21.7 How Is Carbon Dioxide Used to Make Organic Molecules? 653 3-phosphoglycerate. In the course of this metabolic sequence, the NADPH and ATP produced in the light reactions are consumed, as indicated earlier in Equation 21.3. The Calvin cycle of reactions starts with ribulose bisphosphate carboxylase catalyzing formation of 3-phosphoglycerate from CO 2 and RuBP and concludes with ribulose-5- phosphate kinase (also called phosphoribulose kinase), which forms RuBP (Figure 21.24 and Table 21.1). The carbon balance is given at the right side of Table 21.1. Several features of the reactions in this table merit discussion. Note that a total of 18 equiva- lents of ATP consumed in hexose formation are expended (reactions 2 and 15): 12 to form 12 equivalents of 1,3-bisphosphoglycerate from 3-phosphoglycerate by a reversal of the normal glycolytic reaction catalyzed by 3-phosphoglycerate kinase and six to phosphorylate Ru-5-P to regenerate 6 RuBP. All 12 NADPH equivalents are used in reaction 3. Plants possess an NADPH-specific glyceraldehyde-3-phosphate dehydrogenase, which contrasts with its glycolytic counterpart in its specificity for NADP over NAD and in the direction in which the reaction normally proceeds. The Calvin Cycle Reactions Can Account for Net Hexose Synthesis When carbon rearrangements are balanced to account for net hexose synthesis, five of the glyceraldehyde-3-phosphate molecules are converted to dihydroxyacetone phosphate (DHAP). Three of these DHAPs then condense with three glyceraldehyde- 3-P via the aldolase reaction to yield three hexoses in the form of fructose bisphos- phate (Figure 21.24). (Recall that the ⌬G°Ј for the aldolase reaction in the glycolytic direction is ϩ23.9 kJ/mol. Thus, the aldolase reaction running “in reverse” in the Calvin cycle would be thermodynamically favored under standard-state conditions.) Taking one FBP to glucose, the desired product of this scheme, leaves 30 carbons, dis- tributed as 2 fructose-6-phosphates, 4 glyceraldehyde-3-phosphates, and 2 DHAP. These 30 Cs are reorganized into 6 RuBP by reactions 9 through 15. Step 9 and steps 12 through 14 involve carbohydrate rearrangements like those in the pentose phosphate pathway (see Chapter 22). Reaction 11 is mediated by sedoheptulose-1,7- bisphosphatase. This phosphatase is unique to plants; it generates sedoheptulose-7-P, Reactions 1 through 15 constitute the cycle that leads to the formation of one equivalent of glucose. The enzyme catalyzing each step, a concise reaction, and the overall carbon balance are given. Numbers in parentheses show the numbers of carbon atoms in the substrate and product molecules. Prefix numbers indicate in a stoichiometric fashion how many times each step is carried out in order to provide a balanced net reaction. 1. Ribulose bisphosphate carboxylase: 6 CO 2 ϩ 6 H 2 O ϩ 6 RuBP 88n 12 3-PG 6(1) ϩ 6(5) 88n 12(3) 2. 3-Phosphoglycerate kinase: 12 3-PG ϩ 12 ATP 88n 12 1,3-BPG ϩ 12 ADP 12(3) 88n 12(3) 3. NADP ϩ -glyceraldehyde-3-P dehydrogenase: 12 1,3-BPG ϩ 12 NADPH 88n 12 NADP ϩ ϩ 12 G-3-P ϩ 12 P i 12(3) 88n 12(3) 4. Triose-P isomerase: 5 G-3-P 88n 5 DHAP 5(3) 88n 5(3) 5. Aldolase: 3 G-3-P ϩ 3 DHAP 88n 3 FBP 3(3) ϩ 3(3) 88n 3(6) 6. Fructose bisphosphatase: 3 FBP ϩ 3 H 2 O 88n 3 F-6-P ϩ 3 P i 3(6) 88n 3(6) 7. Phosphoglucoisomerase: 1 F-6-P 88n 1 G-6-P 1(6) 88n 1(6) 8. Glucose phosphatase: 1 G-6-P ϩ 1 H 2 O 88n 1 GLUCOSE ϩ 1 P i 1(6) 88n 1(6) The remainder of the pathway involves regenerating six RuBP acceptors (ϭ 30 C) from the leftover two F-6-P (12 C), four G-3-P (12 C), and two DHAP (6 C). 9. Transketolase: 2 F-6-P ϩ 2 G-3-P 88n 2 Xu-5-P ϩ 2 E-4-P 2(6) ϩ 2(3) 88n 2(5) ϩ 2(4) 10.Aldolase: 2 E-4-P ϩ 2 DHAP 88n 2 sedoheptulose-1,7-bisphosphate (SBP) 2(4) ϩ 2(3) 88n 2(7) 11.Sedoheptulose bisphosphatase: 2 SBP ϩ 2 H 2 O 88n 2 S-7-P ϩ 2 P i 2(7) 88n 2(7) 12.Transketolase: 2 S-7-P ϩ 2 G-3-P 88n 2 Xu-5-P ϩ 2 R-5-P 2(7) ϩ 2(3) 88n 4(5) 13.Phosphopentose epimerase: 4 Xu-5-P 88n 4 Ru-5-P 4(5) 88n 4(5) 14.Phosphopentose isomerase: 2 R-5-P 88n 2 Ru-5-P 2(5) 88n 2(5) 15.Phosphoribulose kinase: 6 Ru-5-P ϩ 6 ATP 88n 6 RuBP ϩ 6 ADP 6(5) 88n 6(5) Net: 6 CO 2 ϩ 18 ATP ϩ 12 NADPH ϩ 12 H ϩ ϩ 12 H 2 O 88n glucose ϩ 18 ADP ϩ 18 P i ϩ 12 NADP ϩ 6(1) 88n 1(6) TABLE 21.1 The Calvin Cycle Series of Reactions 654 Chapter 21 Photosynthesis 1 15 2 4 5 6 9 7 10 11 14 13 12 3 8 ATP 12 6 6 5 COO – COO – + CO H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – H 2 COPO 3 2 – Ribulose-1,5-bis- phosphate (RuBP) HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HCOH HOCH HOCH HOCH HCOH HCOH HCOH HCOH HOCH HCOH HCOH Two 3-Phospho- glycerates (3-PG) 6 CO 2 HOCH 1,3-Bisphospho- glycerate (BPG) C O OPO 3 2 – + Glyceraldehyde- 3-phosphate (G-3-P) 12 12 CHO CO H 2 COH H 2 COH H 2 COH H 2 COH H 2 COH Ribulose- 5-phosphate (Ru-5-P) CO Dihydroxyacetone phosphate (DHAP) CO Fructose-1,6- bisphosphate (FBP) Glucose HOCH CO Fructose-6- phosphate (F-6-P) Erythrose-4- phosphate (E4P) CHO H 2 COPO 3 2 – HCOH HCOH HCOH Glucose-6- phosphate (G-6-P) CHO CO Sedoheptulose-1,7- bisphosphate (SBP) HOCH CO Sedoheptulose-7- phosphate (S-7-P) CO Xylulose-5- phosphate (Xu-5-P) Ribose-5- phosphate (R-5-P) CHO 6 12 12 12 12 12 P i P i 3 P i 2 P i 4 2 2 2 2 2 4 22 2 1 2 2 2 2 3 2 3 4 7 ATP ADP ADP NADP + NADPH 6 Phosphoribulose kinase Ribulose bisphos- phate carboxylase Phospho- glycerate kinase Glyceraldehyde- 3-phosphate dehydrogenase Triose phosphate isomerase Aldolase Fructose bisphosphatase Phospho- glucoisomerase Glucose-6- phosphatase Aldolase Sedoheptulose bisphosphatase Phospho- pentose isomerase Phosphopentose epimerase Transketolase Transketolase 21.7 How Is Carbon Dioxide Used to Make Organic Molecules? 655 the seven-carbon sugar serving as the transketolase substrate. Likewise, phosphoribu- lose kinase carries out the unique plant function of providing RuBP from Ru-5-P (reaction 15). The net conversion accounts for the fixation of 6 equivalents of carbon dioxide into 1 hexose at the expense of 18 ATP and 12 NADPH. The Carbon Dioxide Fixation Pathway Is Indirectly Activated by Light Plant cells contain mitochondria and can carry out cellular respiration (glycolysis, the citric acid cycle, and oxidative phosphorylation) to provide energy in the dark. Futile cycling of carbohydrate to CO 2 by glycolysis and the citric acid cycle in one direction, and CO 2 to carbohydrate by the CO 2 fixation pathway in the opposite direction, is thwarted through regulation of the Calvin cycle (Figure 21.25). In this regulation, the activities of key Calvin cycle enzymes are coordi- nated with the output of photosynthesis. In effect, these enzymes respond indi- rectly to light activation. Thus, when light energy is available to generate ATP and NADPH for CO 2 fixation, the Calvin cycle proceeds. In the dark, when ATP and NADPH cannot be produced by photosynthesis, fixation of CO 2 ceases. The light- induced changes in the chloroplast which regulate key Calvin cycle enzymes in- clude (1) changes in stromal pH, (2) generation of reducing power, and (3) Mg 2ϩ efflux from the thylakoid lumen. Light Induces pH Changes in Chloroplast Compartments As discussed in Sec- tion 21.6, illumination of chloroplasts leads to light-driven pumping of pro- tons into the thylakoid lumen, which causes pH changes in both the stroma and the thylakoid lumen (Figure 21.26). The stromal pH rises, typically to pH 8. Because rubisco and rubisco activase are more active at pH 8, CO 2 fixation is activated as stro- mal pH rises. Fructose-1,6-bisphosphatase, ribulose-5-phosphate kinase, and glyceraldehyde-3- phosphate dehydrogenase all have alkaline pH optima. Thus, their activities increase as a result of the light-induced pH increase in the stroma. Light Energy Generates Reducing Power Illumination of chloroplasts initiates pho- tosynthetic electron transport, which generates reducing power in the form of re- duced ferredoxin. In turn, Fd red leads to reduced thioredoxin via ferredoxin–thioredoxin reductase (FTR) (Figure 21.27). Thioredoxin is a small (12 kD) protein possessing in its reduced state a pair of sulfhydryls (OSH HSO), which upon oxidation form a disulfide bridge (OSOSO). Several Calvin cycle enzymes have pairs of cysteine residues that are involved in a disulfide–sulfhydryl transition between an inactive (OSOSO) and an active (OSH HSO) form. These enzymes include fructose-1,6- bisphosphatase (residues Cys 174 and Cys 179 ), NADP ϩ -malate dehydrogenase (residues Cys 10 and Cys 15 ), and ribulose-5-phosphate kinase (residues Cys 16 and Cys 55 ). Thus, light acti- vates these key enzymes through this ferredoxin-dependent, thioredoxin-mediated pathway (Figure 21.27). ᮤ ACTIVE FIGURE 21.24 The Calvin–Benson cycle of reactions.The number associated with the arrow at each step indicates the number of molecules reacting in a turn of the cycle that produces one molecule of glucose. Reactions are numbered as in Table 21.1. Test yourself on the concepts in this figure at www.cengage.com/login. + CO 2 + H 2 O Hexose ADP P i ADP P i Light + + C O 2 f i x a t i o n G l y c o l y s i s , c i t r i c a c i d c y c l e c y c l e ATP ATP FIGURE 21.25 Light regulation of CO 2 fixation prevents a substrate cycle between cellular respiration and hexose synthesis by CO 2 fixation. 8 dark 6 pH Stroma Thylakoid space lightdark ANIMATED FIGURE 21.26 Light- induced pH changes in chloroplast compartments. These pH changes modulate the activity of key Calvin cycle enzymes. See this figure animated at www .cengage.com/login. PSI Fd ox Fd red FTR FTR SH SH S S T T Active enzyme Inactive enzyme S SH SH S S S SH SH FIGURE 21.27 The pathway for light-regulated reduction of Calvin cycle enzymes. Light-generated reducing power (Fd red ϭ reduced ferredoxin) provides e Ϫ for reduction of thioredoxin (T) by FTR. 656 Chapter 21 Photosynthesis Light Induces Movement of Mg 2؉ Ions from the Thylakoid Vesicles into the Stroma When light-driven proton pumping across the thylakoid membrane occurs, a con- comitant efflux of Mg 2ϩ ions from vesicles into the stroma is observed. This efflux of Mg 2ϩ somewhat counteracts the charge accumulation due to H ϩ influx and is one rea- son why the membrane potential change in response to proton pumping is less in chloroplasts than in mitochondria (see Equation 21.5). Both ribulose bisphosphate car- boxylase and fructose-1,6-bisphosphatase are Mg 2ϩ -activated enzymes, and Mg 2ϩ flux into the stroma as a result of light-driven proton pumping stimulates the CO 2 fixation path- way at these key steps. Activity measurements have indicated that fructose bisphos- phatase may be the rate-limiting step in the Calvin cycle. The recurring theme of fruc- tose bisphosphatase as the target of the light-induced changes in the chloroplasts implicates this enzyme as a key point of control in the Calvin cycle. Protein–Protein Interactions Mediated by an Intrinsically Unstructured Protein Also Regulate Calvin–Benson Cycle Activity The 8.5-kD chloroplast protein CP12 is an instrinsically unstructured protein (see Chapter 6) that interacts with ribulose-5-P kinase (R5PK) and glyceraldehyde-3-P de- hydrogenase (GAPDH) to form a complex. When complexed with CP12, R5PK and GAPDH are relatively inactive. Note that the first of these enzymes is ATP-dependent and the second is NADPH-dependent. Thus, formation of this complex constrains the use of ATP and NADPH, the principal products of the light reactions of photosynthe- sis. Reduced thioredoxin, through interactions with CP12, leads to the dissociation of the GAPDH–CP12–R5PK complex. Uncomplexed R5PK and GAPDH are inherently more active, and operation of the Calvin–Benson cycle is enhanced. 21.8 How Does Photorespiration Limit CO 2 Fixation? As indicated, ribulose bisphosphate carboxylase/oxygenase catalyzes an alternative reaction in which O 2 replaces CO 2 as the substrate added to RuBP (Figure 21.28a). The ribulose-1,5-bisphosphate oxygenase reaction diminishes plant productivity because it leads to loss of RuBP, the essential CO 2 acceptor. The K m for O 2 in this oxygenase reaction is about 200 M. Given the relative abundance of CO 2 and O 2 in the at- mosphere and their relative K m values in these rubisco-mediated reactions, the ra- tio of carboxylase to oxygenase activity in vivo is about 3 or 4 to 1. The products of ribulose bisphosphate oxygenase activity are 3-phosphoglycerate and phosphoglycolate. Dephosphorylation and oxidation convert phosphoglycolate to glyoxylate, the ␣-keto acid of glycine (Figure 21.28b). Transamination yields glycine. In mitochondria, two glycines from photorespiration are converted into one serine and CO 2 . This step is the source of the CO 2 evolved during photorespi- ration. Transamination of glyoxylate to glycine by the product serine yields hydroxy- pyruvate; reduction of hydroxypyruvate yields glycerate, which can be phosphory- lated to 3-phosphoglycerate. 3-Phosphoglycerate can fuel resynthesis of ribulose bisphosphate by the Calvin cycle (see Figure 21.24). Other fates of phosphoglycolate are also possible, including oxidation to CO 2 , with the released energy being dissipated as heat. Obviously, agricultural productivity is dramatically lowered by this phenomenon, which, because it is a light-related uptake of O 2 and release of CO 2 , is termed photorespiration. As we shall see, certain plants, particularly tropical grasses, have evolved means to circumvent photorespiration. These plants are more efficient users of light for carbohydrate synthesis. Tropical Grasses Use the Hatch–Slack Pathway to Capture Carbon Dioxide for CO 2 Fixation Tropical grasses are less susceptible to the effects of photorespiration, as noted earlier. Studies using 14 CO 2 as a tracer indicated that the first organic intermediate labeled in these plants was not a three-carbon compound but a four-carbon compound. Hatch 21.8 How Does Photorespiration Limit CO 2 Fixation? 657 and Slack, two Australian biochemists, first discovered this C-4 product of CO 2 fixa- tion, and the C-4 pathway of CO 2 incorporation is named the Hatch–Slack pathway after them. The C-4 pathway is not an alternative to the Calvin cycle series of reactions or even a net CO 2 fixation scheme. Instead, it functions as a CO 2 delivery system, car- rying CO 2 from the relatively oxygen-rich surface of the leaf to interior cells where oxygen is lower in concentration and hence less effective in competing with CO 2 in the rubisco reaction. Thus, the C-4 pathway is a means of avoiding photorespiration by sheltering the rubisco reaction in a cellular compartment away from high [O 2 ]. The C-4 compounds serving as CO 2 transporters are malate or aspartate. Compartmentation of these reactions to prevent photorespiration involves the in- teraction of two cell types: mesophyll cells and bundle sheath cells. The mesophyll cells take up CO 2 at the leaf surface, where O 2 is abundant, and use it to carboxylate phospho- enolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 21.29). This four-carbon dicarboxylic acid is then either reduced to malate by an NADPH- specific malate dehydrogenase or transaminated to give aspartate in the mesophyll cells. The 4-C CO 2 carrier (malate or aspartate) is then transported to the bundle sheath cells, where it is decarboxylated to yield CO 2 and a 3-C product. The CO 2 is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, CO 2 O 2 O 2 + H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– CO HCOH HCOH + O – O – O O – O O – O O – O O – O O – O O – O O C HCOH C C C C C C C H 2 COH H 2 COH P i H 2 COH H 2 COH HCOH C OH H 2 O 2 CO H + + CH 2 NH 3 + NH 3 + HC + 2 Glycine NAD + NADH ATP ADP Ribulose bisphosphate carboxylase/ oxygenase 3-Phospho- glycerate Ribulose bisphosphate Phospho- glycolate Glycerate Glyoxylate Glycolate oxygenase Glycolate Phosphoglycolate phosphatase Serine Hydroxypyruvate Glycine Transamination Serine (a) (b) FIGURE 21.28 The oxygenase reaction of rubisco. (a) The reaction of ribulose bisphosphate carboxylase with O 2 and ribulose bisphosphate yields 3-phosphoglycerate and phosphoglycolate. (b) Conver- sion of two phosphoglycolates to serine ϩ CO 2 . 658 Chapter 21 Photosynthesis and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another CO 2 (Figure 21.29). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of CO 2 uptake (C3 plants). Intercellular Transport of Each CO 2 Via a C-4 Intermediate Costs 2 ATPs The trans- port of each CO 2 requires the expenditure of two high-energy phosphate bonds. The energy of these bonds is expended in the phosphorylation of pyruvate to PEP (phos- phoenolpyruvate) by the plant enzyme pyruvate-P i dikinase; the products are PEP, AMP, and pyrophosphate (PP i ). This represents a unique phosphotransferase reaction in that both the - and ␥-phosphates of a single ATP are used to phosphorylate the two substrates, pyruvate and P i . The reaction mechanism involves an enzyme phosphohisti- dine intermediate. The ␥-phosphate of ATP is transferred to P i , whereas formation of E-His-P occurs by addition of the -phosphate from ATP: EOHis ϩ AMP ␣ OP  OP ␥ ϩ P i ⎯⎯→ EOHisOP  ϩ AMP ␣ ϩ P ␥ P i EOHisOP  ϩ pyruvate ⎯⎯→ PEP ϩ EOHis Net: ATP ϩ pyruvate ϩ P i ⎯⎯→ AMP ϩ PEP ϩ PP i Pyruvate-P i dikinase is regulated by reversible phosphorylation of a threonine residue, the nonphosphorylated form being active. Interestingly, ADP is the phos- phate donor in this interconvertible regulation. Despite the added metabolic ex- pense of two phosphodiester bonds for each equivalent of carbon dioxide taken up, CO 2 fixation is more efficient in C4 plants, provided that light intensities and tem- peratures are both high. (As temperature rises, photorespiration in C3 plants rises and efficiency of CO 2 fixation falls.) Tropical grasses that are C4 plants include sugarcane, maize, and crabgrass. In terms of photosynthetic efficiency, cultivated fields of sugarcane represent the pinnacle of light-harvesting efficiency. Approxi- mately 8% of the incident light energy on a sugarcane field appears as chemical en- ergy in the form of CO 2 fixed into carbohydrate. This efficiency compares dramat- ically with the estimated photosynthetic efficiency of 0.2% for uncultivated plant areas. Research on photorespiration is actively pursued in hopes of enhancing the PEP + P P P Oxaloacetate Pyruvate Malate Pyruvate Malate Ribulose- 1,5-bisphosphate + Calvin cycle 2 3-Phospho- glycerates Mesophyll cell AMP NADP + NADP + NADPH NADPH Bundle sheath cell P CO 2 CO 2 ATP Glucose FIGURE 21.29 Essential features of the compartmentation and biochemistry of the Hatch–Slack pathway of carbon dioxide uptake in C4 plants. Summary 659 efficiency of agriculture by controlling this wasteful process. Only 1% of the 230,000 different plant species known are C4 plants; most are in hot climates. Cacti and Other Desert Plants Capture CO 2 at Night In contrast to C4 plants, which have separated CO 2 uptake and fixation into distinct cells in order to minimize photorespiration, succulent plants native to semiarid and tropical environments separate CO 2 uptake and fixation in time. Carbon dioxide (as well as O 2 ) enters the leaf through microscopic pores known as stomata, and wa- ter vapor escapes from plants via these same openings. In nonsucculent plants, the stomata are open during the day, when light can drive photosynthetic CO 2 fixation, and closed at night. Succulent plants, such as the Cactaceae (cacti) and Crassulaceae, cannot open their stomata during the heat of day because any loss of precious H 2 O in their arid habitats would doom them. Instead, these plants open their stomata to take up CO 2 only at night, when temperatures are lower and water loss is less likely. This carbon dioxide is immediately incorporated into PEP to form OAA by PEP car- boxylase; OAA is then reduced to malate by malate dehydrogenase and stored within vacuoles until morning. During the day, the malate is released from the vac- uoles and decarboxylated to yield CO 2 and a 3-C product. The CO 2 is then fixed into organic carbon by rubisco and the reactions of the Calvin cycle. Because this process involves the accumulation of organic acids (OAA, malate) and is common to succulents of the Crassulaceae family, it is referred to as crassulacean acid metabo- lism, and plants capable of it are called CAM plants. SUMMARY 21.1 What Are the General Properties of Photosynthesis? Photosyn- thesis takes place in membranes. In photosynthetic eukaryotes, the pho- tosynthetic membranes form an inner membrane system within chloro- plasts that is called the thylakoid membrane system. Photosynthesis is traditionally broken down into two sets of reactions: the light reactions, whereby light energy is used to generate NADPH and ATP concomitant with O 2 evolution, and the dark reactions in which NADPH and ATP provide the chemical energy for fixation of CO 2 into glucose. Water is the ultimate e Ϫ donor for NADP ϩ reduction. 21.2 How Is Solar Energy Captured by Chlorophyll? Chlorophyll and various accessory lig ht-harvesting pigments absorb light through- out the visible spectrum and use the light energy to initiate electron- transfer reactions. The absorption of a photon of light by a pigment molecule promotes an electron of the pigment molecule to a higher orbital (and higher energy level). As a result, the pigment molecule is a much better electron donor. Photosynthetic units consist of arrays of hundreds of chlorophyll molecules and accessory light-harvesting pig- ments, but only a single reaction center. The reaction center is formed from a pair of Chl molecules. 21.3 What Kinds of Photosystems Are Used to Capture Light Energy? Photosynthetic bacteria have a single photosystem, but eukaryotic pho- totrophs have two distinct photosystems. Type I photosystems use pro- teins with Fe 4 S 4 clusters as terminal e Ϫ acceptors; type II photosystems reduce quinones, such as plastoquinone or phylloquinone. In oxygenic phototrophs (cyanobacteria, green algae, and higher plants), photo- system II (PSII) generates a strong oxidant that functions in O 2 evolu- tion through the photolysis of water and a weak reductant that reduces plastoquinone to plastohydroquinone (PQH 2 ). Photosystem I (PSI) generates a weak oxidant that accepts electrons from plastohydro- quinone via the cytochrome b 6 f complex and a strong reductant capa- ble of reducing NADP ϩ to NADPH. Overall photosynthetic electron transfer is accomplished by three supramolecular membrane-spanning complexes: PSII, the cytochrome b 6 f complex, and PSI. Oxygen evolu- tion requires the accumulation of four oxidizing equivalents in PSII. The electrons withdrawn from water are used to re-reduce P680 ϩ back to P680, restoring its ability to absorb another photon, become P680*, and transfer an e Ϫ once again. Electrons from P680* traverse PSII and reduce plastoquinone. Plastohydroquinone is oxidized via the cyto- chrome b 6 f complex, with plastocyanin serving as e Ϫ acceptor. The cy- tochrome b 6 f complex catalyzes a Q cycle: It translocates 4 H ϩ from the stroma to the thylakoid lumen for each molecule of PQH 2 that it oxi- dizes. PSI is a light-driven plastocyaninϺferredoxin oxidoreductase hav- ing P700 as its reaction center Chl dimer. Electrons from P700* are transferred to the Fe 4 S 4 cluster of ferredoxin. Reduced ferredoxin reduces NADP ϩ via the ferredoxinϺNADP ϩ reductase flavoprotein. The electron “hole” in P700 ϩ is filled by reduced plastocyanin. 21.4 What Is the Molecular Architecture of Photosynthetic Reaction Centers? All known photosynthetic reaction centers have a universal molecular architecture. The “core” structure is a pair of protein sub- units having (at least) five transmembrane ␣-helical segments that pro- vide a scaffold upon which the reaction center Chl pair and its associ- ated chain of electron transfer cofactors are arrayed in a characteristic spatial pattern that facilitates rapid removal of an electron from the photoactivated RC and efficient transfer of the e Ϫ across the membrane to a terminal acceptor (such as a quinone or a ferredoxin molecule). Photosynthetic electron transport is always coupled to H ϩ translocation across the membrane, creating the potential for ATP synthesis by F 1 F 0 - type ATP synthases. 21.5 What Is the Quantum Yield of Photosynthesis? The absorption of light energy by the photosynthetic apparatus is very efficient. The quantum yield of chemical energy, either in the form of ATP and NADPH, or in the form of glucose, depends on a number of factors that are still subject to investigation, including the H ϩ /e Ϫ ratio and the ATP/H ϩ ratio. 21.6 How Does Light Drive the Synthesis of ATP? Photosynthetic elec- tron transport leads to proton translocation across the photosynthetic membrane and creation of an H ϩ gradient that can be used by an F 1 F 0 - type ATP synthase to drive ATP formation from ADP and P i . Pho- tophosphorylation occurs by either of two modes: noncyclic and cyclic. 660 Chapter 21 Photosynthesis Noncyclic photophosphorylation depends on both PSI and PSII and leads to O 2 evolution, NADP ϩ reduction, and ATP synthesis. In cyclic photophosphorylation, only PSI is used, no NADP ϩ is reduced, and no O 2 is evolved. However, the electron-transfer events of cyclic pho- tophosphorylation lead to H ϩ translocation and ATP synthesis. 21.7 How Is Carbon Dioxide Used to Make Organic Molecules? Ribulose-1,5-bisphosphate is the CO 2 acceptor in the key reaction for conversion of carbon dioxide into organic compounds. The reaction is catalyzed by rubisco (ribulose bisphosphate carboxylase/oxygenase); the products of CO 2 fixation by the rubisco reaction are 2 equivalents of 3-phosphoglycerate. The Calvin–Benson cycle is a series of reactions that converts the 3-phosphoglycerates formed by rubisco into carbohydrates such as glyceraldehyde-3-P, dihydroxyacetone-P, and glucose. CO 2 fixation is activated by light through a variety of mechanisms, including changes in stromal pH, generation of reducing power in the form of ferre- doxin by photosynthetic electron transport, and increased Mg 2ϩ efflux from the thylakoid lumen to the stroma. 21.8 How Does Photorespiration Limit CO 2 Fixation? When O 2 replaces CO 2 in the rubisco, or ribulose bisphosphate carboxylase/ oxygenase, reaction, ribulose-1,5-bisP is destroyed through conversion into 3-phosphoglyerate and phosphoglycolate. Phosphoglycolate is oxi- dized to form CO 2 , with loss of organic substance from the cell. Because O 2 is taken up and CO 2 is released in these reactions, the process is called photorespiration. Tropical grasses carry out the Calvin–Benson cycle of reactions in cells shielded from high O 2 levels. CO 2 is first incorporated into PEP by PEP carboxylase to form oxaloacetate (OAA) in the mesophyll cells on the leaf surface. OAA is then reduced to malate and transported to bundle sheath cells. There, CO 2 is released and taken up by the rubisco reaction to form 3-phosphoglyerate, initiating the Calvin–Benson cycle. Plants capable of doing this are called C4 plants. Succulent plants of semiarid and tropical regions such as Cactaceae and Crassulaceae exchange gases through their stomata only at night in order to avoid precious water loss. CO 2 taken up at night is added to PEP by PEP carboxylase to form OAA, which is then reduced to malate in the dark. During the day, malate is decarboxylated to yield CO 2 , which then enters the Calvin–Benson cycle. This metabolic variation is referred to as crassulacean acid metabolism. PROBLEMS Preparing for an exam? Create your own study path for this chapter at www.cengage.com/login. 1. In photosystem I, P700 in its ground state has an Ᏹ o Ј ϭ ϩ0.4 V. Exci- tation of P700 by a photon of 700-nm light alters the Ᏹ o Ј of P700* to Ϫ0.6 V. What is the efficiency of energy capture in this light reaction of P700? 2. What is the Ᏹ o Ј for the light-generated primary oxidant of photo- system II if the light-induced oxidation of water (which leads to O 2 evolution) proceeds with a ⌬G°Ј of Ϫ25 kJ/mol? 3. (Integrates with Chapters 3 and 20.) Assuming that the concen- trations of ATP, ADP, and P i in chloroplasts are 3, 0.1, and 10 mM, respectively, what is the ⌬G for ATP synthesis under these condi- tions? Photosynthetic electron transport establishes the proton- motive force driving photophosphorylation. What redox potential difference is necessary to achieve ATP synthesis under the forego- ing conditions, assuming 1.3 ATP equivalents are synthesized for each electron pair transferred? 4. (Integrates with Chapter 20.) Write a balanced equation for the Q cycle as catalyzed by the cytochrome b 6 f complex of chloroplasts. 5. If noncyclic photosynthetic electron transport leads to the translo- cation of 3 H ϩ /e Ϫ and cyclic photosynthetic electron transport leads to the translocation of 2 H ϩ /e Ϫ , what is the relative photosyn- thetic efficiency of ATP synthesis (expressed as the number of pho- tons absorbed per ATP synthesized) for noncyclic versus cyclic photophosphorylation? (Assume that the CF 1 CF 0 –ATP synthase yields 3 ATP/14 H ϩ .) 6. (Integrates with Chapter 20.) In mitochondria, the membrane po- tential (⌬) contributes relatively more to ⌬p (proton-motive force) than does the pH gradient (⌬pH). The reverse is true in chloro- plasts. Why do you suppose that the proton-motive force in chloro- plasts can depend more on ⌬pH than mitochondria can? Why is (⌬) less in chloroplasts than in mitochondria? 7. Predict the consequences of a Y161F mutation in the amino acid se- quence of the D1 subunit of PSII. 8. (Integrates with Chapter 20.) Calculate (in Einsteins and in kJ/mol) how many photons would be required by the Rhodopseudomonas viridis photophosphorylation system to synthesize 3 ATPs. (Assume that the R. viridis F 1 F 0 –ATP synthase c-subunit rotor contains 12 c-subunits and that the R. viridis cytochrome bc 1 complex translocates 2 H ϩ /e Ϫ .) 9. (Integrates with Chapters 18 and 20.) Calculate ⌬G°Ј for the NADP ϩ -specific glyceraldehyde-3-P dehydrogenase reaction of the Calvin–Benson cycle. 10. Write a balanced equation for the synthesis of a glucose molecule from ribulose-1,5-bisphosphate and CO 2 that involves the first three reactions of the Calvin cycle and subsequent conversion of the two glyceraldehyde-3-P molecules into glucose. 11. 14 C-labeled carbon dioxide is administered to a green plant, and shortly thereafter the following compounds are isolated from the plant: 3-phosphoglycerate, glucose, erythrose-4-phosphate, sedoheptulose-1,7-bisphosphate, and ribose-5-phosphate. In which carbon atoms will radioactivity be found? 12. The photosynthetic CO 2 fixation pathway is regulated in response to specific effects induced in chloroplasts by light. What is the nature of these effects, and how do they regulate this metabolic pathway? 13. Write a balanced equation for the conversion of phosphoglycolate to glycerate-3-P by the reactions of photorespiration. Does this balanced equation demonstrate that photorespiration is a wasteful process? 14. The overall equation for photosynthetic CO 2 fixation is 6 CO 2 ϩ 6 H 2 O ⎯⎯→ C 6 H 12 O 6 ϩ 6 O 2 All the O atoms evolved as O 2 come from water; none comes from car- bon dioxide. But 12 O atoms are evolved as 6 O 2 , and only 6 O atoms appear as 6 H 2 O in the equation. Also, 6 CO 2 have 12 O atoms, yet there are only 6 O atoms in C 6 H 12 O 6 . How can you account for these discrepancies? (Hint: Consider the partial reactions of photosynthe- sis: ATP synthesis, NADP ϩ reduction, photolysis of water, and the overall reaction for hexose synthesis in the Calvin–Benson cycle.) 15. The number of c-subunits in F 1 F 0 -type ATP synthases shows some variation from organism to organism. For example, the yeast ATP synthase contains 10 c-subunits, the spinach CF 1 CF 0 –ATP synthase has 14, and the cyanobacterium Spirulina platensis enzyme appar- ently has 15. a. What is the consequence of c-subunit stoichiometry for the H ϩ /ATP ratio? b. What is the relationship between c-subunit stoichiometry and the magnitude of ⌬p (the proton-motive force)? 16. The reduction of membrane-associated quinones, such as coen- zyme Q and plastoquinones, is a common feature of photosystems (see Figures 21.15, 21.20, and 21.21). Assume Ᏹ o Ј for PQ/PQH 2 ϭ 0.07 V and the potential of the ground-state chlorophyll molecule ϭ 0.5 V, calculate ⌬G for the reduction of plastoquinone by a. 870-nm light. b. 700-nm light. c. 680-nm light. Further Reading 661 17. Plastoquinone oxidation by cytochrome bc 1 and cytochrome b 6 f complexes apparently leads to the translocation of 4 ϩ /2e Ϫ . If Ᏹ o Ј for cytochrome f ϭ 0.365 V (Table 20.1) and E o Ј for PQ/PQH 2 ϭ 0.07 V, calculate ⌬G for the coupled reaction: 2 h ϩ 4 H ϩ in ⎯⎯→4 H ϩ out (Assume a value of 23 kJ/mol for the free energy change (⌬G) as- sociated with moving protons from inside to outside.) 18. What is the overall free energy change (⌬G) for noncyclic photo- synthetic electron transport? 4 (700-nm photons) ϩ 4 (680-nm photons) ϩ 2 H 2 O ϩ 2 NADP ϩ ⎯⎯→ O 2 ϩ 2 NADPH ϩ 2 H ϩ Preparing for the MCAT Exam 19. From Figure 21.5, predict the spectral properties of accessory light- harvesting pigments found in plants. 20. Draw a figure analogous to Figure 21.26, plotting [Mg 2ϩ ] in the stroma and thylakoid lumen on the y-axis and dark-light-dark on the x-axis. FURTHER READING General References Blankenship, R. E., 2002. Molecular Mechanisms of Photosynthesis. Malden, MA: Blackwell Science. Buchanan, B. B., Gruissem, W., and Jones, R. I., 2000. Biochemistry and Molecular Biology of Plants. Rockville, MD: American Society of Plant Physiologists. Cramer, W. A., and Knaff, D. B., 1990. Energy Transduction in Biological Membranes—A Textbook of Bioenergetics. New York: Springer-Verlag. Harold, F. M., 1987. The Vital Force: A Study of Bioenergetics. Chapter 8: Harvesting the Light. San Francisco: Freeman & Company. Heathcote, P., Fyfe, P. K., and Jones, M. R., 2002. Reaction centers: The structure and evolution of biological solar power. Trends in Biochem- ical Sciences 27:79–87. Photosynthetic Pigments Glazer, A. N., 1983. Comparative biochemistry of photosynthetic light- harvesting pigments. Annual Review of Biochemistry 52:125–157. Green, B. R., and Durnford, D. G., 1996. The chlorophyll-carotenoid proteins of oxygenic photosynthesis. Annual Review of Plant Physiol- ogy and Plant Molecular Biology 47:685–714. Hoffman, E., et al., 1996. Structural basis of light harvesting by carote- noids: Peridinin-chlorophyll protein from Amphidinium carterae. Sci- ence 272:1788–1791. Properties of the Thylakoid Membranes Anderson, J. M., 1986. Photoregulation of the composition, function and structure of the thylakoid membrane. Annual Review of Plant Physiology 37:93–136. Anderson, J. M., and Anderson, B., 1988. The dynamic photosynthetic membrane and regulation of solar energy conversion. Trends in Bio- chemical Sciences 13:351–355. Photosynthetic Reaction Centers of Photosynthetic Bacteria Deisenhofer, J., and Michel, H., 1989. The photosynthetic reaction cen- ter from the purple bacterium Rhodopseudomonas viridis. Science 245: 1463–1473. Deisenhofer, J., Michel, H., and Huber, R., 1985. The structural basis of light reactions in bacteria. Trends in Biochemical Sciences 10:243–248. Deisenhofer, J., et al., 1985. Structure of the protein subunits in the pho- tosynthetic reaction center of Rhodopseudomonas viridis at 3 Å reso- lution. Nature 318:618–624; also Journal of Molecular Biology (1984) 180:385–398. Structure and Function of Photosystems I and II and the Cytochrome b 6 f Complex Amunts, A., Drory, O., and Nelson, N., 2007. The structure of a plant photosystem I supercomplex at 3.4 Å resolution. Nature 447:58–63. Barber, J., 2003. Photosystem II: The engine of life. Quarterly Review of Biophysics 36:71–89. Cramer, W. A., et al., 2006. Transmembrane traffic in the cytochrome b 6 f complex. Annual Review of Biochemistry 75:769–790. Merchant, S., and Sawaya, M. R., 2005. The light reactions: A guide to recent acquisitions for the picture gallery. Plant Cell 17:648–663. Nelson, N., and Yocum, C. F., 2006. Structure and function of photosys- tems I and II. Annual Review of Plant Biology 57:521–566. Yano, J., et al., 2006. Where water is oxidized to dioxygen: Structure of the photosynthetic Mn 4 Ca cluster. Science 314:821–825. Zouni, A., et al., 2001. Crystal structure of photosystem II from Syne- chococcus elongatus at 3.8 Å resolution. Nature 409:739–743. Photophosphorylation Allen, J. F., 2002. Photosynthesis of ATP—electrons, proton pumps, ro- tors, and poise. Cell 110:273–276. Arnon, D. I., 1984. The discovery of photosynthetic phosphorylation. Trends in Biochemical Sciences 9:258–262. Avenson, T. J., et al., 2005. Regulating the proton budget of higher plant photosynthesis. Proceedings of the National Academy of Sciences U.S.A. 102:9709–9713. Jagendorf, A. T., and Uribe, E., 1966. ATP formation caused by acid-base transition of spinach chloroplasts. Proceedings of the National Academy of Sciences U.S.A. 55:170–177. Remy, A., and Gerwert, K., 2003. Coupling of light-induced electron transfer to proton uptake in photosynthesis. Nature Structural Biology 10:637–644. Seelert, H., Dencher, N., and Müller, D. J., 2003. Fourteen protomers compose the oligomer III of the proton-rotor in spinach chloroplast ATP synthase. Journal of Molecular Biology 333:337–344. Shikanai, T., 2007. Cyclic electron transport around photosystem I: Genetic approaches. Annual Review of Plant Biology 58:199–217. Carbon Dioxide Fixation Burnell, J. N., and Hatch, M. D., 1985. Light–dark modulation of leaf pyruvate, P 1 dikinase. Trends in Biochemical Sciences 10:288–291. Cushman, J. C., and Bohnert, H. J., 1999. Crassulacean acid metabolism: Molecular genetics. Annual Review of Plant Physiology and Plant Molec- ular Biology 50:305–332. Graciet, E., Lebreton, S., and Gontero, B, 2004. Emergence of new reg- ulatory mechanisms in the Benson–Calvin pathway via protein– protein interactions: A glyceraldehyde-3-phosphate dehydrogenase/ CP12/phosphoribulokinase complex. Journal of Experimental Botany 55:1245–1254. Hatch, M. D., 1987. C 4 photosyntheis: A unique blend of modified bio- chemistry, anatomy, and ultrastructure. Biochimica Biophysica Acta 895:81–106. Kaplan, A., and Reinhold, L., 1999. CO 2 -concentrating mechanisms in photosynthetic organisms. Annual Review of Plant Physiology and Plant Molecular Biology 50:539–570. Knaff, D. B., 1989. The regulatory role of thioredoxin in chloroplasts. Trends in Biochemical Sciences 14:433–434. Portis, A. R., Jr., 1992. Regulation of ribulose 1,5-bisphosphate carboxy- lase/oxygenase activity. Annual Review of Plant Physiology and Plant Molecular Biology 43:415–437. Spreitzer, R. J., and Salvucci, M. E., 2002. Rubisco: Structure, regulatory interactions, and possibilities for a better enzyme. Annual Review of Plant Biology 53:449–475. Wingler, A., et al., 2000. Photorespiration: Metabolic pathways and their role in stress protection. Philosophical Transactions of the Royal Society of London B 355:1517–1529. © Jason Alan/iStockphoto.com 22 Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway 22.1 What Is Gluconeogenesis, and How Does It Operate? The ability to synthesize glucose from common metabolites is very important to most organisms. Human metabolism, for example, consumes about 160 Ϯ 20 grams of glucose per day, about 75% of this in the brain. Body fluids carry only about 20 grams of free glucose, and glycogen stores normally can provide only about 180 to 200 grams of free glucose. Thus, the body carries only a little more than a 1-day supply of glucose. If glucose is not obtained in the diet, the body must pro- duce new glucose from noncarbohydrate precursors. The term for this activity is gluconeogenesis, which means the generation (genesis) of new (neo) glucose. Furthermore, muscles consume large amounts of glucose via glycolysis, produc- ing large amounts of pyruvate. In vigorous exercise, muscle cells become anaerobic and pyruvate is converted to lactate. Gluconeogenesis salvages this pyruvate and lac- tate and reconverts it to glucose. Another pathway of glucose catabolism, the pentose phosphate pathway, is the pri- mary source of NADPH, the reduced coenzyme essential to most reductive biosyn- thetic processes. For example, NADPH is crucial to the biosynthesis of fatty acids (see Chapter 24) and amino acids (see Chapter 25). The pentose phosphate path- way also results in the production of ribose-5-phosphate, an essential component of ATP, NAD ϩ , FAD, coenzyme A, and particularly DNA and RNA. This important pathway will also be considered in this chapter. The Substrates for Gluconeogenesis Include Pyruvate, Lactate, and Amino Acids In addition to pyruvate and lactate, other noncarbohydrate precursors can be used as substrates for gluconeogenesis in animals. These include most of the amino acids, as well as glycerol and all the TCA cycle intermediates. On the other hand, fatty acids are not substrates for gluconeogenesis in animals, because most fatty acids yield only acetyl-CoA upon degradation, and animals cannot carry out net synthesis of sugars from acetyl-CoA. Lysine and leucine are the only amino acids that are not substrates for gluconeogenesis. These amino acids produce only acetyl-CoA upon degradation. Note also that acetyl-CoA can be a substrate for glu- coneogenesis in plants when the glyoxylate cycle is operating (see Chapter 19). Nearly All Gluconeogenesis Occurs in the Liver and Kidneys in Animals Interestingly, the mammalian organs that consume the most glucose, namely, brain and muscle, carry out very little glucose synthesis. The major sites of gluconeogen- A basket of fresh bread. Carbohydrates such as these provide a significant portion of human caloric intake. Con pan y vino se anda el camino. (With bread and wine you can walk your road.) Spanish proverb KEY QUESTIONS 22.1 What Is Gluconeogenesis, and How Does It Operate? 22.2 How Is Gluconeogenesis Regulated? 22.3 How Are Glycogen and Starch Catabolized in Animals? 22.4 How Is Glycogen Synthesized? 22.5 How Is Glycogen Metabolism Controlled? 22.6 Can Glucose Provide Electrons for Biosynthesis? ESSENTIAL QUESTIONS As shown in Chapters 18 and 19, the metabolism of sugars is an important source of energy for cells. Animals, including humans, typically obtain significant amounts of glucose and other sugars from the breakdown of starch in their diets. Glucose can also be supplied via breakdown of cellular reserves of glycogen (in animals) or starch (in plants). What is the nature of gluconeogenesis, the pathway that synthesizes glucose from noncarbohydrate precursors; how is glycogen synthesized from glucose; and how are electrons from glucose used in biosynthesis? Create your own study path for this chapter with tutorials, simulations, animations, and Active Figures at www.cengage.com/login. . www.cengage.com/login. 1. In photosystem I, P700 in its ground state has an Ᏹ o Ј ϭ ϩ0.4 V. Exci- tation of P700 by a photon of 700-nm light alters the Ᏹ o Ј of P700 * to Ϫ0.6 V. What is the efficiency. PSI is a light-driven plastocyaninϺferredoxin oxidoreductase hav- ing P700 as its reaction center Chl dimer. Electrons from P700 * are transferred to the Fe 4 S 4 cluster of ferredoxin. Reduced. ferredoxin reduces NADP ϩ via the ferredoxinϺNADP ϩ reductase flavoprotein. The electron “hole” in P700 ϩ is filled by reduced plastocyanin. 21.4 What Is the Molecular Architecture of Photosynthetic