The transmembrane proton gradient predicted by chemiosmotic theory has been experimentally measured. When intact mitochondria are suspended with an oxidizable substrate such as succinate in a lightly buf fered medium, the addition of O₂ results in acidification of the suspending medium (Fig. 18-19). The stoichiometry of proton pumping can be estimated from the pH changes, after correcting for buffering effects. The measurement is difficult, and there is no universal agreement on the stoichiometry; about ten protons are pumped out for each pair of electrons transferred from NADH to O₂. The result is a steady state with a difference of about one pH unit between the matrix and the medium, inside (matrix) alkaline. Indirect measurements of the transmembrane electrical potential in respiring mitochondria yield a value of 0.1 to 0.2 V, inside negative. Substituting these values into the equation for proton-motive force (Eqn 18-3) gives a value of 15 to 25 kJ/mol, the free-energy change for the transmembrane movement of one equivalent of protons back into the mitochondrial matrix. This free-energy change is large enough to account for the synthesis of one ATP when two to three protons flow into the matrix.

Uncouplers are hydrophobic weak acids (Fig. 18-14). Their hydrophobicity allows them to diffuse readily across mitochondrial membranes. After entering the mitochondrial matrix in the protonated form, they can release a proton (dissociate), thus dissipating the proton gradient. Ionophores uncouple electron transfer from oxidative phosphorylation by creating electrical short circuits across the mitochondrial membrane (Fig. 18-20). The toxic ionophore valinomycin forms a lipid-soluble complex with K+, which is abundant in the cytosol (see Fig. 10-26). Whereas K+ penetrates the mitochondrial inner membrane only very slowly, the K+-valinomycin complex readily passes through. The influx of positive ions neutralizes the excess of negative charge inside the matrix, diminishing the electrical component of the proton-motive force. Valinomycin slows mitochondrial ATP synthesis without blocking electron transfer to O2 (Table 18-4).

Figure 18-19 Isolated mitochondria are suspended in a medium containing ADP, Pi, and succinate, but initially no O2. When a small amount of O2 is injected into the reaction mixture, succinate oxidation and electron transfer to O2 begin immediately. A pH electrode registers a sudden decrease in the pH of the medium, indicating that protons are moving out of the mitochondria. As the injected O2 is consumed, protons slowly leak back into the mitochondria, and the external pH returns to the initial level. From the known amount of O2 added and the measured pH change, one can in principle calculate the number of protons extruded per molecule of O2 consumed.

Figure 18-20 Ionophores such as valinomycin uncouple oxidative phosphorylation by dissipating ion gradients across the inner mitochondrial membrane, eliminating the contribution of Δψ to the proton-motive force. (a) A transmembrane electrical potential (Δψ) exists because of the unequal distribution of protons on either side. (b) Valinomycin combines reversibly with K⁺ ions to form a membrane-permeable complex that diffuses across the inner membrane and releases K⁺ on the inside. This movement of charge reduces the value of Δψ, and uncouples electron transfer from ATP synthesis.

If the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the electrochemical potential of the proton-motive force, an artificially created proton gradient should be able to replace electron transfer in driving ATP synthesis. This prediction of the chemiosmotic theory has been experimentally tested and confirmed (Fig. 18-21). Mitochondria manipulated so as to impose a difference of proton concentration and a separation of charge across the inner membrane (Fig. 18-21) carry out the synthesis of ATP in the absence of an oxidizable substrate; the proton-motive force alone suf fices to drive ATP synthesis.

Chemiosmotic theory also accounts for a third condition that uncouples oxidation from phosphorylation-mechanical disruption of the mitochondrial membrane. Without an intact membrane there can be no proton gradient, hence no energy conservation and no ATP synthesis.

The Detailed Mechanism of ATP Formation ftemains Elusive Although it is clear that a transmembrane proton gradient provides the energy for ATP synthesis, it is not clear how this energy is transmitted to the ATP synthase. This enzyme catalyzes the conversion of enzyme-bound ADP and Pi into bound ATP even in the absence of a proton gradient. Remarkably, it appears that the reaction ADP + Pi ATP + H2O is readily reversible-that the free-energy change for ATP synthesis on the enzyme surface is close to zero. However, the ATP formed in this reaction remains tightly bound to the active site, preventing further catalysis at that site. The proton-motive force apparently supplies the energy needed to force dissociation of the tightly bound ATP from the enzyme, allowing another catalytic cycle to begin. How is it possible that ATP synthesis, known to be strongly endergonic in aqueous solution (ΔG°' = 30.5 kJ/mol), is readily reversible on the enzyme surface? The very tight noncovalent binding of enzyme to ATP may supply enough binding energy (Chapter 8) to make bound ATP about as stable as its hydrolysis products (Fig. 18-22). Alternatively, the reaction may occur in a very hydrophobic pocket in the enzyme interior, where the energetics of hydrolysis in water do not apply.

Figure 18-21 An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. In this two-step experiment, isolated mitochondria are first incubated in a pH 9 buffer containing 0.1 M KCl (a). The slow leakage of buffer and KCl into the mitochondria eventually brings the matrix into equilibrium with the surrounding medium. No oxidizable substrates are present. (b) In the second step, mitochondnia are separated from the pH 9 buffer and resuspended in pH 7 buffer containing valinomycin but no KCl. The change in buffer creates a difference of two pH units. The outward flow of K+, carried (without a counterion) down its concentration gradient by valinomycin, creates a charge imbalance across the membrane (matrix negative). The sum of the chemical potential provided by the pH difference and the electrical potential provided by the separation of charges is a proton-motive force large enough to support ATP synthesis in the absence of an oxidizable substrate.

Fignre 18-22 Reaction coordinate diagram for the condensation of ADP and Pi to form ATP on the surface of ATP synthase. Although the free-energy change for this reaction in aqueous solution (ΔGaq) is large and positive, the very tight binding of ATP to the enzyme provides binding energy (ΔGB) that brings the free energy of enzyxne-bound ATP close to that of ADP + Pi. On the enzyme surface, the reaction is therefore readily reversible; the equilibrium constant is believed to be near 1.

Each F1 complex has the subunit composition α3β3γδε (Fig. 18-15). A tight binding site for ATP, apparently identical to the catalytic site, is located on each β subunit, or perhaps between each β and its associated a subunit. Every F1 complex therefore has three ATP-synthesizing sites, which interact with F0 through the single copies of γ, δ, and ε subunits. On the basis of detailed kinetic and binding studies of the reactions catalyzed by F0F1, Paul Boyer has suggested a mechanism (Fig. 18-23) in which the three active sites on F1 alternate in catalyzing ATP synthesis. The limiting step in the process is the release of newly synthesized ATP from the enzyme. A conformational transition driven by the proton-motive force reduces the enzyme's affinity for ATP. Figure 18-23 The "binding change" model for ATP synthase action. The enzyme has three equivalent adenine nucleotide binding sites, one for each pair of α and β subunits. At any given moment, one of these sites is in the T (tight-binding) conformation, a second is in the L (loose-binding) conformation, and a third is in the O (open; very loose-binding) conformation. At the beginning of a catalytic cycle, the T site is occupied by ATP, and ADP and Pi bind loosely to the L site. The proton-motive force causes, perhaps by the flow of protons through the F0 channel, a cooperative conformational change in which the T site is converted to an O site, and ATP dissociates from it; the L site is converted to a T site, where ADP and P; quickly condense to form ATP; and the O site becomes an L site, where ADP and Pi loosely bind. The experimental results require that at least two of the three catalytic sites alternate in activity; ATP cannot be released from one site unless and until ADP and Pi are bound at the other.

The transition induced by the proton-motive force may be envisioned as a 120° rotation of the α₃β₃ portion of F₁ (Fig. 18-23), placing one of the three α-β pairs in a special position relative to the proton channel of F₀. However, no physical rotation of F₁ has been demonstrated, and the model does not require it; the conformational change that interconverts the three types of sites may be an allosteric transition, in which changes at one α-β pair force compensating changes in the other two pairs.

What makes the mechanism of this enzyme particularly difficult to solve is the Uectorial nature of the process it catalyzes. Somehow, the enzyme must sense not merely a certain concentration of protons, but a difference of proton concentration in two regions of space. Determination of the detailed structure of F₁ by x-ray crystallography should shed light on the mechanism of its action.

The primary role of electron transfer in mitochondria is to furnish energy for the synthesis of ATP during oxidative phosphorylation, but this energy serves also to drive several transport processes essential to oxidative phosphorylation. We have seen that the inner mitochondrial membrane is generally impermeable to charged species, but two specific systems in the inner mitochondrial membrane transport ADP and Pi into the matrix and ATP out to the cytosol (Fig. 18-24). The adenine nucleotide translocase, which extends across the inner membrane, binds ADP^3- on the outside (cytosolic) surface of the inner membrane and transports it inward in exchange for an ATP^4- molecule simultaneously transported outward (see Fig. 13-1 for the ionic forms of ATP and ADP). Because this antiporter moves four negative charges out for each three moved in, its activity is favored by the transmembrane electrochemical gradient, which gives the matrix a net negative charge; the proton-motive force drives ATP-ADP exchange.

Figure 18-24 Transport systems of the mitochondrial inner membrane carry ADP and Pi into the matrix and allow the newly synthesized ATP to leave. The ATP-ADP translocase is an antiporter; the same protein moves ADP into the matrix and ATP out. The effect of replacing ATP4- with ADP3- is the net efflux of one negative charge, which is favored because the matrix is electrically negative relative to the outside. At pH 7, Pi is present as both HPO42- and H2PO4-; the transport system that carries Pi into the matrix is specific for H2PO4-. There is no net flow of charge during symport of H2PO4- and H+, but the relatively low proton concentration in the matrix favors the inward movement of H+. Thus the proton-motive force is responsible both for providing the energy for ATP synthesis by ATP synthase and for transporting substrates (ADP and Pi) in and product (ATP) out of the mitochondrial matrix..

Adenine nucleotide translocase is specifically inhibited by atractyloside, a toxic glycoside formed by a species of thistle; for centuries it has been known that grazing cattle are poisoned when they ingest this plant. If the transport of ADP into and ATP out of the mitochondria is inhibited, cytosolic ATP cannot be regenerated from ADP, explaining the toxicity of atractyloside (Table 18-4).

A second membrane transport system essential to oxidative phosphorylation is the phosphate translocase, which promotes symport of one H₂P0₄^- and one H⁺ into the matrix. This transport process, too, is favored by the transmembrane proton gradient (Fig. 18-24).

The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix. Given that the inner membrane is not permeable to cytosolic NADH, how can the NADH generated by glycolysis outside mitochondria be reoxidized to NAD⁺ by O₂ via the respiratory chain? Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route. The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle (Fig. 18-25). The reducing equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to yield malate by the action of cytosolic malate dehydrogenase. The malate thus formed passes through the inner membrane into the matrix via the malatea-ketoglutarate transport system. Within the matrix the reducing equivalents are passed by the action of matrix malate dehydrogenase to matrix NAD⁺, forming NADH; this NADH can then pass electrons directly to the respiratory chain in the inner membrane. Three molecules of ATP are generated as this pair of electrons passes to ⁰². Cytosolic oxaloacetate must be regenerated via transamination reactions (see Fig. 17-5) and the activity of membrane transporters (Fig. 18-25) to start another cycle of the shuttle.

Figure 18-25 The malate-aspartate shuttle for transporting reducing equivalents from cytosolic NADH into the mitochondrial matrix. 1. NADH in the cytosol (intermembrane space) passes two reducing equivalants to oxaloacetate, producing malate. 2. Malate is transported across the inner membrane by the malate-a-ketoglutarate transporter. 3. In the matrix, malate passes two reducing equivalants to NAD⁺; the resulting matrix NADH is oxidized by the mitochondrial respiratory chain. The oxaloacetate formed from malate cannot pass directly into the cytosol. It is first transaminated to form aspartate 4. which can leave via the glutamate-aspartate transporter 5. Oxaloacetate is regenerated in the cytosol 6, completing the cycle.

In skeletal muscle and brain, another type of NADH shuttle, the glycerol-3-phosphate shuttle, occurs (Fig. 18-26). It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH into Complex III, not Complex I (Fig. 18-9), providing only enough energy to synthesize two ATP molecules per pair of electrons. The mitochondria of higher plants have an externally oriented NADH dehydrogenase that is able to transfer electrons directly from cytosolic NADH into the respiratory chain.

Fignre 18-26 The glycerol-3-phosphate shuttle, an alternative means of moving reducing equivalents from the cytosol to the mitochondrial matrix. Dihydroxyacetone phosphate in the cytosol accepts two reducing equivalents from cytosolic NADH in a reaction catalyzed by cytosolic glycerol-3-phosphate dehydrogenase. A membrane-bound isozyme of glycerol-3-phosphate dehydrogenase, located on the outer face of the inner membrane, transfers two reducing equivalents from glycerol-3-phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems.

The complete oxidation of a molecule of glucose to CO₂ yields two ATP and two NADH from glycolysis in the cytosol (Chapter 14); two NADH from pyruvate oxidation in the mitochondrial matrix (Chapter 15); and two ATP, six NADH, and two FADH₂ from citric acid cycle reactions in the matrix (Chapter 15). Each NADH produced in the matrix yields three ATP from mitochondrial oxidative phosphorylation, and for each FADH₂, two ATP are generated. Cytosolic NADH, after shuttling into the matrix, yields two or three ATP, depending on which shuttle is used. The total yield of glucose oxidation is therefore 36 or 38 ATP per glucose (Table 18-5).

By comparison, glycolysis under anaerobic conditions (lactate fermentation) yields only two ATP per glucose. Clearly, the evolution of oxidative phosphorylation provided a tremendous increase in the energetic efficiency of catabolism.

The oxidation of fatty acids and amino acids also takes place within the mitochondrial matrix, and the FADH₂ and NADH produced by these oxidative pathways also serve as electron donors for oxidative phosphorylation. Complete oxidation of the 16-carbon saturated fatty acid palmitate to CO₂ produces eight acetyl-CoAs, seven FADH₂, and seven NADH (Chapter 16). The oxidation of each acetyl-CoA via the citric acid cycle produces three NADH, one FADH₂, and one ATP (GTP). The gain in ATP is therefore 131 ATP (Table 18-6); but, because the activation of palmitate to palmitoyl-CoA costs two ATP equivalents, the net gain is 129 ATP per palmitate. A similar calculation may be made for the ATP yield upon oxidation of each of the amino acids (Chapter 17). The important conclusion here is that aerobic oxidative pathways that result in electron transfer to O₂ accompanied by oxidative phosphorylation account for the vast majority of the ATP produced in catabolism.