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▲ EXPERIMENTAL FIGURE 8-14 Electron transfer from NADH or FADH2 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), the pH of the surrounding medium drops sharply—a change that corresponds to an increase in protons outside the mitochondria. (The presence of a large amount of valinomycin and K+ in the reaction dissipates the voltage gradient generated by H + translocation, so that all pumped H+ ions contribute to the pH change.) 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.

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