Harnessing the Proton Motive Force for Energy Requiring Processes

The hypothesis that a proton-motive force across the inner mitochondrial membrane is the immediate source of energy for ATP synthesis was proposed in 1961 by Peter Mitchell. Virtually all researchers working in oxidative phosphoryla-tion and photosynthesis initially opposed this chemiosmotic mechanism. They favored a mechanism similar to the wellelucidated substrate-level phosphorylation in glycolysis, in which oxidation of a substrate molecule is directly coupled to ATP synthesis. By analogy, electron transport through the membranes of chloroplasts or mitochondria was believed to generate an intermediate containing a high-energy chemical bond (e.g., a phosphate linked to an enzyme by an ester bond), which was then used to convert Pi and ADP to ATP. Despite intense efforts by a large number of investigators, however, no such intermediate was ever identified.

Definitive evidence supporting the role of the protonmotive force in ATP synthesis awaited development of techniques to purify and reconstitute organelle membranes and membrane proteins. The experiment with chloroplast thy-lakoid vesicles containing F0F1 particles, outlined in Figure 8-23, was one of several demonstrating that the F0F1 complex is an ATP-generating enzyme and that ATP generation is

▲ EXPERIMENTAL FIGURE 8-23 Synthesis of ATP by F0F1 depends on a pH gradient across the membrane. Isolated chloroplast thylakoid vesicles containing F0F-| particles were equilibrated in the dark with a buffered solution at pH 4.0. When the pH in the thylakoid lumen became 4.0, the vesicles were rapidly mixed with a solution at pH 8.0 containing ADP and Pj. A burst of ATP synthesis accompanied the transmembrane movement of protons driven by the 10,000-fold H+ concentration gradient (10~4 M versus 10~8 M). In similar experiments using "inside-out" preparations of submitochondrial vesicles, an artificially generated membrane electric potential also resulted in ATP synthesis.

dependent on proton movement down an electrochemical gradient. With general acceptance of Mitchell's chemios-motic mechanism, researchers turned their attention to the structure and operation of the F0F1 complex.

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