Proton pump: Electron transport is coupled to ADP phosphorylation by the pumping of H+ across the inner mitochondrial membrane, from the matrix to the intermembrane space, at Complexes I, III, and IV. For each pair of electrons transferred from NADH to O2, 10 H+ are pumped. This creates an electrical gradient (with more positive charges on the cytosolic side of the membrane than on the matrix side) and a pH (chemical) gradient (the cytosolic side of the membrane is at a lower pH than the matrix side), as shown in Figure 6.13. The energy (proton-motive force) generated by these gradients is sufficient to drive ATP synthesis. Thus, the H+ gradient serves as the common intermediate that couples oxidation to phosphorylation.
2.
ATP synthase: The multisubunit enzyme ATP synthase ([Complex V] Fig. 6.14) synthesizes ATP using the energy of the H+ gradient. It contains a membrane domain (Fo) that spans the inner mitochondrial membrane and an extramembranous domain (F1) that appears as a sphere that protrudes into the mitochondrial matrix (see Fig. 6.13). The chemiosmotic hypothesis proposes that after H+ have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a H+ channel in the Fo domain, driving rotation of the c ring of Fo and, at the same time, dissipating the pH and electrical gradients. Rotation in Fo causes conformational changes in the three β subunits of F1 that allow them to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. One complete rotation of the c ring produces three ATP. [Note: ATP synthase is also called F1/Fo-ATPase because the enzyme can also catalyze the hydrolysis of ATP to ADP and Pi.] contains eight subunits. One complete turn of the ring is driven by eight H+ (protons) moving through the Fo domain. The resulting conformational changes in the three β subunits of the F1 domain allow phosphorylation of three adenosine diphosphates (ADP) to three ATP.] Pi = inorganic phosphate.
a. Coupling in oxidative phosphorylation: In normal mitochondria, ATP synthesis is coupled to electron transport through the H+ gradient. Increasing (or decreasing) one process has the same effect on the other. For example, hydrolysis of ATP to ADP and Pi in energy-requiring reactions increases the availability of substrates for ATP synthase and, thus, increases H+ flow through the enzyme. Electron transport and H+ pumping by the ETC increase to maintain the H+ gradient and allow ATP synthesis.
b. Oligomycin: This drug binds to the Fo (hence the letter “o”) domain of ATP synthase, closing the H+ channel and preventing reentry of H+ into the matrix, thereby inhibiting phosphorylation of ADP to ATP. Because the pH and electrical gradients cannot be dissipated in the presence of this phosphorylation inhibitor, electron transport stops because of the difficulty of pumping any more H+ against the steep gradient. This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control and is the consequence of the tight coupling of these processes.
c.