Conversion of Glucose-6-Phosphate into Free Glucose Is the Third Bypass

The third bypass is the imal reaction of gluconeogenesis, the dephosphorylation of glucose-6-phosphate to yield free glucose (Fig. 19-2). Because the hexokinase reaction of glycolysis is irreversible (Table 19-1, step l ), the hydrolytic reaction is catalyzed by another enzyme, glucose-6-phosphatase:

Glucose-6-phosphate + H₂O -------------> glucose + Pi ΔG°' = -13.8 kJ/mol

This Mg²⁺-dependent enzyme is found in the endoplasmic reticulum of hepatocytes. Glucose-6-phosphatase is not present in muscles or in the brain, and gluconeogenesis does not occur in these tissues. Instead, glucose produced by gluconeogenesis in the liver or ingested in the diet is delivered to brain and muscle through the bloodstream.

Gluconeogenesis Is Energetically Costly

The sum of the biosynthetic reactions leading from pyruvate to free blood glucose (Table 19-2) is

2Pyruvate +4ATP +2GTP +2NADH +4H₂O --------------> glucose +4ADP +2GDP +6Pi +2NAD+ +2H⁺

For each molecule of glucose formed from pyruvate, six high-energy phosphate groups are required, four from ATP and two from GTP. In addition, two molecules of NADH are required for the reduction of two molecules of 1,3-bisphosphoglycerate. This equation is clearly not the simple reverse of the equation for the conversion of glucose into pyruvate by glycolysis, which yields only two molecules of ATP:

Glucose + 2ADP + 2Pi + 2NAD⁺ -----------------> 2 pyruvate + 2ATP + 2NADH⁺ 2H⁺ + 2H₂O

Thus the synthesis of glucose from pyruvate is a relatively costly process. Much of this high energy cost is necessary to ensure that gluconeogenesis is irreversible. Under intracellular conditions, the overall free-energy change of glycolysis is at least -63 kJ/mol. Under the same conditions the overall free-energy change of gluconeogenesis from pyruvate is also highly negative. Thus glycolysis and gluconeogenesis are both essentially irreversible processes under intracellular conditions.

Citric Acid Cycle Intermediates and Many Amino Acids Are Glucogenic

The biosynthetic pathway to glucose described above allows the net synthesis of glucose not only from pyruvate but also from the citric acid cycle intermediates citrate, isocitrate, α-ketoglutarate, succinate, fumarate, and malate. All may undergo oxidation in the citric acid cycle to yield oxaloacetate. However, only three carbon atoms of oxaloacetate are converted into glucose; the fourth is released as CO₂ in the conversion of oxaloacetate to phosphoenolpyruvate by PEP carboxykinase (Fig. 19-3).

that some or all of the carbon atoms of many of the amino acids derived from proteins are ultimately converted by mammals into either pyruvate or certain intermediates of the citric acid cycle. Such amino acids can therefore undergo net conversion into glucose and are called glucogenic amino acids (Table 19-3). Alanine and glutamine make especially important contributions in that they are the principal molecules used to transport amino groups from extrahepatic tissues to the liver. After removal of their amino groups in liver mitochondria, the carbon skeletons remaining (pyruvate and a-ketoglutarate, respectively) are readily funneled into gluconeogenesis.

In contrast, there is no net conversion of even-carbon fatty acids into glucose by mammals because such fatty acids yield only acetylCoA on oxidative cleavage. Acetyl-CoA cannot be used as a precursor of glucose by mammals. The pyruvate dehydrogenase reaction is irreversible under intracellular conditions, and no other pathway exists by which acetyl-CoA can be converted into pyruvate. For every two carbons of acetate that enter the citric acid cycle, two carbons are lost as CO₂ (Fig. 19-5); therefore, there can be no net conversion of acetate to oxaloacetate or pyruvate. However, fatty acids make an important contribution to gluconeogenesis in another way. Oxidation of fatty acids delivered to the liver during starvation provides much of the ATP and NADH needed to drive gluconeogenesis energetically.

Futile Cycles in Carbohydrate Metabolism Consume ATP

At the three points where a glycolytic reaction is bypassed by a different type of enzymatic reaction in gluconeogenesis, simultaneous operation of both pathways would be wasteful. For example, phosphofructokinase-1 and fructose-1,6-bisphosphatase catalyze opposing reactions:

ATP + fructose-6-phosphate -------------> ADP + fructose-1,6-bisphosphate + H⁺

Fructose-1,6-bisphosphate + H₂O -----------------> fructose-6-phosphate + Pi

The sum of these two reactions is

ATP + H₂O -----------------> ADP + Pi + H⁺ + heat

an energy-wasting rection resulting in the hydrolysis of ATP without any net metabolic work being done. Clearly, if these two reactions were allowed to proceed simultaneously at a high rate in the same cell, a large amount of chemical energy would be dissipated as heat. Such an ATP-degrading cycle is called a futile cycle. A similar futile cycle could occur with the other two sets of bypass reactions.
Under normal circumstances futile cycles probably do not take place at a significant rate, because they are prevented by reciprocal regulatory mechanisms (as discussed below). However, futile cycling sometimes occurs physiologically to produce heat. For example, in cold weather bumblebees cannot fly until they have warmed their muscles to about 30 °C by futile cycling of fructose-6-phosphate and fructose1,6-bisphosphate and the consequent heat-generating hydrolysis of ATP.

Gluconeogenesis and Glycolysis Are Reciprocally Regulated

To assure that futile cycling does not occur under normal circumstances, gluconeogenesis and glycolysis are regulated separately and reciprocally. The first control point occurs in the reactions catalyzed by the pyruvate dehydrogenase complex of glycolysis and pyruvate carboxylase of gluconeogenesis (Fig. 19-6); the latter enzyme requires the positive allosteric modulator acetyl-CoA for its activity. As a consequence, the biosynthesis of glucose from pyruvate is promoted only when excess mitochondrial acetyl-CoA builds up beyond the immediate needs of the cell for the citric acid cycle. When the cell's energetic needs are being met, oxidative phosphorylation slows, NADH accumulation inhibits the citric acid cycle, and acetyl-CoA accumulates. The increased concentration of acetyl-CoA inhibits the pyruvate dehydrogenase complex, slowing the formation of acetyl-CoA from pyruvate, and stimulates gluconeogenesis by activating pyruvate carboxylase. This allows excess pyruvate to be converted to glucose.

The second control point in gluconeogenesis is the reaction catalyzed by fructose-1,6-bisphosphatase, which is strongly inhibited by AMP. The corresponding glycolytic enzyme, phosphofructokinase-1, is stimulated by AMP and ADP but inhibited by citrate and ATP, so that these opposing steps in the two pathways are regulated in a coordinated or reciprocal manner. When sufficient concentrations of acetylCoA or of the product of acetyl-CoA condensation with oxaloacetate (citrate) are present, or when a high proportion of the cell's adenylate is in the form of ATP, gluconeogenesis is favored, thus promoting formation of glucose and its storage as glycogen (described later).

The special role of liver in maintaining a constant blood glucose level requires additional regulatory mechanisms to coordinate glucose production and consumption. When the blood glucose level decreases, the hormone glucagon signals the liver to produce and release more glucose. One source of glucose is glycogen stored in the liver; another source is gluconeogenesis.

The hormonal regulation of glycolysis and gluconeogenesis in liver is mediated by fructose-2,6-bisphosphate, an allosteric effector for the enzymes phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (FBPase-1) (Fig. 19-7). When fructose-2,6-bisphosphate binds to its allosteric site on PFK-l, it increases that enzyme's afFmity for its substrate fructose-6-phosphate and reduces its affinity for the allosteric inhibitors ATP and citrate. Fructose-2,6-bisphosphate therefore activates PFK-1 and stimulates glycolysis in liver. Fructose-2,6bisphosphate also inhibits FBPase-l, thereby slowing gluconeogenesis.

Although structurally related to fructose-1,6-bisphosphate, fructose-2,6-bisphosphate is clearly not an intermediate in gluconeogenesis or glycolysis; it is a regulator whose cellular level reflects the level of glucagon in the blood (see below), which in turn varies with the blood glucose level. Because fructose-2,6-bisphosphate is effective at low concentrations (in the micromolar range), very little fructose is diverted from metabolic pathways for its synthesis.
The cellular concentration of fructose-2,6-bisphosphate is set by the relative rates of its formation and breakdown. Fructose-2,6-bisphosphate is formed by phosphorylation of fructose-6-phosphate, catalyzed by phosphofructokinase-2 (PFK-2), and is broken down by fructose-2,6-bisphosphatase (FBPase-2) (Fig. 19-8). (Note that these enzymes are distinct from PFK-1 and FBPase-l, which catalyze the formation and breakdown, respectively, of fructose-1,6-bisphosphate.) PFK-2 and FBPase-2 are two distinct enzymatic activities, but both are part of a single, bifunctional protein. The balance of these two activities in the liver, and therefore the cellular level of fructose-2,6-bisphosphate, is regulated by glucagon. Glucagon stimulates adenylate cyclase, an enzyme that synthesizes 3',5'-cyclic AMP (cAMP) from ATP (see Fig. 14-18). Cyclic AMP in turn stimulates a cAMP-dependent protein kinase, which transfers a phosphate group from ATP to the bifunctional protein PFK-2/FBPase-2 (Fig. 19-8). Phosphorylation of this protein enhances its FBPase-2 activity and inhibits PFK-2. Glucagon thereby lowers the cellular level of fructose-2,6-bisphosphate, inhibiting glycolysis and stimulating gluconeogenesis. The resulting production of more glucose enables the liver to replenish blood glucose in response to glucagon.

Gluconeogenesis Converts Fats and Proteins to Glucose in Germinating Seeds

Many plants store lipids and proteins in their seeds, to be used as sources of energy and biosynthetic precursors during germination, before photosynthetic mechanisms can supply both. Active gluconeogenesis occurs in germinating seeds, providing glucose for the synthesis of polysaccharides and many metabolites derived from hexoses, and for the production of sucrose (the transport form of carbon in plants).

Unlike animals, plants can convert acetyl-CoA derived from fatty acid oxidation into glucose. Triacylglycerols are converted into glucose by the combined action of the β-oxidation pathway (Chapter 16), the glyoxylate cycle (Chapter 15), and gluconeogenesis. Hydrolysis of storage triacylglycerols produces glycerol-3-phosphate, which can enter the gluconeogenic pathway after its oxidation to dihydroxyacetone phosphate (Fig. 19-9). Fatty acids, the other product of lipolysis, are activated, then oxidized to acetyl-CoA in glyoxysomes by isozymes of the β-oxidation pathway (Fig. 19-10). The separation of this process from the citric acid cycle, the enzymes of which are in mitochondria but not glyoxysomes, prevents the further oxidation of acetyl-CoA to CO₂.Instead, the acetyl-CoA is converted, via the glyoxylate cycle, into succinate. Succinate passes to the mitochondrial matrix, where it is converted by citric acid cycle enzymes into oxaloacetate, which passes out of the mitochondrion and into the cytosol. Cytosolic oxaloacetate is converted by gluconeogenesis to fructose-6-phosphate, the precursor to sucrose. Thus, the integration of reaction sequences occurring in three subcellular compartments is required for the production of fructose-6phosphate or sucrose from stored lipids. About 75% of the carbon in the stored lipids of seeds is converted into carbohydrate by this means; the other 25% is lost as CO₂ in the conversion of oxaloacetate to phosphoenolpyruvate.

Amino acids derived from the breakdown of stored seed proteins also yield precursors for gluconeogenesis. They are deaminated and oxidized, via pathways discussed in Chapter 17, to succinyl-CoA, pyruvate, oxaloacetate, fumarate, and α-ketoglutarate-all good starting materials for gluconeogenesis.



Carbohydrate Biosynthesis

We have now reached a turning point in the study of cellular metabolism. The preceding chapters of Part III have described how the major foodstuffs-carbohydrates, fatty acids, and amino acids-are degraded via converging catabolic pathways to enter the citric acid cycle and yield their electrons to the respiratory chain. The exergonic flow of electrons to oxygen is coupled to the endergonic synthesis of ATP. We now turn to anabolic pathways, which use chemical energy in the form of ATP and NADH or NADPH to synthesize cell components from simple precursor molecules. Anabolic pathways are generally reductive rather than oxidative. Catabolism and anabolism proceed simultaneously in a dynamic steady state, so that the energy-yielding degradation of cell components is counterbalanced by biosynthetic processes, which create and maintain the intricate orderliness of living cells.
Several organizing principles of biosynthesis deserve emphasis at the outset. The first principle is that the pathway taken in the synthesis of a biomolecule is usually different from the pathway taken in its degradation. Although the two opposing pathways may share many reversible reactions, there is always at least one enzymatic step that is unique to each pathway. If the reactions of catabolism and anabolism were catalyzed by the same set of enzymes acting reversibly, the flow of carbon through these pathways would be dictated exclusively by mass action (p. 371), not by the cell's changing needs for energy, precursors, or macromolecules.
Second, corresponding anabolic and catabolic pathways are controlled by different regulatory enzymes. These opposing pathways are regulated in a coordinated, reciprocal manner, so that stimulation of the biosynthetic pathway is accompanied by inhibition of the corresponding degradative pathway, and vice versa. Biosynthetic pathways are usually regulated at their initial steps, so that the cell avoids wasting precursors to make unneeded intermediates; intrinsic economy prevails in the molecular logic of living cells.
Third, energy-requiring biosynthetic processes are coupled to the energy-yielding breakdown of ATP in such a way that the overall process is essentially irreversible in vivo. Thus the total amount of ATP (and NAD(P)H) energy used in a given biosynthetic pathway always exceeds the minimum amount of free energy required to convert the precursor into the biosynthetic product. The resulting large, negative, free-energy change for the overall process assures that it will occur even when the concentrations of precursors are relatively low.
This chapter provides many opportunities for elaboration of the three principles outlined above, as we describe pathways for carbohydrate biosynthesis. The chapter is divided into four parts. First we consider gluconeogenesis, the ubiquitous pathway for synthesis of glucose. We then describe how glucose is converted into a variety of polysaccharides: glycogen in animals and many microorganisms, starch and sucrose in plants. At this point the focus shifts entirely to plants. The third topic is the incorporation of CO₂ into more complex molecules (CO₂ fixation), a process that represents the ultimate source of reduced carbon compounds for all organisms. The chapter ends with a discussion of the regulation of carbohydrate metabolism in plants. The overall regulation of carbohydrate metabolism in mammals is covered separately

Gluconeogenesis We begin our survey of biosynthetic processes with the central pathway that leads to the formation of different carbohydrates from noncarbohydrate precursors in animal tissues (Fig. 19-1). The biosynthesis of glucose is an absolute necessity in all mammals, because the brain and nervous system, as well as the kidney medulla, testes, erythrocytes, and embryonic tissues, require glucose from the blood as their sole or major fuel source. The human brain alone requires over 120 g of glucose per day. Mammalian cells constantly make glucose directly from simpler precursors, such as pyruvate and lactate, and then pass the glucose into the blood. Other important carbohydrates, including glycogen, are also made from simple precursors (Fig. 19-1). The formation of glucose from nonhexose precursors is called gluconeogenesis ("formation of new sugar"). Gluconeogenesis is a universal pathway, found in all animals, plants, fungi, and microorganisms. The reactions are the same in every case. The important precursors of glucose in animals are lactate, pyruvate, glycerol, and most of the amino acids (Fig. 19-1). In higher animals gluconeogenesis occurs largely in the liver and to a much smaller extent in kidney cortex. In plant seedlings, stored fats and proteins are converted into the disaccharide sucrose for transport throughout the developing plant. Glucose and its derivatives are precursors in the synthesis of plant cell walls, nucleotides and coenzymes, and a variety of other essential metabolites. Many microorganisms are able to grow on simple organic compounds such as acetate, lactate, and propionate, which they convert to glucose by gluconeogenesis.

Although the reactions of gluconeogenesis are the same in all organisms, the metabolic context and the regulation of the pathway dif fer from organism to organism, and from tissue to tissue. In this chapter we focus iirst on gluconeogenesis as it occurs in the mammalian liver. Later, its role and regulation in plants will be considered.

.Just as the glycolytic conversion of glucose into pyruvate is a central pathway for catabolism of carbohydrates, the conversion of pyruvate into glucose is a central pathway in gluconeogenesis. These pathways are not identical, although they share several steps (Fig. 19-2). Seven of the ten enzymatic reactions of gluconeogenesis are the reverse of glycolytic reactions However, three steps in glycolysis are essentially irreversible in vivo and cannot be used in gluconeogenesis: the conversion of glucose to glucose-6-phosphate by hexokinase, the phosphorylation of fructose6-phosphate to fructose-1,6-bisphosphate by phosphofructokinase-l, and the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase (Fig. 19-2). With pathway intermediates at concentrations typically found in an erythrocyte, these three reactions are characterized by a large and negative free-energy change, ΔG, whereas other glycolytic reactions have a ΔG near 0 These three steps are bypassed by a separate set of enzymes, catalyzing different reactions with different equilibria; they function in gluconeogenesis but not in glycolysis and are irreversible in the direction of glucose synthesis. Thus, both glycolysis and gluconeogenesis are irreversible processes in cells. Gluconeogenesis and glycolysis are independently regulated through controls exerted on specific enzymatic steps that are not common to the two pathways.

We begin by considering the three bypass reactions of gluconeogenesis.

Conversion of Pyruvate into Phosphoenolpyruvate Requires a Bypass

The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate into phosphoenolpyruvate (Fig. 19-2). This reaction cannot occur by reversal of the pyruvate kinase reaction (p. 413), which has a large, negative, standard free-energy change and has been found to be irreversible under the conditions that exist in intact cells (Table 19-l, step 10 ). Instead, the phosphorylation of pyruvate is achieved by a roundabout sequence of reactions that in mammals and some other organisms requires the cooperation of enzymes in both the cytosol and mitochondria. As we will see, the pathway shown in Figure 19-2 and described in more detail below is one of two paths from pyruvate to phosphoenolpyruvate; it is the predominant one when pyruvate or alanine is the gluconeogenic precursor. A second pathway, described later, predominates when lactate is the gluconeogenic precursor.
First, pyruvate is transported from the cytosol to the mitochondria, or generated within mitochondria by deamination of alanine. Then, pyruvate carboxylase, a mitochondrial enzyme that requires biotin, converts the pyruvate to oxaloacetate:

Pyruvate + HCO₃^- + ATP ------> oxaloacetate + ADP + Pi + H⁺ .............(19-1)

Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway; acetyl-CoA is a required positive effector for the enzyme. This is also an anaplerotic reaction (Chapter 15), and as one of the major entry points in the citric acid cycle it affects many metabolic pathways. The reaction mechanism is described in Figure 15-13b.
The oxaloacetate formed from pyruvate in the mitochondrion is reversibly reduced to malate by mitochondrial malate dehydrogenase at the expense of NADH:

Oxaloacetate + NADH + H⁺ ------> L-malate + NAD⁺ (19-2)

The malate then leaves the mitochondrion via the malate-α-ketoglutarate transporter in the inner mitochondrial membrane (see Fig. 18-25). In the cytosol, malate is reoxidized to oxaloacetate, with the production of cytosolic NADH:

Malate + NAD⁺ ------> oxaloacetate + NADH + H⁺ ................ (19-3)

The oxaloacetate is then converted to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase in a Mg²⁺-dependent reaction in which GTP serves as the phosphate donor (Fig. 19-3):

Oxaloacetate + GTP ------>phosphoenolpyruvate + CO₂ + GDP .............. (19-4)

This reaction is reversible under intracellular conditions.
The overall equation for this set of bypass reactions, the sum of Equations 19-1 through 19-4, is

Pyruvate+ATP+GTP+HCO₃^- phosphoenolpyruvate+ADP+GDP+Pi+H⁺+CO₂ .................(19-5)

ΔG°' = 0.9 kJ/mol

Two high-energy phosphate groups (one from ATP and one from GTP), each yielding 30.5 kJ/mol under standard conditions, must be expended to phosphorylate one molecule of pyruvate to PEP, thus requiring an input of 61 kJ/mol under standard conditions. In contrast, when PEP is converted into pyruvate during glycolysis, only one ATP is generated from ADP. Although the standard free-energy change (ΔG°') of the net reaction leading to PEP is 0.9 kJ/mol, the actual free-energy change (ΔG), calculated from measured cellular concentrations of intermediates, is very strongly negative (ΔG = -25 kJ/mol); this results from the ready consumption of PEP in other reactions, such that its concentration remains low. The reaction is thus irreversible in the cell.

Note that the CO2 lost in the PEP carboxykinase reaction is the same molecule that is added to pyruvate in the pyruvate carboxylase step (Fig. 19-3). This carboxylation-decarboxylation sequence represents a way of "activating" pyruvate in that the decarboxylation of oxaloacetate facilitates PEP formation. In Chapter 20 we will see that a similar carboxylation step is used to activate acetyl-CoA for fatty acid biosynthesis. The path of these reactions through the mitochondria is not coincidental. The [NADH]/[NAD+] ratio in the cytosol is 8 × 10-4, about 105 times lower than in mitochondria. Because cytosolic NADH is consumed in gluconeogenesis (in the conversion of 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate; Fig. 19-2), glucose biosynthesis cannot proceed unless NADH is made available. The transport of malate from the mitochondrion to the cytosol and its reconversion there to oxaloacetate has the effect of moving reducing equivalents in the form of NADH to the cytosol, where they are scarce. This path from pyruvate to PEP therefore provides an important balance between NADH produced and consumed in the cytosol during gluconeogenesis. A second and shorter pyruvate ------> PEP bypass predominates when lactate is the gluconeogenic precursor (Fig. 19-4). This pathway makes use of lactate produced by glycolysis in erythrocytes or muscle, for example, and it is particularly important in larger vertebrates after vigorous exercise (see Box 14-1). The conversion of lactate to pyruvate in the hepatocyte cytosol yields NADH, and the export of malate from mitochondria is no longer necessary. After the pyruvate produced by the lactate dehydrogenase reaction is transported into the mitochondrion, it is converted to oxaloacetate by pyruvate carboxylase as described above. This oxaloacetate, however, is converted directly to PEP by a mitochondrial form of PEP carboxykinase. The PEP is then transported out of the mitochondrion and continues on the gluconeogenic path. The mitochondrial and cytosolic forms of PEP carboxykinase are encoded by separate nuclear genes, providing an example of two distinct enzymes catalyzing the same reaction but having different cellular locations and metabolic roles.

Conversion of Fructose-1,6-Bisphosphate into Fructose-6-Phosphate Is the Second Bypass

The second reaction of the catabolic glycolytic sequence that cannot participate in the anabolic process of gluconeogenesis is the phosphorylation of fructose-6-phosphate by phosphofructokinase-1 (Table 19-l, Step 3 ). Because this reaction is irreversible in intact cells, the generation of fructose-6-phosphate from fructose-1,6-bisphosphate (Fig. 19-2) is catalyzed by a different enzyme, Mg²⁺-dependent fructose1,6-bisphosphatase, which promotes the essentially irreversible hydrolysis of the C-1 phosphate:

Fructose-1,6-bisphosphate + H₂O --------------> fructose-6-phosphate + Pi
        ΔG°' = -16.3 kJ/mol

Coupling ATP Synthesis to Light-Driven Electron Flow

We have now seen how one of the two energy-rich products formed in the light reactions, NADPH, is generated by photosynthetic electron transfer from H₂O to NADP⁺. What about the other energy-rich product, ATP?

In 1954 Daniel Arnon and his colleagues discovered that ATP is generated from ADP and Pi during photosynthetic electron transfer in illuminated spinach chloroplasts. Support for these findings came from the work of Albert Frenkel who detected light-dependent ATP production in membranous pigment-containing structures called chromatophores, derived from photosynthetic bacteria. They concluded that some of the light energy captured by the photosynthetic systems of these organisms is transformed into the phosphate bond energy of ATP. This process is called photophosphorylation or photosynthetic phosphorylation, to distinguish it from oxidative phosphorylation in respiring mitochondria.

Recall that oxidative phosphorylation of ADP to ATP in mitochondria occurs at the expense of the free energy released as high-energy electrons flow downhill along the electron transfer chain from substrates to O₂. In a similar way, photophosphorylation of ADP to ATP is coupled to the energy released as high-energy electrons flow down the photosynthetic electron transfer chain from excited photosystem II to the electron-deficient photosystem I. The direct effect of electron flow is the formation of a proton gradient, which then provides the energy for ATP synthesis by an ATP synthase.
ATP synthesis in chloroplasts can be coupled to two types of electron flow-cyclic and noncyclic-as we shall see. We will also turn our attention to photophosphorylation in organisms other than green plants, and to the possible bacterial origins of chloroplasts. The chapter concludes with the development of an overall equation for photosynthesis in plants.

A Proton Gradient Couples Electron Flow and Phosphorylation

Several properties of photosynthetic electron transfer and photophosphorylation in chloroplasts show a role for a proton gradient as in mitochondrial oxidative phosphorylation: (1) the reaction centers, electron carriers, and ATP-forming enzymes are located in a membrane-the thylakoid membrane; (2) photophosphorylation requires intact thylakoid membranes; (3) the thylakoid membrane is impermeable to protons; (4) photophosphorylation can be uncoupled from electron flow by reagents that promote the passage of protons through the thylakoid membrane; (5) photophosphorylation can be blocked by venturicidin and similar agents that inhibit the formation of ATP from ADP and Pi by ATP synthase of mitochondria (see Fig. 18-13); and (6) ATP synthesis is catalyzed by F₀F₁ complexes, located on the outer surface of the thylakoid membranes, that are very similar in structure and function to the F₀F₁ complexes of mitochondria.
Electron-transferring molecules in the connecting chain between photosystem II and photosystem I are oriented asymmetrically in the thylakoid membrane, so that photoinduced electron flow results in the net movement of protons across the membrane, from the outside of the thylakoid membrane to the inner compartment (Fig. 18-45).

In 1966 Andre Jagendorf showed that a pH gradient across the thylakoid membrane (alkaline outside) could furnish the driving force to generate ATP. Jagendorf's early observations provided some of the most important experimental evidence in support of Mitchell's chemiosmotic hypothesis. In the dark, he soaked chloroplasts in a pH 4 buffer, which slowly penetrated into the inner compartment of the thylakoids, lowering their internal pH. He added ADP and Pi to the dark suspension of chloroplasts and then suddenly raised the pH of the outer medium to 8, momentarily creating a large pH gradient across the membrane. As protons moved out of the thylakoids into the medium, ATP was generated from ADP and Pi. Because the formation of ATP occurred in the dark (with no input of energy from light), this experiment showed that a pH gradient across the membrane is a highenergy state that can, as in mitochondrial oxidative phosphorylation, mediate the transduction of energy from electron transfer into the chemical energy of ATP.

The stoichiometry for this process (protons transported per electron) is not well established. Electron transfer between photosystems II and I through the cytochrome bf complex contributes to the proton gradient an amount of proton-motive force roughly equivalent to one to two ATP formed per pair of electrons.

The ATP Synthase of Chloroplasts Is Like That of Mitochondria

The enzyme responsible for ATP synthesis in chloroplasts is a large complex with two functional components, CF₀ and CF_l (the C denoting chloroplast origin). CF₀ is a transmembrane proton pore composed of several integral membrane proteins and is homologous with mitochondrial F₀. CF_l is a peripheral membrane protein complex very similar in subunit composition, structure, and function to mitochondrial F₁ (Table 18-8). Together these proteins constitute the ATP synthase of chloroplasts. (Bacteria also contain ATP synthases remarkably similar in structure and function to those of chloroplasts and mitochondria
Electron microscopy of sectioned chloroplasts shows ATP synthase complexes as knoblike projections on the outside (stromal) surface of thylakoid membranes; these correspond to the ATP synthase complexes seen to project on the inside (matrix) surface of the inner mitochondrial membrane (see Fig. 18-15). Thus both the orientation of the ATP synthase and the direction of proton pumping in chloroplasts are opposite to those in mitochondria. In both cases, the F_l portion of ATP synthase is located on the more alkaline side of the membrane through which protons flow down their concentration gradient; the direction of proton flow relative to F₁ is the same in both cases (Fig. 18-46).
The mechanism of chloroplast ATP synthase is also believed to be essentially identical to that of its mitochondrial analog; ADP and Pi readily condense to form ATP on the enzyme surface, but the release of this enzyme-bound ATP requires a proton-motive force (see Fig. 1823). As for the mitochondrial ATP synthase, the details of this mechanism remain to be determined.

Cyclic Electron Flow Produces ATP but Not NADPH or O₂

There is an alternative path of light-induced electron flow that allows chloroplasts to vary the ratio of NADPH and ATP formed during illuminations; this is called cyclic electron flow to differentiate it from the normally unidirectional or noncyclic electron flow that proceeds from H₂O to NADP⁺, as we have discussed thus far. Cyclic electron flow involves only photosystem I (Fig. 18-44). Electrons passed from P700 to ferredoxin do not continue to NADP⁺, but move back through the cytochrome bf complex to plastocyanin. Plastocyanin donates electrons to P700, the illumination of which promotes electron transfer to ferredoxin. Thus illumination of photosystem I can cause electrons to cycle continuously out of the reaction center of photosystem I and back into it, each electron being propelled around the cycle by the energy yielded by absorption of one photon. Cyclic electron flow is not accompanied by net formation of NADPH or the evolution of O₂. However, it is accompanied by proton pumping and by the phosphorylation of ADP to ATP, referred to as cyclic photophosphorylation. The overall reaction equation for cyclic electron flow and photophosphorylation is simply
Cyclic electron flow and photophosphorylation are believed to occur when the plant cell is already amply supplied with reducing power in the form of NADPH but requires additional ATP for other metabolic needs. By regulating the partitioning of electrons between NADP⁺ reduction and cyclic photophosphorylation, a plant adjusts the ratio of NADPH and ATP produced in the light reactions to match the needs for these products in the carbon fixation reactions and in other energy-requiring processes.

Chloroplasts Probably Evolved from Endosymbiotic Cyanobacteria

Like mitochondria, chloroplasts contain their own DNA and proteinsynthesizing machinery. Some chloroplast proteins are encoded by chloroplast genes and synthesized in the chloroplast; others are encoded by nuclear genes, synthesized outside the chloroplast, and imported. When plant cells grow and divide, chloroplasts give rise to new chloroplasts by division, during which their DNA is replicated and divided between daughter chloroplasts.
Cyanobacteria are photosynthetic prokaryotes (formerly called blue-green algae) in which the machinery and mechanism for light capture, electron flow, and ATP synthesis are similar in many respects to those in the chloroplasts of higher plants. The bacteriumlike division of chloroplasts, and their similarities to cyanobacteria in photosynthetic components and mechanisms, support the hypothesis that chloroplasts arose during evolution from endosymbiotic prokaryotes that also gave rise to modern cyanobacteria (see Fig. 2-17). Studies of photosynthesis in lower eukaryotes and prokaryotes may therefore yield insight into photosynthetic mechanisms that is generalizable to the higher plants.

Diverse Photosynthetic Organisms Use Hydrogen Donors Other Than H20

Photosynthesis occurs not only in green plants but in lower eukaryotic organisms such as algae, euglenoids, dinoflagellates, and diatoms, and also in certain prokaryotes. The photosynthetic prokaryotes include the cyanobacteria, the green sulfur bacteria found in mountain lakes, the purple bacteria common in the ocean, and the purple sulfur bacteria of sulfur springs. The cyanobacteria, found in both fresh and salt waters, are perhaps the most versatile of photosynthetic organisms. Because they can also iix atmospheric nitrogen (Chapter 21), cyanobacteria are among the most self sufficient organisms in the biosphere. At least half of the photosynthetic activity on earth occurs in the many different microorganisms that constitute the phytoplankton in oceans, rivers, and lakes.
With the exception of the cyanobacteria, which have an 02-producing photosynthetic system resembling that of plants, photosynthetic bacteria do not produce O₂. Many are obligate anaerobes that cannot tolerate O₂. Some photosynthetic bacteria use inorganic compounds as hydrogen donors. For example, the green sulfur bacteria use hydrogen sulfide, according to the equation
These bacteria, instead of giving off molecular O₂, produce elemental sulfur as the oxidation product of H₂S. Other photosynthetic bacteria use organic compounds as hydrogen donors, for example, lactate:

Plant and bacterial photosynthesis are fundamentally similar processes despite the differences in the hydrogen donors they employ. This similarity becomes obvious when the equation of photosynthesis is written in a more general form:

in which H₂O symbolizes a hydrogen donor and D is its oxidized form. H₂D thus may be water, hydrogen sulfide, lactate, or some other organic compound, depending upon the species.

The Structure of a Bacterial Photosystem fteaction Center Has Been Determined

The three-dimensional structure of the photoreaction center of a photosynthetic bacterium, Rhodopseudomonas viridis, which is analogous in some ways to photosystem II in higher plants, is known from x-ray crystallographic studies (Fig. 18-47). This structure sheds light on how phototransduction takes place in the bacterium, and presumably in higher plants as well.
The bacterial reaction center has four types of proteins: a c-type cytochrome, two subunits (L and M) associated with bacteriochlorophyll, and a fourth protein, the H subunit. A single reaction center contains f'our hemes (cytochromes), four bacteriochlorophylls that are similar to the chlorophylls of chloroplasts, two bacteriopheophytins, one inorganic Fe, and a quinone. From a variety of physical studies, the extremely rapid sequence of events shown in Figure 18-47b has been deduced.
A pair of closely spaced bacteriochlorophylls (the "special pair") constitutes the site of the initial photochemistry in the bacterial reaction center. The "electron hole" that develops in the chlorophyll is filled with electrons from the c-type cytochrome, a role played by H20 in photosystem II of higher plants.
The bacteriochlorophylls, bacteriopheophytins, and quinone are held rigidly in a fixed orientation relative to each other by the proteins of the reaction center. The photochemical reactions among these components therefore take place in a virtually solid state, accounting for the high efficiency and rapidity of the reactions; nothing is left to chance collision or dependent upon random diffusion.
Although the reaction centers of plants have not yet been seen in such detail, the similarities between the bacterial and eukaryotic reaction centers suggest that the principles deduced from studies of the geometry and mechanism of the bacterial reaction center will also apply to the reaction centers of plants.

Salt-Loving Bacteria Use Light Energy to Make ATP

The halophilic ("salt-loving") bacterium Halobacterium halobium conserves energy derived from absorbed sunlight by an interesting variation on the principle employed by true photosynthetic organisms. These unusual bacteria live only in brine ponds and salt lakes (Great Salt Lake and the Dead Sea, for example), where the high salt concentration results from water loss by evaporation; indeed, they cannot live in NaCl concentrations lower than 3 M. Halobacteria are aerobes and normally use O₂ to oxidize organic fuel molecules. However, the solubility of O₂ is so low in brine ponds, in which the NaCl concentration may exceed 4 M, that these bacteria must sometimes call on another source of energy, namely sunlight. The plasma membrane of H. halobium contains patches of light-absorbing pigments, called purple patches. These patches are made up of closely packed molecules of the protein bacteriorhodopsin (see Fig. 10-10), which contains retinal (vitamin A aldehyde; see Fig. 9-18) as a prosthetic group. When the cells are illuminated, the bacteriorhodopsin molecules are excited by an absorbed photon. As the excited molecules revert to their initial ground state, an induced conformational change results in the release of protons outside the cell, forming an acid-outside pH gradient across the plasma membrane. Protons tend to diffuse back into the cell through an ATP synthase complex in the membrane, very similar to that of mitochondria and of chloroplasts, supplying the energy for ATP synthesis (Fig. 18-48). Thus halobacteria can use light to supplement the ATP synthesized by oxidative phosphorylation with the O₂ that is available. However, halobacteria do not evolve O₂, nor do they carry out photoreduction of NADP⁺; their phototransducing machinery is therefore much simpler than that of cyanobacteria or higher plants.
Bacteriorhodopsin, with only 247 amino acid residues, is the simplest light-driven proton pump known. The determination of its molecular structure should yield important insights into light-dependent energy transduction and the action of the proton pumps that function in respiration and photosynthesis.

Photosynthesis Uses the Energy in Light Very Efficiently

The standard free-energy change for the synthesis of glucose from CO₂ and H₂O during photosynthesis by the reaction

6 CO2+6 H2O

light

C6H12O6 + 6 O2

............(18-9)

is 2,840 kJ/mol. (Recall that oxidation of glucose by the reverse of this equation proceeds with a decrease of 2,840 kJ/mol.) Now let us compare this energy requirement with the energy yielded by the light reactions of plant photosynthesis.
Recall that two photons must be absorbed, one by each photosystem, to cause flow of one electron from H₂O to NADP⁺. To generate one molecule of O₂, four electrons must be transferred. Therefore, production of six molecules of O₂ (as in Eqn 18-9) requires the absorption and use of 48 photons: (2 photons/e^-)(4e^-/O₂)(6 0₂) = 48 photons. Because the energy of one einstein may range from 300 kJ at 400 nm to about 170 kJ at 700 nm (Fig. 18-35), anywhere from 8,160 to 14,400 kJ (depending upon the wavelength of the absorbed light) is required under standard conditions to make 1 mol of glucose "costing" 2,840 kJ.
In the next chapter we turn to a consideration of the carbon fixation reactions by which photosynthetic organisms use the ATP and NADPH produced in the light reactions to carry out the reduction of CO₂ to carbohydrates.

Light-Driven Electron Flow

Thylakoid membranes have two different kinds of photosystems, each with its own type of photochemical reaction center and a set of antenna molecules. The two systems have distinct and complementary functions. Photosystem I has a reaction center designated P700 and a high ratio of chlorophyll a to chlorophyll b. Photosystem II, with its reaction center P680, contains roughly equal amounts of chlorophyll a and b and may also contain a third type, chlorophyll c. The thylakoid membranes of a single spinach chloroplast have many hundreds of each kind of photosystem. All O₂-evolving photosynthetic cells-those of higher plants, algae, and cyanobacteria-contain both photosystems I and II; all other species of photosynthetic bacteria, which do not evolve O₂, contain only photosystem I.
It is between photosystems I and II that light-driven electron flow occurs, producing NADPH and a transmembrane proton gradient.

Light Absorption by Photosystem II Initiates Charge Separation

How can light energy captured by chloroplasts induce electrons to flow energetically "uphill"? Excitation briefly creates a chemical species of very low standard reduction potential-an excellent electron donor. Excited P680, designated P680*, within picoseconds transfers an electron to pheophytin (a chlorophyll-like accessory pigment lacking Mg2+), giving it a negative charge (designated Ph- ) (Fig. 18-41a). With the loss of its electron, P680* is transformed into a radical cation, designated P680+. Thus excitation, in creating Ph- and P680* , causes charge separation. Ph- very rapidly passes its extra electron to a protein-bound plastoquinone, QA, which in turn passes its electron to another, more loosely bound quinone, QB (Fig. 18-42; see also Fig. 1844). When QB has acquired two electrons in two such transfers from QA and two protons from the solvent water, it is in its fully reduced quinol form, QBH2. This molecule dissociates from its protein and diffuses away from the photochemical reaction center, carrying in its chemical bonds some of the energy of the photons that originally excited P680. The overall reaction initiated by light in photosystem II is therefore 4 P680 + 4H+ + 2QB + light (4 photons) --------> 4 P680+ + 2QBH2 ........... (18-4)Eventually, the electrons in QBH2 are transferred through a chain of membrane-bound carriers to NADP+, reducing it to NADPH and releasing H+ (as described later). The potent herbicide DCMU (Fig. 1842) competes with QB for the QB binding site in photosystem II, thus blocking photosynthetic electron transfer (Table 18-4). In the meantime, P680+ must acquire an electron to return to its ground state in preparation for the capture of another photon of energy (Fig. 18-41a). In principle, the required electron might come from any number of organic or inorganic compounds. Photosynthetic bacteria can use a variety of electron donors for this purpose-acetate, succinate, malate, or sulfide-depending on what is available in a particular ecological niche. About 3 billion years ago, evolution of primitive photosynthetic bacteria (the progenitors of the modern cyanobacteria) produced a photosystem capable of taking electrons from a donor that is always available-water. In this process two water molecules are split, yielding four electrons, four protons, and molecular oxygen: 2H2O 4H+ + 4e- + O2. A single photon of visible light does not possess enough energy to break the bonds in water; four photons are required in this photolytic cleavage reaction. The four electrons abstracted from water do not pass directly to P680+, which can only accept one electron at a time. Instead, a remarkable molecular device, the water-splitting complex, passes four electrons one at a time to P680+. The immediate electron donor to P680+ is a Tyr residue (often represented by the symbol Z) in protein D1 of the photosystem II reaction center: This Tyr residue regains its missing electron by oxidizing a cluster of four manganese ions in the water-splitting complex. With each singleelectron transfer, this Mn cluster becomes more oxidized; four singleelectron transfers, each corresponding to the absorption of one photon, produce a charge of +4 on the Mn complex (Fig. 18-43): In this state, the Mn complex can take four electrons from a pair of water molecules, releasing 4H+ and 02: [Mn complex]4+ + 2 H2O [Mn complex]0 + 4H+ + O2 The sum of Equations 18-4 through 18-7 is The water-splitting activity is an integral part of the photosystem II reaction center, and it has proved exceptionally difficult to purify. The detailed structure of the Mn cluster is not yet known. Manganese can exist in stable oxidation states from +2 to +7, so a cluster of four Mn ions can certainly donate or accept four electrons; the chemical details of this process, however, remain to be clarified. Light Absorption by Photosystem I Creates a Powerful fteducing Agent The photochemical events that follow excitation of photosystem I (P700) are formally similar to those in photosystem II (following the general scheme of Figure 18-40). Light is first captured by any one of about 200 chlorophyll a and b molecules, or additional accessory pigments, that serve as antennae, and the absorbed energy moves to P700 by resonance energy transfer. The excited reaction center P700* loses an electron to an acceptor, Ao (believed to be a special form of chlorophyll, functionally analogous to the pheophytin of photosystem II), creating Ao and P700+ (Fig. 18-41b); again, excitation has resulted in charge separation at the photochemical reaction center. P700+ is a strong oxidizing agent, which quickly acquires an electron from plastocyanin, a soluble Cu-containing electron transfer protein. A0- is an exceptionally strong reducing agent, which passes its electron through a chain of carriers that leads to NADP+ (Fig. 18-44). First, phylloquinone (A1) accepts an electron from A0 and passes it on to an iron-sulfur protein. From here, the electron moves to ferredoxin (Fd), another iron-sulfur protein loosely associated with the thylakoid membrane. Spinach ferredoxin (Mr 10,700), which has been isolated and crystallized, contains a 2Fe-2S center (Fig. 18-5). The Fe atoms of ferredoxin transfer electrons via one-electron Fe2+ to Fe3+ valence changes The fourth electron carrier in the chain is a flavoprotein called ferredoxin-NADP+ oxidoreductase. It transfers electrons from reduced ferredoxin (Fd2+red) to NADP+, reducing the latter to NADPH: Photosystems I and II Cooperate to Carry Electrons from H2O to NADP+ Early studies by Robert Emerson established that maximum rates of photosynthesis, measured as O2 evolution, required light of at least two wavelengths, now known to excite photosystems I and II. The two photosystems must function together in the O2-evolving light reactions of photosynthesis. The diagram in Figure 18-44, often called the Z scheme because of its overall form, outlines the pathway of electron flow between the two photosystems as well as the energy relationships in the light reactions. When photons are absorbed by photosystem I, electrons are expelled from the reaction center and flow down a chain of electron carriers to NADP+ to reduce it to NADPH. P700+, transiently electrondeficient, accepts an electron expelled by illumination of photosystem II, which arrives via a second connecting chain of electron carriers. This leaves an "electron hole" in photosystem II, which is filled by electrons from H2O. We have described how water is split to yield: (1) electrons, which are donated to the electron-deficient photosystem II; (2) H+ ions (protons), which are released inside the thylakoid lumen; and (3) O2, which is released into the gas phase. The Z scheme thus describes the complete route by which electrons flow from H2O to NADP+ according to the equation 2H2O + 2 NADP+ + 8 photons ----------> O2 + 2NADPH + 2H+ For each electron transferred from H2O to NADP+, two photons are absorbed, one by each photosystem. To form one molecule of O2, which requires transfer of four electrons from two H2O to two NADP+, a total of eight photons must be absorbed, four by each photosystem. The Cytochrome bf Complex Links Photosystems II and I Electrons stored in QBH2 as a result of the excitation of P680 in photosystem II (Fig. 18-42c) are carried to P700 of photosystem I via an assembly of several integral membrane proteins known as the cytochrome bf complex and the soluble protein plastocyanin (Fig. 18-44). The purified cytochrome bf complex contains a b-type cytochrome with two heme groups (cytochrome b563), an iron-sulfur protein (Mr 20,000), and cytochrome f (named for the Latin frons, meaning "leaf"), also called cytochrome c552?Electrons flow through the cytochrome bf complex from QBH2 to cytochrome f; the detailed path is uncertain. Cytochrome f passes its electron to plastocyanin, the donor for P700 reduction (Figs. 18-41b, 18-44). The cytochrome bf complex of plants is remarkably similar to the cytochrome bc1 complex (Complex III) of the mitochondrial electron transfer chain, and it carries out a similar function. Both complexes convey electrons from a reduced quinone, a mobile, lipid-soluble carrier of two electrons (UQ in mitochondria, QB in chloroplasts), to a watersoluble protein that carries one electron (cytochrome c in mitochondria, plastocyanin in chloroplasts). As in mitochondria, the function of this complex involves a Q cycle (see Fig. 18-10) in which electrons pass from QBH2 to cytochrome b one at a time. As in the mitochondrial Complex III, this cycle results in the pumping of protons across the membrane; in chloroplasts, the direction of proton movement is from the stromal compartment to the thylakoid lumen. The result is the production of a proton gradient across the thylakoid membrane as electrons pass from photosystem II to photosystem I (Fig. 18-45). Because the volume of the flattened thylakoid lumen is small, the influx of a small number of protons has a relatively large effect on lumenal pH. The measured difference in pH between the stroma (pH 8) and the thylakoid lumen (pH 4.5) represents a 3,000-fold difference in proton concentration-a powerful driving force for ATP synthesis.

Photosynthesis: Harvesting Light Energy

We now turn to another reaction sequence in which the flow of electrons is coupled to the synthesis of ATP: light-driven phosphorylation. The capture of solar energy by photosynthetic organisms and its conversion into the chemical energy of reduced organic compounds is the ultimate source of nearly all biological energy. Photosynthetic and heterotrophic organisms live in a balanced steady state in the biosphere (Fig. 18-32). Photosynthetic organisms trap solar energy and form ATP and NADPH, which they use as energy sources to make carbohydrates and other organic components from CO₂ and H₂O; simultaneously, they release O₂ into the atmosphere. Aerobic heterotrophs (we humans, for example) use the O₂ so formed to degrade the energy-rich organic products of photosynthesis to CO₂ and H₂O, generating ATP for their own activities. The CO₂ formed by respiration in heterotrophs returns to the atmosphere, to be used again by photosynthetic organisms. Solar energy thus provides the driving force for the continuous cycling of atmospheric CO₂ and O₂ through the biosphere and provides the reduced substrates (fuels), such as glucose, on which nonphotosynthetic organisms depend.

Enormous amounts of energy are stored as products of photosynthesis. Each year at least 1017 kJ of free energy from sunlight is captured and used for biosynthesis by photosynthetic organisms. This is more than ten times the fossil-fuel energy used each year by people the world over. Even fossil fuels (coal, oil, and natural gas) are the products of photosynthesis that took place millions of years ago. Because of our global dependence upon solar energy, past and present, for both energy and food, discovering the mechanism of photosynthesis is a central goal of biochemical research. The overall equation of photosynthesis describes an oxidationreduction reaction in which H20 donates electrons (as hydrogen) for the reduction of COZ to carbohydrate (CH2O): CO2 + H2O  ------> O2 + (CH2O) Unlike NADH (the hydrogen donor in oxidative phosphorylation), H2O is a poor electron donor; its standard reduction potential is 0.82 V, compared with -0.32 V for NADH. The central difference between photophosphorylation and oxidative phosphorylation is that the latter process begins with a good electron donor, whereas the former requires the input of energy in the form of light to create a good electron donor. Except for this crucial difference, the two processes are remarkably similar. In photophosphorylation, electrons flow through a series of membrane-bound carriers including cytochromes, quinones, and ironsulfur proteins, while protons are pumped across a membrane to create an electrochemical potential. This potential is the driving force for ATP synthesis from ADP and Pi by a membrane-bound ATP synthase complex closely similar to that which functions in oxidative phosphorylation. Photosynthesis encompasses two processes: the light reactions, which occur only when plants are illuminated, and the carbon fixation reactions, or so-called dark reactions, which occur in both light and darkness (Fig. 18-33). In the light reactions, chlorophyll and other pigments of the photosynthetic cells absorb light energy and conserve it in chemical form as the two energy-rich products ATP and NADPH; simultaneously, O2 is evolved. In the carbon fixation reactions, ATP and NADPH are used to reduce CO2 to form glucose and other organic products. The formation of O2, which occurs only in the light, and the reduction of CO2, which does not require light, thus are distinct and separate processes. In this chapter we are concerned only with the light reactions; the reduction of CO2 is described in Chapter 19. In photosynthetic eukaryotic cells, both the light and carbon fixation reactions take place in the chloroplasts (Fig. 18-34). When solar energy is not available, mitochondria in the plant cell generate ATP by oxidizing carbohydrates originally produced in chloroplasts in the light. Chloroplasts may assume many different shapes in different species, and they usually have a much larger volume than mitochondria. They are surrounded by a continuous outer membrane, which, like the outer mitochondrial membrane, is permeable to small molecules and ions. An inner membrane system encloses the internal compartment, in which there are many flattened, membrane-surrounded vesicles or sacs, called thylakoids, which are usually arranged in stacks called grana. The thylakoid membranes are separate from the inner chloroplast membrane. Embedded in the thylakoid membranes are the photosynthetic pigments and all the enzymes required for the primary light reactions. The fluid in the compartment surrounding the thylakoids, the stroma, contains most of the enzymes required for the carbon fixation reactions, in which CO2 is reduced to form triose phosphates and, from them, glucose. Our discussion here focuses on the nature of the light-absorbing systems and their roles in photosynthesis. Light Produces Electron Flow in Chloroplasts How do the pigment molecules of the thylakoid membranes transduce absorbed light energy into chemical energy? The key to answering this question came from a discovery made in 1937 by Robert Hill, a pioneer in photosynthesis research. He found that when leaf extracts containing chloroplasts were supplemented with a nonbiological hydrogen acceptor and then illuminated, evolution of O2 and simultaneous reduction of the hydrogen acceptor took place, according to an equation now known as the Hill reaction: 2H2O + 2A light  > 2AH2 + O2 where A is the artiiicial hydrogen acceptor. One of the nonbiological hydrogen acceptors used by Hill was the dye 2,6-dichlorophenolindophenol, now called a Hill reagent, which in its oxidized form (A) is blue and in its reduced form (AH2) is colorless. When the leaf extract supplemented with the dye was illuminated, the blue dye became colorless and O2 was evolved. In the dark neither O2 evolution nor dye reduction took place. This was the iirst specific clue to how absorbed light energy is converted into chemical energy: it causes electrons to flow from H2O to an electron acceptor. Moreover, Hill found that CO2 was not required for this reaction, nor was it reduced to a stable form under these conditions. He therefore concluded that O2 evolution can be dissociated from CO2 reduction. Several years later it was found that NADP+ is the biological electron acceptor in chloroplasts, according to the equation 2H2O + 2NADP+ light  > 2 NADPH + 2H+ + O2 This equation shows an important distinction between mitochondrial oxidative phosphorylation and the analogous process in chloroplasts: in chloroplasts electrons flow from H2O to NADP+, whereas in mitochondrial respiration electrons flow in the opposite direction, from NADH or NADPH to O2, with the release of free energy. Because lightinduced electron flow in chloroplasts is in the reverse or "uphill" direction, from H2O to NADP+, it cannot occur without the input of free energy; this energy comes from light. To understand how this occurs, we must first consider the effects of light absorption on molecular structure. Absorption of Light Excites Molecules Visible light is electromagnetic radiation of wavelengths 400 to 700 nm, a small part of the electromagnetic spectrum (Fig. 18-35), ranging from violet to red. The energy of a single photon (a quantum of light) is greater at the violet end of the spectrum than at the red end. The energy in a "mole" of photons (one einstein; 6 × 1023 photons) is 170 to 300 kJ, about an order of magnitude more than the energy required to synthesize a mole of ATP from ADP and Pi (about 30 kJ under standard conditions). The energy of an einstein in the infrared or microwave regions of the spectrum is too small to be useful in the kinds of photochemical events that occur in photosynthesis. UV light and x rays, on the other hand, have so much energy that they damage proteins and nucleic acids and induce mutations that are often lethal. The ability of a molecule to absorb light depends upon the arrangement of electrons around the atomic nuclei in its structure. When a photon is absorbed, an electron is lifted to a higher energy level. This happens on an all-or-none basis; to be absorbed, the photon must contain a quantity of energy (a quantum) that exactly matches the energy of the electronic transition. A molecule that has absorbed a photon is in an excited state, which is generally unstable. The electrons lifted into higher-energy orbitals usually return rapidly to their normal lower-energy orbitals; the excited molecule reverts (decays) to the stable ground state, giving up the absorbed quantum as light or heat or using it to do chemical work. The light emitted upon decay of excited molecules, called fluorescence, is always of a longer wavelength (lower energy) than the absorbed light. Excitation of molecules by light and their fluorescent decay are extremely fast processes, occurring in about 10-15 and 10-12 s, respectively. The initial events in photosynthesis are therefore very rapid. Chlorophylls Absorb Light Energy for Photosynthesis The most important light-absorbing pigments in the thylakoid membranes are the chlorophylls, green pigments with polycyclic, planar structures resembling the protoporphyrin of hemoglobin (see Fig. 718), except that Mg2+, not Fe2+, occupies the central position (Fig. 1836). Chlorophyll a, present in the chloroplasts of all green plant cells, contains four substituted pyrrole rings, one of which (ring IV) is reduced, and a fifth ring that is not a pyrrole. All chlorophylls have a long phytol side chain, esterified to a carboxyl-group substituent in ring IV. The four inward-oriented nitrogen atoms of chlorophyll a are coordinated with the Mg2+.  The heterocyclic five-ring system that surrounds the Mg2+ has an extended polyene structure, with alternating single and double bonds. Such polyenes characteristically show strong absorption in the visible region of the spectrum (Fig. 18-37); the chlorophylls have unusually high molar absorption coefficients (see Box 5-1) and are therefore particularly well-suited for absorbing visible light during photosynthesis. Chloroplasts of higher plants always contain two types of chlorophyll. One is invariably chlorophyll a, and the second in many species is chlorophyll b, which has an aldehyde group instead of a methyl group attached to ring II (Fig. 18-36). Although both are green, their absorption spectra are slightly different (Fig. 18-37), allowing the two pigments to complement each other's range of light absorption in the visible region. Most higher plants contain about twice as much chlorophyll a as chlorophyll b. The bacterial chlorophylls differ only slightly from the plant pigments (Fig. 18-36). Accessory Pigments Also Absorb Light In addition to chlorophylls, the thylakoid membranes contain secondary light-absorbing pigments, together called the accessory pigments, the carotenoids and phycobilins. Carotenoids may be yellow, red, or purple. The most important are β-carotene (Fig. 18-36), a red-orange isoprenoid compound that is the precursor of vitamin A in animals, and the yellow carotenoid xanthophyll. The carotenoid pigments absorb light at wavelengths other than those absorbed by the chlorophylls (Fig. 18-37) and thus are supplementary light receptors. Phycobilins are linear tetrapyrroles that have the extended polyene system found in chlorophylls, but not their cyclic structure or central Mg2+. Examples are phycoerythrin and phycocyanin (Fig. 18-36). The relative amounts of the chlorophylls and the accessory pigments are characteristic for different plant species. It is variation in the proportions of these pigments that is responsible for the range of colors of photosynthetic organisms, which vary from the deep bluegreen of spruce needles, to the greener green of maple leaves, to the red, brown, or even purple color of different species of multicellular algae and the leaves of some decorative plants. Experimental determination of the effectiveness of light of different colors in promoting photosynthesis yields an action spectrum (Fig. 18-38), often useful in identifying the pigment primarily responsible for a biological effect of light. By capturing light in a region of the spectrum not used by other plants, a photosynthetic organism can claim its unique ecological niche. For example, the phycobilins, present only in red algae and cyanobacteria, absorb in the region 520 to 630 nm, allowing these organisms to live in niches where light of lower or higher wavelength has been filtered out by the pigments of other organisms living in the water above them, or by the water itsel? Chlorophyll Funnels Absorbed Energy to Reaction Centers The light-absorbing pigments of thylakoid membranes are arranged in functional sets or arrays called photosystems. In spinach chloroplasts each photosystem contains about 200 molecules of chlorophylls and about 50 molecules of carotenoids. The clusters can absorb light over the entire visible spectrum but especially well between 400 to 500 nm and 600 to 700 nm (Fig. 18-37). All the pigment molecules in a photosystem can absorb photons, but only a few can transduce the light energy into chemical energy. A transducing pigment consists of several chlorophyll molecules combined with a protein complex also containing tightly bound quinones; this complex is called a photochemical reaction center. The other pigment molecules in a photosystem are called light-harvesting or antenna molecules. They function to absorb light energy and transmit it at a very high rate to the reaction center where the photochemical reactions occur (Fig. 18-39, p. 578), which will be described in detail later. The chlorophyll molecules in thylakoid membranes are bound to integral membrane proteins (chlorophyll a/b-binding, or CAB, proteins) that orient the chlorophyll relative to the plane of the membrane and confer light absorption properties that are subtly different from those of free chlorophyll. When isolated chlorophyll molecules in vitro are excited by light, the absorbed energy is quickly released as fluorescence and heat, but when chlorophyll in intact spinach leaves is excited by visible light (Fig. 18-40 (step l ), p. 579), very little fluorescence is observed. Instead, a direct transfer of energy from the excited chlorophyll (an antenna chlorophyll) to a neighboring chlorophyll molecule occurs, exciting the second molecule and allowing the first to return to its ground state (step 2). This resonance energy transfer is repeated to a third, fourth, or subsequent neighbor, until the chlorophyll at the photochemical reaction center becomes excited (step 3). In this special chlorophyll molecule, an electron is promoted by excitation to a higher-energy orbital. This electron then passes to a nearby electron acceptor that is part of the electron transfer chain of the chloroplast, leaving the excited chlorophyll molecule with an empty orbital (an "electron hole") (step 4 ). The electron acceptor thus acquires a negative charge. The electron lost by the reaction-center chlorophyll is replaced by an electron from a neighboring electron donor molecule (step 5 ), which becomes positively charged. In this way, excitation by Light causes electric charge separation and initiates an oxidationreduction chain. Coupled to the light-dependent electron flow along this chain are processes that generate ATP and NADPH.

Oxidative Phosphorylation Is Regulated by Cellular Energy Needs

The rate of respiration (O₂ consumption) in mitochondria is under tight regulation; it is generally limited by the availability of ADP as a substrate for phosphorylation. As we saw in Figure 18-13b, the respiration rate in isolated mitochondria is low in the absence of ADP and increases strikingly with the addition of ADP; this phenomenon is part of the definition of coupling of oxidation and phosphorylation. The dependence of the rate of O₂ consumption on the concentration of the Pi acceptor ADP, called acceptor control of respiration, can be dramatic. In some animal tissues the acceptor control ratio, the ratio of the maximal rate of ADP-induced O₂ consumption to the basal rate in the absence of ADP, is at least 10.
The intracellular concentration of ADP is one measure of the energy status of cells. Another, related measure is the mass-action ratio of the ATP-ADP system: [ATP]/([ADP][Pi]). Normally this ratio is very high, so that the ATP-ADP system is almost fully phosphorylated. When the rate of some energy-requiring process in cells (protein synthesis, for example) increases, there is an increased rate of breakdown of ATP to ADP and Pi, lowering the mass-action ratio. With more ADP available for oxidative phosphorylation, the rate of respiration increases, causing regeneration of ATP. This continues until the massaction ratio returns to its normal high level, at which point respiration slows again. The rate of oxidation of cell fuels is regulated with such sensitivity and precision that the ratio [ATP]/([ADP][Pi] fluctuates only slightly in most tissues, even during extreme variations in energy demand. In short, ATP is formed only as fast as it is used in energyrequiring cell activities.

Uncoupled Mitochondria in Brown Fat Produce Heat

There is a remarkable and instructive exception to the general rule that respiration slows when the ATP supply is adequate. In most mammals, including humans, newborns have a type of adipose tissue called brown fat in which fuel oxidation serves not to produce ATP, but to generate heat to keep the newborn warm. Hibernating mammals (grizzly bears, for example) also have large amounts of brown fat. This specialized adipose tissue, located at the back of the neck of human infants, is brown because of the presence of large numbers of mitochondria and thus large amounts of cytochromes, whose heme groups are strong absorbers of visible light.

The mitochondria of brown fat oxidize fuels (particularly fatty acids) normally, passing electrons through the respiratory chain to O2. This electron transfer is accompanied by proton pumping out of the matrix, as in other mitochondria. The mitochondria of brown fat, however, have a unique protein in their inner membrane: thermogenin, also called the uncoupling protein (UCP) (Table 18-4). This protein, an integral membrane protein, provides a path for protons to return to the matrix without passing through the F0F1 complex (Fig. 18-27). As a result of this short-circuiting of protons, the energy of oxidation is not conserved by ATP formation but is dissipated as heat, which contributes to maintaining the body temperature. For the hairless newborn infant, maintaining body heat is an important use of metabolic energy. Hibernating animals depend on uncoupled mitochondria of brown fat to generate heat during their long winter period of dormancy ATP-Producing Pathways Are Coordinately Regulated The major catabolic pathways (glycolysis, the citric acid cycle, fatty acid and amino acid oxidation, and oxidative phosphorylation) have interlocking and concerted regulatory mechanisms that allow them to function together in an economical and self regulating manner to produce ATP and biosynthetic precursors. The relative concentrations of ATP and ADP control not only the rates of electron transfer and oxidative phosphorylation but also the rates of the citric acid cycle, pyruvate oxidation, and glycolysis (Fig. 18-28). Whenever there is an increased drain on ATP, the rate of electron transfer and oxidative phosphorylation increases. Simultaneously, the rate of pyruvate oxidation via the citric acid cycle increases, thus increasing the flow of electrons into the respiratory chain. These events can in turn evoke an increase in the rate of glycolysis, increasing the rate of pyruvate formation. When the conversion of ADP to ATP lowers the ADP concentration, acceptor control will slow electron transfer and thus oxidative phosphorylation. Glycolysis and the citric acid cycle will also slow, because ATP is an allosteric inhibitor of phosphofructokinase-1 (see Fig. 14-20) and of pyruvate dehydrogenase (see Fig. 15-14). Phosphofructokinase-1 is inhibited not only by ATP but by citrate, the first intermediate of the citric acid cycle. When both ATP and citrate are elevated they produce a concerted allosteric inhibition that is greater than the sum of their individual effects. In many types of tumor cells, this interlocking coordination appears to be defective: glycolysis proceeds at a higher rate than required by the citric acid cycle. As a result, cancer cells use far more blood glucose than normal cells, but cannot oxidize the excess pyruvate formed by rapid glycolysis, even in the presence of O2. To reoxidize cytoplasmic NADH, most of the pyruvate is reduced to lactate (Chapter 14), which passes from the cells into the blood. The high glycolytic rate may result in part from smaller numbers of mitochondria in tumor cells. In addition, some tumor cells overproduce an isozyme of hexokinase that associates with the cytosolic face of the mitochondrial inner membrane and is insensitive to feedback inhibition by glucose-6phosphate (p. 432). This enzyme may monopolize the ATP produced in mitochondria, using it to produce glucose-6-phosphate and committing the cell to continue glycolysis. Mutations in Mitochondrial Genes Cause Human Disease Mitochondria contain their own genome, a circular, double-stranded DNA molecule. There are 37 genes (16,569 base pairs) in the human mitochondrial chromosome (Fig. 18-29), including 13 that encode proteins of the respiratory chain (Table 18-7); the remaining genes code for ribosomal and transfer RNA molecules essential to the proteinsynthesizing machinery of mitochondria. Many of the mitochondrial proteins are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, then imported and assembled within mitochondria. Mutations in the mitochondrial DNA are known to cause the human disease called Leber's hereditary optic neuropathy (LHON). This rare genetic disease affects the central nervous system, including the optic nerves, causing bilateral loss of vision, of rapid onset, in early adulthood. The disease is invariably inherited from the female parent, a consequence of the fact that all of the mitochondria of the developing embryo are derived from the mother. The unfertilized egg contains many mitochondria, and the few mitochondria in the much smaller sperm cell do not enter the egg at the time of fertilization. Mitochondria arise only from the division of preexisting mitochondria. A single base change in the mitochondrial gene ND4 (Fig. 18-29a) changes an Arg residue to a His residue in one of the proteins of Complex I, and the result is mitochondria partially defective in electron transfer from NADH to ubiquinone. Although these mitochondria are able to produce some ATP by electron transfer from succinate, they apparently cannot supply ATP in sufficient quantity to support the very active metabolism of neurons. One result is damage to the optic nerve, leading to blindness. A single base change in the mitochondrial gene for cytochrome b, a component of Complex III, also produces LHON, demonstrating that the pathology of the disease results from a general reduction of mitochondrial function, not just from a defect in electron transfer through Complex I. Another serious human genetic disease, myoclonic epilepsy and ragged-red fiber disease (MERRF) is caused by a mutation in the mitochondrial gene that encodes a transfer RNA (Fig. 18-29). This disease, characterized by uncontrollable muscular jerking, apparently results from defective production of several of the proteins synthesized using mitochondrial transfer RNAs. Skeletal muscle fibers of individuals with MERRF have abnormally shaped mitochondria that sometimes contain paracrystalline structures (Fig. 18-29b). Mitochondria Probably Evolved from Endosymbiotic Bacteria The fact that mitochondria contain their own DNA, ribosomes, and transfer RNAs supports the theory of the endosymbiotic origin of mitochondria (see Fig. 2-17). This theory supposes that the first organisms capable of aerobic metabolism, including respiration-linked ATP production, were prokaryotes. Primitive eukaryotes that lived anaerobically (by fermentation) acquired the ability to carry out oxidative phosphorylation when they established a symbiotic relationship with bacteria living in their cytosol. After much evolution and the movement of many bacterial genes into the nucleus of the "host" eukaryote, the endosymbiotic bacteria eventually became mitochondria. This theory presumes that early free-living prokaryotes had the enzymatic machinery for oxidative phosphorylation, and it predicts that their modern prokaryotic descendents have respiratory chains closely similar to those of modern eukaryotes. They do. Aerobic bacteria carry out NAD-linked electron transfer from substrates to O2, coupled to the phosphorylation of cytosolic ADP. The dehydrogenases are located in the bacterial cytosol, but the electron carriers of the respiratory chain are in the plasma membrane. The electron carriers are similar to those of mitochondria, act in the same sequence (Fig. 18-30), and translocate protons outward across the plasma membrane concomitantly with electron transfer to O2. We noted earlier that bacteria such as E. coli have F0F1 complexes in their plasma membranes; the F1 portions protrude into the cytosol and catalyze ATP synthesis from ADP and Pi as protons flow back into the cell through proton channels formed by F0. Figure 18-30 Respiratory chain in the inner membrane of E. coli. Electrons from NADH pass to menaquinone (MQ), at which point the chain branches. The upper path is dominant in cells grown under normal aerobic conditions, but when O2 is the limiting factor in growth, the lower route predominates. Certain bacterial transport systems bring about uptake of extracellular nutrients (lactose, for example) against a concentration gradient, in symport with protons (see Fig. 10-25). The respiration-linked transmembrane proton extrusion provides the driving force for this uptake. The rotary motion of bacterial flagella, which move cells through their surroundings, is provided by "proton turbines," molecular rotary motors driven not by ATP but directly by the transmembrane electrochemical potential generated by respiration-linked proton pumping (Fig. 18-31). It appears likely that the chemiosmotic mechanism evolved early (before the emergence of eukaryotes). The protonmotive force can clearly be used to power processes other than ATP synthesis.

Strong Experimental Evidence Implicates the Proton-Motive Force in ATP Synthesis

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 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. 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. 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 Proton-Motive Force Energizes Active Transport 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 ADP3- on the outside (cytosolic) surface of the inner membrane and transports it inward in exchange for an ATP4- 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. 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 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). Shuttle Systems Are Required for Mitochondrial Oxidation of Cytosolic NADH 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. 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. Oxidative Phosphorylation Produces Most of the ATP Made in Aerobic Cells 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  and two ATP, six NADH, and two FADH2 from citric acid cycle reactions in the matrix 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 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. 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 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. 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.