NADH AND FADH₂ are generated during the oxidation of glucose:

Nearly all of the energy available from oxidizing carbohydrates, fats, and other foodstuffs in glycolysis and the Krebs cycle is initially stored in the form of high-energy electrons carried by NADH and FADH₂. How is this energy used to generate ATP? Of the various hypotheses advanced to explain how ATP is synthesized in the mitochondrion and chloroplast, one has gained much favor in recent years. Known as the chemiosmotic hypothesis, it was proposed by Peter Mitchell of Glynn Research Laboratories in England, who was awarded the Nobel Prize in 1978 for his contribution. According to this hypothesis, the transfer of the high-energy electrons from NADH and FADH₂ along the electron transport chain in the inner membrane of the mitochondrion results in the pumping of H+ ions across the membrane. This creates an electrochemical gradient across the inner membrane. It is the gradient that provides the energy to drive the synthesis of ATP.

Let us look at the chemiosmotic process in more detail.

Electrons are transferred from NADH and FADH₂ to oxygen through large respiratory enzyme complexes:

Built into the inner mitochondrial membrane are four large enzyme complexes (Complex I, the NADH dehydrogenase complex; II, the succinate reductase complex; III, the b-c1 complex; and IV, the cytochrome oxidase complex). Mobile electron carriers (e.g. Q and Cyt c) transport the electrons from one complex to the next. The NADH passes its two electrons and a H+ ion to Complex I, becoming oxidized to NAD+, and the electrons are passed from one transport molecule to the next in a precise sequence with oxygen acting as the final electron acceptor. The electrons from FADH₂ are passed to Complex II, and then to the rest of the electron transport chain.

A proton (H+) gradient is generated at three sites:

As the electrons flow through the chain from NADH or FADH₂ to oxygen, energy is released and is used to pump H+ ions across the inner mitochondrial membrane, from the matrix into the outer compartment. H+ ions are pumped across the membrane at three sites in the chain. The transport of H+ ions has two major consequences: (1) it generates a pH gradient across the inner mitochondrial membrane, with the pH higher in the matrix than in the outer compartment, and (2) it generates an electrical gradient across the inner mitochondrial membrane with the inside negative and the outside positive (as a result of the outflow of the positive H+ ions). The net result is a steep electrochemical gradient across the membrane, with the concentration of H+ ions higher in the outer compartment than in the matrix.

ATP is synthesized as protons flow back to the matrix through a proton channel:

The electrochemical gradient functions rather like a battery--the flow of H+ ions (rather than electrons as in an ordinary battery) move back across the membrane and drive the synthesis of ATP. How does this happen? Recall that the mitochondrial membrane contains numerous enzyme complexes, the ATP synthetases, which look rather like lollipops inserted into the membrane. The synthetase complexes act both as H+ ion channels and as enzymes that catalyze the synthesis of ATP. When the H+ ions move through the channels down their concentration gradient (i.e., from a region of high H+ concentration in the outer compartment to one of low H+ ion concentration in the matrix), ATP is synthesized from ADP and P_i.

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