EXPERIMENTAL FIGURE 12-19 Electron transfer from NADH to O₂ is coupled to proton transport across the mitochondrial membrane. If NADH is added to a suspension of mitochondria depleted of O₂, no NADH is oxidized. When a small amount of O₂ is added to the system (arrow), there is a sharp rise in the concentration of protons in the surrounding medium outside the mitochondria (decrease in pH). Thus the oxidation of NADH by O₂ is coupled to the movement of protons out of the matrix. Once the O₂ is depleted, the excess protons slowly move back into the mitochondria (powering the synthesis of ATP), and the pH of the extracellular medium returns to its initial value.