Figure 5

Analysis of Complex IV enzymatic activity. (A) Complex IV monomer structure from bovine (PDB 1 V54) with the catalytic core (blue), supernumerary subunits (green), and subunits absent in yeast (yellow). (B) Schematic of the Complex IV redox pathway with the color scheme as in panel A, showing redox centers [heme (square) and copper (circle)], paths of electron (thin grey line) and proton (thick grey line) transfer, and site of cyanide inhibition (yellow X). (C) Representative time courses of Complex IV catalytic activity monitored as the kinetics of ferrocytochrome c oxidation (decrease in absorbance at 550 nm) for DDM-solubilized mitochondria (left panel), SMA nanoparticles (center panel), and mitochondria subjected to mock SMA treatment (right panel). Reactions were performed in the absence of inhibitor (blue) or in the presence of potassium cyanide (red). (D) Representative difference spectra (reduced versus oxidized) of cytochromes in DDM-solubilized mitochondria (upper trace) and in SMA nanoparticles (lower trace). The positions of the α-absorption bands of cytochromes cc ₁ , b, and aa ₃ are indicated. The 604 nm absorbance peaks of hemes a and a ₃ , both coordinated by the Complex IV Cox1 subunit, were used to calculate the concentration of Complex IV in DDM- and SMA-treated samples.