In 1961, Peter Mitchell proposed a hypothesis to explain how the energy stored in the proton electrochemical gradient is used to synthesize ATP. In 1978, he was awarded the Nobel Prize in Chemistry for work that fundamentally changed the way we understand how energy is harnessed by a cell.

According to Mitchell’s hypothesis, the gradient of protons provides a source of potential energy that is converted into chemical energy stored in ATP. First, for the potential energy of the proton gradient to be released, there must be an opening in the membrane for the protons to flow through. Mitchell suggested that protons in the intermembrane space diffuse down their electrical and concentration gradients through a transmembrane protein channel into the mitochondrial matrix. Second, the movement of protons through the enzyme must be coupled with the synthesis of ATP. This coupling is made possible by ATP synthase, a remarkable enzyme composed of two distinct subunits called F_o and F₁ (Fig. 7.11). F_o forms the channel in the inner mitochondrial membrane through which protons flow; F₁ is the catalytic unit that synthesizes ATP. Proton flow through the channel (F_o) makes it possible for the enzyme (F₁) to synthesize ATP.

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Proton flow through the F_o channel causes it to rotate, converting the energy of the proton gradient into mechanical rotational energy, a form of kinetic energy. The rotation of the F_o subunit leads to rotation of the F₁ subunit in the mitochondrial matrix (Fig. 7.11). The rotation of the F₁ subunit in turn causes conformational changes that allow it to catalyze the synthesis of ATP from ADP and P_i. In this way, mechanical rotational energy is converted into the chemical energy of ATP.

Direct experimental evidence for Mitchell’s idea, called the chemiosmotic hypothesis, did not come for over a decade. One of the key experiments that provided support for his idea is illustrated in Fig. 7.12.

Quick Check 5 Uncoupling agents are proteins spanning the inner mitochondrial membrane that allow protons to pass through the membrane and bypass the channel of ATP synthase. Describe the consequences to the proton gradient and ATP production.

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Approximately 2.5 molecules of ATP are produced for each NADH that donates electrons to the chain and 1.5 molecules of ATP for each FADH₂. Therefore, overall, the complete oxidation of glucose yields about 32 molecules of ATP from glycolysis, pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation (Table 7.1). Some of the energy held in the bonds of glucose is now available in a molecule that can be readily used by cells.

It is worth taking a moment to follow the flow of energy in cellular respiration, illustrated in its full form in Fig. 7.13. We began with glucose and noted that it holds chemical potential energy in its covalent bonds. This energy is released in a series of reactions and captured in chemical form. Some of these reactions generate ATP directly by substrate-level phosphorylation. Others are redox reactions that transfer energy to the electron carriers NADH and FADH₂. These electron carriers donate electrons to the electron transport chain, which uses the energy stored in the electron carriers to pump protons across the inner membrane of the mitochondria. In other words, the energy of the reduced electron carriers is transformed into energy stored in a proton electrochemical gradient. ATP synthase then converts the energy of the proton gradient to rotational energy, which drives the synthesis of ATP. The cell now has a form of energy that it can use in many ways to perform work.