Most of the free energy released during the oxidation of glucose to CO₂ is retained in the reduced coenzymes NADH and FADH₂ generated during glycolysis and the citric acid cycle. During respiration electrons are released from NADH and FADH₂ and eventually are transferred to O₂, forming H₂O according to the following overall reactions:

The free energy released during oxidation of a single NADH or FADH₂ molecule by O₂ is sufficient to drive the synthesis of several molecules of ATP from ADP and P_i, a reaction with a ΔG°′ of +7.3 kcal/mol. The mitochondrion maximizes the production of ATP by transferring electrons from NADH and FADH₂ through a series of electron carriers all but one of which are integral components of the inner membrane. This step-by-step transfer of electrons via the electron transport chain (also known as the respiratory chain) allows the free energy in NADH and FADH₂ to be released in small increments. At several sites during electron transport from NADH to O₂, protons from the mitochondrial matrix are transported uphill across the inner mitochondrial membrane and a proton concentration gradient forms across it (Figure 16-17). Because the outer membrane is freely permeable to protons, the pH of the mitochondrial matrix is higher (i.e., the proton concentration is lower) than that of the cytosol and intermembrane space. An electric potential across the inner membrane also results from the uphill pumping of positively charged protons outward from the matrix, which becomes negative with respect to the intermembrane space. Thus free energy released during the oxidation of NADH or FADH₂ is stored both as an electric potential and a proton concentration gradient — collectively, the proton-motive force — across the inner membrane. The movement of protons back across the inner membrane, driven by this force, is coupled to the synthesis of ATP from ADP and P_i by the F₀F₁ complex.

The synthesis of ATP from ADP and P_i, driven by the transfer of electrons from NADH or FADH₂ to O₂, is the major source of ATP in aerobic nonphotosynthetic cells. Much evidence shows that in mitochondria and bacteria this process, called oxidative phosphorylation, depends on generation of an electrochemical proton gradient (i.e., proton-motive force) across the inner membrane, with electron transport, proton pumping, and ATP formation occurring simultaneously. In the laboratory, for instance, addition of O₂ and an oxidizable substrate such as pyruvate or succinate to isolated intact mitochondria results in a net synthesis of ATP if the inner mitochondrial membrane is intact. In the presence of minute amounts of detergents that make the membrane leaky, the oxidation of these metabolites by O₂ still occurs, but no ATP is made. Under these conditions, no transmembrane proton concentration gradient or membrane electric potential can be maintained.

Although bacteria lack mitochondria, aerobic bacteria nonetheless carry out oxidative phosphorylation by the same processes that occur in eukaryotic mitochondria. Enzymes that catalyze the reactions of both the glycolytic pathway and the citric acid cycle are present in the cytosol of bacteria; enzymes that oxidize NADH to NAD⁺ and transfer the electrons to the ultimate acceptor O₂ are localized to the bacterial plasma membrane.

The movement of electrons through these membrane carriers is coupled to the pumping of protons out of the cell (see Figure 16-2). The movement of protons back into the cell, down their concentration gradient, is coupled to the synthesis of ATP. Bacterial F₀F₁ complexes are essentially identical in structure and function with the mitochondrial F₀F₁ complex, which we examine in some detail below. The proton-motive force across the bacterial plasma membrane is also used to power the uptake of nutrients such as sugars and the rotation of bacterial flagella (see Figure 16-1).

The F₀F₁ complex, or ATP synthase, has two principal components, F₀ and F₁, both of which are oligomeric proteins (Figure 16-28). F₀ is located within the membrane and contains a transmembrane channel through which protons flow toward F₁, most of which extends into the mitochondrial matrix (or cytosol in bacteria).

The F₀ component contains three types of subunits, a, b, and c; in bacteria, the subunit composition is a₁b₂c_9–12. The c subunits form a donut-shaped ring in the plane of the membrane. Each a subunit is thought to span the membrane eight times, each b once, and each c twice. In mitochondria, each F₀ complex also contains, depending on the species, two to five additional peptides of unknown function. When F₀ is experimentally incorporated into liposomes, the permeability of the vesicles to H⁺ is greatly stimulated, indicating that it indeed forms a proton channel. Each copy of subunit c contains two membrane-spanning α helices; an aspartate residue in one of these helices is thought to participate in proton movement, since chemical modification of this aspartate by dicyclohexylcarbodiimide or its mutation specifically blocks H⁺ translocation. The proton channel lies at the interface of the a and c subunits (see Figure 16-28).

The F₁ portion is a water-soluble complex of five distinct polypeptides with the composition α₃β₃γδε. The α and β subunits associate in alternating order to form a hexamer, αβαβαβ or (αβ)₃. This hexamer rests atop the single long γ subunit, whose lower part is a coiled coil that fits into the c-subunit ring of F₀. The ε subunit is attached to γ and probably also contacts the c subunits of F₀. The δ subunit of the F₁ complex contacts the b subunit of the F₀ complex; together these subunits form a rigid “stator” that prevents the (αβ)₃ hexamer from rotating while it rests on the γ subunit (see Figure 16-28).

F₁ forms the knobs that protrude from the matrix side of the inner membrane. When physically separated from the membrane by mechanical agitation, F₁ is capable only of catalyzing ATP hydrolysis. Hence, it has been called the F₁ ATPase, but its natural function is synthesis of ATP. Submitochondrial vesicles from which F₁ is removed cannot catalyze ATP synthesis; when F₁ particles reassociate with these vesicles, they once again become fully active in ATP synthesis.

Each of the three β subunits in the complete F₀F₁ complex can bind ATP, ADP, and P_i, and catalyze ATP synthesis. However, the coupling between proton flow and ATP synthesis must be indirect, since the nucleotide-binding sites on the β subunits of F₁, where ATP synthesis occurs, are 9 – 10 nm from the surface of the mitochondrial membrane. In the next section, we examine this coupling mechanism.

The most widely accepted model for ATP synthesis by the F₀F₁ complex — the binding-change mechanism — posits that the energy released by the downhill movement of protons through F₀ directly powers rotation of the γ subunit and attached ε subunit. Most likely the γ and ε subunits rotate together with the ring of c subunits, relative to the fixed a subunit, but it is possible that the γ subunit rotates in the center of a fixed ring of c subunits. In either case, the γ subunit acts as cam, a rotating shaft within F₁ whose movement causes cyclical changes in the conformations of the β subunits. As schematically depicted in Figure 16-30, rotation of the γ subunit relative to the fixed (αβ)₃ hexamer causes the nucleotide-binding site of each β subunit to cycle through three conformational states in the following order:
1. An O state that binds ATP very poorly and ADP and P_i weakly

2. An L state that binds ADP and P_i more strongly

3. A T state that binds ADP and P_i so tightly that they spontaneously form ATP and that binds ATP very strongly

A final rotation of γ returns the β subunit to the O state, thereby releasing ATP and beginning the cycle again. (ATP or ADP also bind to regulatory or allosteric sites on the three α subunits; this binding modifies the rate of ATP synthesis according to the level of ATP and ADP in the matrix, but is not directly involved in synthesis of ATP from ADP and P_i.)