EXPERIMENTAL FIGURE 12-28 Electron transfer from reduced cytochrome c to O₂ via cytochrome c oxidase (complex IV) is coupled to proton transport. The cytochrome c oxidase complex is incorporated into liposomes with the binding site for cytochrome c positioned on the outer surface. (a) When O₂ and reduced cytochrome c are added, electrons are transferred to O₂ to form H₂O, and protons are transported from the inside to the medium outside the vesicles. A drug called valinomycin is added to the medium to dissipate the voltage gradient generated by the translocation of H⁺, which would otherwise reduce the number of protons moved across the membrane. (b) Monitoring of the medium’s pH reveals a sharp drop in pH following addition of O₂. As the reduced cytochrome c becomes fully oxidized, protons leak back into the vesicles, and the pH of the medium returns to its initial value. Measurements show that two protons are transported per O atom reduced. Two electrons are needed to reduce one O atom, but cytochrome c transfers only one electron; thus two molecules of cytochrome c²⁺ are oxidized for each O reduced. See B. Reynafarje et al., 1986, J. Biol. Chem. 261:8254.