membrane. B. Cytochrome oxidase is assayed spectrophotometrically by measuring using a double-wavelength spectrophotometer (550–540 nm) the oxidation of reduced cytochrome c in skin fibroblasts permeabilized by 2 successive freeze/thaw cycles. The reaction is first order with respect to substrate concentration and is thus diminished by half when half of the reduced cytochrome c is consumed. Subsequent sequential addition of rotenone, cyanide, oxidized cytochrome c and succinate measures reduction of cytochrome c, first by the succinate-cytochrome c reductase (CII plus CIII). The activity is essentially rate controlled by CII and can be inhibited by malonate, a competitive inhibitor of CII. Further addition of glycerol-3 phosphate measures the activity from the glycerol-3 phosphate dehydrogenase (G3Pdh) to CIII. This activity can be selectively inhibited by iGP1(143). Finally, addition of decylubiquinol in the presence of EDTA is used to measure antimycin-sensitive CIII activity. Abbreviations: The RC complexes are abbreviated as, CI, CII, CIII, CIV, and the ATP synthase as CV; c, cytochrome c; COX, cytochrome oxidase; Ddh, the dihydroorotate dehydrogenase which catalyze the production of uridine, an essential step for the synthesis of nucleic acids; EDTA, ethylenediamine tetraacetic acid; ETF, the electron transfer flavoprotein involved in the oxidation of fatty acids; G3Pdh, the glycerol 3-phosophate dehydrogenase; GCCR, iGP1-sensitive glycerol 3-phosphate; IM, inner membrane; KCN, potassium cyanide; OM, outer membrane; QCCR, antimycin-sensitive decylubiquinol-cytochrome c reductase; SCCR, malonate-sensitive cytochrome c reductase; UQ, ubiquinone 50, or coenzyme Q10.