12.5 Harnessing the Proton-Motive Force to Synthesize ATP
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 studying oxidative phosphorylation and photosynthesis initially rejected his proposal (called the chemiosmotic hypothesis). They favored a mechanism similar to the then well-elucidated substrate-level phosphorylation in glycolysis, in which chemical transformation of a substrate molecule (like phosphoenolpyruvate in glycolysis) is directly coupled to ATP synthesis. Despite intense efforts by a large number of investigators, however, compelling evidence for such a substrate-level phosphorylation–mediated mechanism was never observed.
Definitive evidence supporting Mitchell’s hypothesis depended on developing techniques to purify and reconstitute organelle membranes and membrane proteins. An experiment with vesicles made from chloroplast thylakoid membranes (equivalent to the inner membranes of mitochondria) containing ATP synthase, outlined in Figure 12-29, was one of several demonstrating that ATP synthase is an ATP-generating enzyme and that ATP generation is dependent on proton movement down an electrochemical gradient. It turns out that the protons actually move through ATP synthase as they traverse the membrane.
EXPERIMENTAL FIGURE 12-29 Synthesis of ATP by ATP synthase depends on a pH gradient across the membrane. Isolated chloroplast thylakoid vesicles containing ATP synthase (F0F1 particles) were equilibrated in the dark with a buffered solution at pH 4.0. When the pH in the thylakoid lumen reached 4.0, the vesicles were rapidly mixed with a solution at pH 8.0 containing ADP and Pi. 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 mitochondrial membrane vesicles, an artificially generated membrane electric potential also resulted in ATP synthesis.
As we shall see, ATP synthase is a multiprotein complex that can be subdivided into two subcomplexes, called F0 (containing the transmembrane portions of the complex) and F1 (containing the globular portions of the complex that sit above the membrane and point into the matrix in mitochondria). Thus ATP synthase is often called the F0F1 complex; we will use the two terms interchangeably.