20.4 CO2-Assimilation Reactions in Chapter 20 Photosynthesis and Carbohydrate Synthesis in Plants

20.4 ${CO}_{2}$ $bold upper C upper O bold 2$ -Assimilation Reactions

Photosynthetic organisms use the ATP and NADPH produced in the light-dependent reactions of photosynthesis to synthesize all of the thousands of components that make up the organism. Plants (and other autotrophs) can reduce atmospheric ${CO}_{2}$ $CO Subscript 2$ to trioses, then use the trioses as precursors for the synthesis of sucrose and starch, lipids and proteins, and the many other organic components of plant cells (Fig. 20-25). Lacking these synthetic capacities, humans and other animals are ultimately dependent on photosynthetic organisms to provide the reduced fuels and organic precursors essential to life.

A figure shows the products of photosynthesis. — FIGURE 20-25 Products of photosynthesis.

Green plants contain in their chloroplasts the enzymatic machinery that catalyzes the conversion of ${CO}_{2}$ $CO Subscript 2$ to simple (reduced) organic compounds, a process called ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation. This process has also been called ${CO}_{2}$ $bold upper C upper O bold 2$ fixation, but we reserve this term for the specific reaction in which ${CO}_{2}$ $CO Subscript 2$ is incorporated (fixed) into a three-carbon organic compound, the triose phosphate 3-phosphoglycerate. This simple product of photosynthesis is the precursor of more-complex biomolecules, including sugars, polysaccharides, and the metabolites derived from them, all of which are synthesized by metabolic pathways similar to those of animal tissues. Carbon dioxide is assimilated via a cyclic pathway, its key intermediates constantly regenerated. The pathway was elucidated in the early 1950s by Melvin Calvin, Andrew Benson, and James A. Bassham and is often called the Calvin cycle or, more descriptively, the reductive pentose phosphate pathway. It is essentially the reversal of a central pathway of glucose oxidation, the pentose phosphate pathway, which we described in Section 14.6.

Carbohydrate metabolism is more complex in plant cells than in animal cells or in nonphotosynthetic microorganisms. In addition to the universal pathways of glycolysis, gluconeogenesis, and the pentose phosphate pathway, plants have the unique reaction sequences for reduction of ${CO}_{2}$ $CO Subscript 2$ to triose phosphates and the associated reductive pentose phosphate pathway — all of which must be coordinately regulated to ensure proper allocation of carbon to energy production and synthesis of starch and sucrose. Key enzymes are regulated, as we shall see, by (1) reduction of disulfide bonds by electrons flowing from photosystem I and (2) changes in pH and ${Mg}^{2 +}$ $Mg Superscript 2 plus$ concentration that result from illumination. When we look at other aspects of plant carbohydrate metabolism, we also find enzymes that are modulated by (3) conventional allosteric regulation by one or more metabolic intermediates and (4) covalent modification (phosphorylation).

Carbon Dioxide Assimilation Occurs in Three Stages

The first stage in the assimilation of ${CO}_{2}$ $CO Subscript 2$ into biomolecules (Fig. 20-26) is the ${CO}_{2}$ $CO Subscript 2$ -fixation reaction: condensation of ${CO}_{2}$ $CO Subscript 2$ with a five-carbon acceptor, ribulose 1,5-bisphosphate, to form two molecules of 3-phosphoglycerate. In the second stage, the 3-phosphoglycerate is reduced to triose phosphates. Overall, three molecules of ${CO}_{2}$ $CO Subscript 2$ are fixed to three molecules of ribulose 1,5-bisphosphate to form six molecules of glyceraldehyde 3-phosphate (18 carbons). In the third stage, five of the six molecules of triose phosphate (15 carbons) are used to regenerate three molecules of ribulose 1,5-bisphosphate (15 carbons), the starting material. The sixth molecule of triose phosphate, the net product of photosynthesis, can be used to make hexoses for fuel and building materials, sucrose for transport to nonphotosynthetic tissues, or starch for storage. Thus, the overall process is cyclical, with the continuous conversion of ${CO}_{2}$ $CO Subscript 2$ to triose and hexose phosphates.

A figure shows the three stages of C O 2 assimilation in photosynthetic organisms: fixation, reduction, and regeneration of acceptor. — FIGURE 20-26 The three stages of ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation in photosynthetic organisms. Stoichiometries of three key intermediates (numbers in parentheses) reveal the fate of carbon atoms entering and leaving the photosynthetic carbon-reduction cycle (Calvin cycle). Three ${CO}_{2}$ $CO Subscript 2$ are fixed for the net synthesis of one molecule of glyceraldehyde 3-phosphate.

FIGURE 20-26 The three stages of ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation in photosynthetic organisms. Stoichiometries of three key intermediates (numbers in parentheses) reveal the fate of carbon atoms entering and leaving the photosynthetic carbon-reduction cycle (Calvin cycle). Three ${CO}_{2}$ $CO Subscript 2$ are fixed for the net synthesis of one molecule of glyceraldehyde 3-phosphate.

A circular series of reactions is shown. Ribulose 1,5-bisphosphate (3) is at the twelve o’clock position and is a five-carbon vertical chain with C 1 bonded to 2 H and O bonded to a circle labeled P; C 2 double bonded to O; C 3 and C 4 bonded to H and O H; and C 5 bonded to 2 H and O bonded to a circle labeled P. A blue arrow points clockwise and is joined by a blue arrow showing the addition of a yellow box labeled C O 2 (3). This is labeled stage 1: fixation and yields 3-phosphoglycerate (6) at the three o’clock position. 3-phosphoglycerate is a three-carbon vertical molecule with C 1 forming C O O minus, C 2 bonded to H and O H, and C 3 bonded to 2 H and O bonded to a circle labeled P. A blue arrow points down to the six o’clock position and is met by a curved blue arrow from an orange oval labeled A T P (5) to A D P (6). A second arrow points from her to glyceraldehyde 3-phospahte (6) at the nine o’clock position accompanied by a curved arrow to show the addition of an orange oval labeled N A D P H (6) and H plus to yield N A D P plus (6) and a branched arrow to show the loss of P subscript i end subscript (6). This is labeled stage 2: reduction. Glyceraldehyde 3-phosphate is a three-carbon vertical chain with C 1 forming C H O, C 2 bonded to H and O H, and C 3 bonded to 2 H and O bonded to a circle labeled P. A blue arrow labeled 1 points to a yellow box containing text reading, energy production via glycolysis; starch or sugar synthesis. A blue arrow labeled five points up to another blue arrow that meets a blue arrow accompanied by a curved blue arrow showing the addition of an orange oval labeled A T P (3) and low of A D P (3 ). This yields ribulose 1,5-bisphosphate (3). The series of steps between the nine o’clock and twelve o’clock positions are labeled stage 3: regeneration of acceptor. All data are approximate.

Fructose 6-phosphate is a key intermediate in stage 3 of ${CO}_{2}$ $CO Subscript 2$ assimilation; it stands at a branch point, leading either to regeneration of ribulose 1,5-bisphosphate or to synthesis of starch. The pathway from hexose phosphate to pentose bisphosphate involves many of the same reactions used in animal cells for the conversion of pentose phosphates to hexose phosphates during the nonoxidative phase of the pentose phosphate pathway (see Fig. 14-31). In the photosynthetic assimilation of ${CO}_{2}$ $CO Subscript 2$ , essentially the same set of reactions operates in the reverse direction, converting hexose phosphates to pentose phosphates. This reductive pentose phosphate cycle uses the same enzymes as the oxidative pathway, and several additional enzymes that make the reductive cycle irreversible. All 13 enzymes of the pathway are in the chloroplast stroma.

Stage 1: Fixation of ${CO}_{2}$ $bold upper C upper O bold 2$ into 3-Phosphoglycerate

An important clue to the nature of the ${CO}_{2}$ $CO Subscript 2$ -assimilation mechanisms in photosynthetic organisms came in the late 1940s. Calvin and his associates illuminated a suspension of green algae in the presence of radioactive carbon dioxide $(^{14} {CO}_{2})$ $left-parenthesis Superscript 14 Baseline CO Subscript 2 Baseline right-parenthesis$ for just a few seconds, then quickly killed the cells, extracted their contents, and used chromatographic methods to search for the metabolites in which the labeled carbon first appeared. The first compound that became labeled was 3-phosphoglycerate, with the $^{14} C$ $Superscript 14 Baseline upper C$ predominantly located in the carboxyl carbon atom. These experiments strongly suggested that 3-phosphoglycerate is an early intermediate in photosynthesis.

The many plants in which this three-carbon compound is the first intermediate are called $C_{3}$ $bold upper C bold 3$ plants, in contrast to the $C_{4}$ $upper C Subscript 4$ plants described below. Most plant species — 80% to 90% — are $C_{3}$ $upper C Subscript 3$ , including most trees, wheat, oats, rice, beans, peas, and spinach. The enzyme that catalyzes incorporation of ${CO}_{2}$ $CO Subscript 2$ into an organic form is ribulose 1,5-bisphosphate carboxylase/oxygenase, a name mercifully shortened to rubisco. As a carboxylase, rubisco catalyzes the covalent attachment of ${CO}_{2}$ $CO Subscript 2$ to the five-carbon sugar ribulose 1,5-bisphosphate and cleavage of the unstable six-carbon intermediate to form two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as ${CO}_{2}$ $CO Subscript 2$ in its carboxyl group (Fig. 20-26). The enzyme’s oxygenase activity is discussed in Section 20.5.

There are two distinct forms of rubisco. The rubisco of vascular plants, algae, and cyanobacteria is a crucial enzyme in the production of biomass from ${CO}_{2}$ $CO Subscript 2$ . It has a complex form I structure (Fig. 20-27a), with eight identical large catalytic subunits ( $M_{r} 53,000$ $upper M Subscript r Baseline 53,000$ ; encoded in the chloroplast genome), and eight identical small subunits ( $M_{r} 14,000$ $upper M Subscript r Baseline 14,000$ ; encoded in the nuclear genome) of uncertain function. The form II rubisco of photosynthetic bacteria is simpler, having two subunits that in many respects resemble the large subunits of the plant enzyme (Fig. 20-27b). The plant enzyme has an exceptionally low turnover number; only three molecules of ${CO}_{2}$ $CO Subscript 2$ are fixed per second per molecule of rubisco at 25 °C. To achieve high rates of ${CO}_{2}$ $CO Subscript 2$ fixation, plants therefore need large amounts of this enzyme. Rubisco is present at about 250 mg/mL in the chloroplast stroma, corresponding to an extraordinarily high concentration of active sites (~4 mm). In fact, rubisco makes up almost 50% of soluble protein in chloroplasts and is probably one of the most abundant enzymes in the biosphere.

A two-part figure shows the structure of form Roman numeral 1 rubisco in part a and form Roman numeral 2 rubisco in part b. — FIGURE 20-27 Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). (a) A ribbon model of form I rubisco from spinach. The enzyme has eight large (blue) and eight small (gray) subunits, tightly packed into a structure of $M_{r} > 500,000$ $upper M Subscript r Baseline greater-than 500,000$ . A transition-state analog, 2-carboxyarabinitol bisphosphate (yellow), is shown bound to each of the eight substrate-binding sites. ${Mg}^{2 +}$ $Mg Superscript 2 plus$ in the active site is shown in green. (b) Ribbon model of form II rubisco from the bacterium *Rhodospirillum rubrum*. The identical subunits are in gray and blue. [Data from (a) PDB ID 8RUC, I. Andersson, *J. Mol. Biol.* 259:160, 1996; (b) PDB ID 9RUB, T. Lundqvist and G. Schneider, *J. Biol. Chem.* 266:12,604, 1991.]

FIGURE 20-27 Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). (a) A ribbon model of form I rubisco from spinach. The enzyme has eight large (blue) and eight small (gray) subunits, tightly packed into a structure of $M_{r} > 500,000$ $upper M Subscript r Baseline greater-than 500,000$ . A transition-state analog, 2-carboxyarabinitol bisphosphate (yellow), is shown bound to each of the eight substrate-binding sites. ${Mg}^{2 +}$ $Mg Superscript 2 plus$ in the active site is shown in green. (b) Ribbon model of form II rubisco from the bacterium *Rhodospirillum rubrum*. The identical subunits are in gray and blue. [Data from (a) PDB ID 8RUC, I. Andersson, *J. Mol. Biol.* 259:160, 1996; (b) PDB ID 9RUB, T. Lundqvist and G. Schneider, *J. Biol. Chem.* 266:12,604, 1991.]

Part a is labeled form Roman numeral 1 rubisco and shows a circular, symmetrical structure that is open in the center. It has gray helices and strands at the twelve o’clock, three o’clock, six o’clock, and nine o’clock positions that form linear gray regions. In between, there are four roughly round blue regions of helices and strands. Each of these four regions contains a structure in the middle made of yellow spheres with some green spheres visible. The yellow spheres are labeled 2-Craboxyarabinitol biphosphate, and the green spheres are labeled M g 2 plus. Part b shows form Roman numeral 2 rubisco. Two almost horizontal gray helices are at the lower left and two almost vertical gray helices run up the center. Additional gray helices are to the left side and upper left. Between these gray pieces and a blue region of helices and strands at the center left, there is a linear structure made up of yellow spheres labeled ribulose 1,5-bisphosphate. A green sphere below these yellow spheres is labeled M g 2 plus and a structure made of red spheres below with a wireframe chain extending down is labeled carbamoyl-L y s residue. The right side has a similar yellow structure above a green sphere above a red structure with gray helices and strands to its left and above. A blue helix runs up the right center next to the gray helix running up the left of the center. Two blue helices run almost vertically across the bottom and more blue helixes are to the upper right. Blue strands are visible between the helices and the red structure. All data are approximate.

Central to the proposed mechanism for plant rubisco is a carbamoylated Lys side chain with a bound ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion. The ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion brings together and orients the reactants at the active site (Fig. 20-28), setting up for a nucleophilic attack by the five-carbon enediolate reaction intermediate formed on the enzyme (Fig. 20-29). The resulting six-carbon intermediate breaks down to yield two molecules of 3-phosphoglycerate.

A figure shows the position of M g 2 plus in the active site of rubisco. — FIGURE 20-28 Central role of ${Mg}^{2 +}$ $bold upper M g Superscript bold 2 plus$ in the active site of rubisco. ${Mg}^{2 +}$ $bold upper M g Superscript bold 2 plus$ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on ${Lys}^{201}$ $Lys Superscript 201$ ; two in the carboxyl groups of ${Glu}^{204}$ $Glu Superscript 204$ and ${Asp}^{203}$ $Asp Superscript 203$ ; two at C-2 and C-3 of the substrate, ribulose 1,5-bisphosphate; and one in the other substrate, ${CO}_{2}$ $CO Subscript 2$ . A water molecule occupies the ${CO}_{2}$ $CO Subscript 2$ -binding site in the crystal structure. In this figure, a ${CO}_{2}$ $CO Subscript 2$ molecule is modeled in its place. (Residue numbers refer to the spinach enzyme.) [Data from PDB ID 1RXO, T. C. Taylor and I. Andersson, *J. Mol. Biol.* 265:432, 1997.]

FIGURE 20-28 Central role of ${Mg}^{2 +}$ $bold upper M g Superscript bold 2 plus$ in the active site of rubisco. ${Mg}^{2 +}$ $bold upper M g Superscript bold 2 plus$ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on ${Lys}^{201}$ $Lys Superscript 201$ ; two in the carboxyl groups of ${Glu}^{204}$ $Glu Superscript 204$ and ${Asp}^{203}$ $Asp Superscript 203$ ; two at C-2 and C-3 of the substrate, ribulose 1,5-bisphosphate; and one in the other substrate, ${CO}_{2}$ $CO Subscript 2$ . A water molecule occupies the ${CO}_{2}$ $CO Subscript 2$ -binding site in the crystal structure. In this figure, a ${CO}_{2}$ $CO Subscript 2$ molecule is modeled in its place. (Residue numbers refer to the spinach enzyme.) [Data from PDB ID 1RXO, T. C. Taylor and I. Andersson, *J. Mol. Biol.* 265:432, 1997.]

A green sphere labeled M g 2 plus has six dashed vertical lines extending outward. A line extending from just right of the twelve o’clock position meets a red sphere at the end of G l u superscript 204 end superscript that extends to the left. A dashed line from the two o’clock position extends to the gray center of C O 2, which is a linear structure that is red on each end. A dashed line from the five o’clock position extends to a red sphere bonded to a white sphere bonded to carbon at the center of ribulose-1,5-bisphosphate, which extends horizontally to the right and bends down to the left. A dashed line from M g just left of the six o’clock position extends to a red sphere connected to the adjacent carbon of ribulose 1,5-bisphosphate. A dashed line from just above the nine o’clock position extends to a red sphere at the end of carbamoyl-L y s superscript 201 end superscript, which extends linearly to the left. A dashed line from the eleven o’clock position extends to a red sphere at the right end of A s p superscript 203 end superscript, which extends to the left. All data are approximate.

A figure shows the first stage of C O 2 assimilation by showing the carboxylase activity of rubisco. — MECHANISM FIGURE 20-29 First stage of ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation: rubisco’s carboxylase activity. The ${CO}_{2}$ $CO Subscript 2$ -fixation reaction is catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase. The overall reaction accomplishes the combination of one ${CO}_{2}$ $CO Subscript 2$ and one ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate, one of which contains the carbon atom from ${CO}_{2}$ $CO Subscript 2$ (red). Additional proton transfers (not shown), involving ${Lys}^{201} {, Lys}^{175}$ $Lys Superscript 201 Baseline comma Lys Superscript 175$ , and ${His}^{294}$ $His Superscript 294$ , occur in several of these steps.

Rubisco is shown as a blue region with a square opening to its right. A green sphere labeled M g 2 plus is shown in this opening with dashed lines extending away from it. M g has a dashed line extending vertically up to G l u in rubisco; a dashed line extending from the ten o’clock position to A s p in rubisco; and a dashed line extending from the seven o’clock position to O minus further bonded to C below bonded to O minus to the lower right and double bonded to N plus to the lower left at the border of rubisco that is bonded to H and further bonded to L y s superscript 201 end superscript and labeled carbamoylated L y s side chain. Additionally, a dashed line extends from the five-o’clock position to O double bonded to the bottom C of a two-carbon vertical chain that is further bonded to C H 2 to the lower right bonded to O below bonded to a circle labeled P to the right and further bonded to the upper C of the chain bonded to H to the lower right; C to the upper right bonded to H to the left, O H to the right, and C H 2 above bonded to O bonded to a circle labeled P; and bonded to O to the left bonded to H above and connected by a dashed line to M g 2 plus at the three o’clock position. Red arrows point from the O minus on the carbamoylated L y s side chain to H bonded to the upper C of the two-carbon chain, from the bond between the same H and C to the bond between the same C and the C below in the same vertical chain, and from the bond between the C below and its double bond to O. At the top center of rubisco, a five-membered ring has N substituted for C at the bottom vertex with a red pair of electrons and within the open are, double bonds at the lower left and upper right sides, an upper right vertex bonded to H i s superscript 294 end superscript, and N substituted for C at the upper left vertex and bonded to H. Step 1: Ribulose 1,5-bisphosphate forms an enediolate at the active site. An arrow points right accompanied by a curved arrow showing the addition of red highlighted C O 2. This yields an enediolate intermediate on the right. Mg 2 plus has similar bonds to A s p, G l u, and O minus to the lower left except that O minus of the carbamoylated L y s side chain is now bonded to C bonded to O H to the lower right instead of to O minus to the lower right. The dashed line from the five o’clock position of M g 2 plus is now bonded to red highlighted O connected by a red double bond to red highlighted C further connected by a red double bond to red highlighted O. The dashed line from the three o’clock position of M g 2 plus is now connected to O bonded to H above and to C to the lower right that is the upper C of the two-carbon chain, still bonded to the same group to the upper right, and double-bonded to the bottom C of the chain, which is now bonded to O to the lower left further bonded to H and still bonded to C H 2 to the lower right further bonded to O bonded to a circle labeled P. A red arrow points from the red pair of electrons on N in the five-membered ring at the top of the opening in rubisco to the H below and a red arrow points from the bond between this H and O connected by a dashed line to the three o’clock position of M g 2 plus to the bond between the same O and C to its right at the top of the two-carbon chain. Red arrow point from the double bond between the top and bottom carbons of the two-carbon vertical chain to the red highlighted C to the left and from the double bond between the same C and red highlighted O above to the same O, which is connected by a dashed line to the five o’clock position of M g 2 plus. Step 2: C O 2, polarized by the proximity of the M g 2 plus ion, undergoes nucleophilic attack by the enediolate, generating a branched 6-carbons sugar. An arrow points down accompanied by a curved arrow showing the loss of H plus and a curved line showing the addition of H 2 O. In the carbamoylated L y s side chain, O H at the lower right has become O minus again. The five-membered ring at the top of the opening in rubisco is no longer present. A beta-keto acid intermediate is formed. The dashed line from the three o’clock position connects to O double bonded to C at the top of the two-carbon vertical chain, which is bonded to the same substituent to the upper right and bonded to the bottom C of the chain below that is still bonded to the same substituents to the lower left and right but also bonded to red highlighted C to the upper left further connected by a red double bond to O to the upper left and by a red bond to red highlighted O minus to the lower left that is connected by a dashed vertical line to the five o’clock position of M g 2 plus. O with a red pair of electrons is bonded to H to the upper and lower right. Red arrows point from the red pair of electron on O to the top C of the two-carbon vertical chain and from the double bond between the same C and O to the same O, which is connected by a dashed line to the three o’clock position on M g 2 plus. Step 3: Hydroxylation occurs at the C-3 carbonyl. An arrow points down accompanied by a curved arrow showing the loss of H plus. The product is similar except that the molecule on the right side of M g 2 plus is now a hydrated intermediate. The dashed line from the three o’clock position on M g 2 plus is now bonded to O minus bonded to C to the lower right, which is the upper C of the two-carbon vertical chain and which is further bonded to O to the lower right further bonded to H. Red arrows point from the bond between this H and O to the bond between the same O and C and from the bond between the same C and the C below to the same C below, which is the bottom bond of the two-carbon vertical chain. Step 4: Cleavage produces one molecule of 3-phosphoglycerate. An arrow points right accompanied by a curved arrow showing the loss of H plus and a second curved arrow showing the loss of 3-phosphoglycerate, which is a three-carbon chain with the top C bonded to 2 H, to O further bonded to a circle labeled P, and to C below bonded to H on the left, O H on the right, and C below further bonded to O minus to the lower left and double bonded to O to the lower right. This yields a product in which there is no dashed line from the three o’clock position of M g 2 plus and the dashed line from the five o’clock position of M g 2 plus connects to red highlighted O minus connected by a red bond to red highlighted C connected by a red double bond to O to the upper right and bonded to C minus below further bonded to O to the lower left further bonded to H below and to C H 2 to the lower right further bonded to C H 2 below further bonded to O bonded to a circle labeled P. Beneath L y s superscript 2 0 1 end superscript on the left side of the opening in rubisco, L y s superscript 175 end superscript is bonded to N plus at the boundary of rubisco bonded to H above, to the right, and below. A red arrow points from C minus to the right-hand H bonded to N plus. Step 5: Carbanion protonation generates a second molecule of 3-phosphoglycerate. This yields 3-phosphoglycerate, shown as red highlighted C connected by a red bond to red highlighted O minus to the upper left, connected by a red double bond to red highlighted O to the upper right, and bonded to C below bonded to H to the upper right, bonded to O to the lower left further bonded to H below, and bonded to C H 2 to the lower right further bonded O below bonded to a circle labeled P. All data are approximate.

As the catalyst for the first step of photosynthetic ${CO}_{2}$ $CO Subscript 2$ assimilation, rubisco is a prime target for regulation. The enzyme is inactive until carbamoylated on the ε-amino group of ${Lys}^{201}$ $Lys Superscript 201$ (Fig. 20-30). Ribulose 1,5-bisphosphate inhibits carbamoylation by binding tightly to the active site and locking the enzyme in the “closed” conformation, in which ${Lys}^{201}$ $Lys Superscript 201$ is inaccessible. Rubisco activase overcomes the inhibition by promoting ATP-dependent release of the ribulose 1,5-bisphosphate, exposing the Lys amino group to nonenzymatic carbamoylation by ${CO}_{2}$ $CO Subscript 2$ ; this is followed by ${Mg}^{2 +}$ $Mg Superscript 2 plus$ binding, which activates the rubisco.

A figure shows the role of rubisco activase in the carbamoylation of L y s superscript 201 end superscript of rubisco. — FIGURE 20-30 Role of rubisco activase in carbamoylation of ${Lys}^{201}$ $bold upper L y s Superscript bold 201$ of rubisco.

FIGURE 20-30 Role of rubisco activase in carbamoylation of ${Lys}^{201}$ $bold upper L y s Superscript bold 201$ of rubisco.

Rubisco is shown as a blue region with a square opening to its right. N H 3 with a positive charge on N is at the lower left boundary of rubisco and further bonded to L y s superscript 201 end superscript within rubisco. Within the opening, a vertical four-carbon chain is present. Numbered from bottom to top, it has C 1 double bonded to O on the left and bonded to C H 2 on the right further bonded to O below bonded to a circle labeled P; C 2 and C 3 bonded to H on the left and O H on the right; C 4 bonded to 2 H and to O above further bonded to a circle labeled P. Text below reads, Rubisco with unmodified L y s superscript 201 end superscript and bound ribulose 1,5-bisphosphate is inactive. An arrow points right accompanied by a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P with rubisco activase at the inflection point, followed by an arrow showing the loss of ribulose 1,5-bisphosphate. Text below reads, A T P-dependent removal of ribulose 1,5-bisphosphate uncovers epsilon-amino group of L y s superscript 201 end superscript. A second arrow points right accompanied by curved lines showing the addition of red highlighted C O 2 follows by the addition of a green sphere labeled M g 2 plus. This yields N plus H at the boundary of rubisco bonded to L y s superscript 201 end superscript to the left and double bonded to red highlighted C connected by red bonds to red highlighted O to the upper and lower right with a dashed curved line connecting the oxygens to indicate partial double bond character. Dashed lines connected each O with a green sphere labeled M g 2 plus. Text below reads, Epsilon-amino group of L y s superscript 201 end superscript is carbamoylated by C O 2; M g 2 plus binds to carbamoyl-L y s, activating rubisco.

Stage 2: Conversion of 3-Phosphoglycerate to Glyceraldehyde 3-Phosphate

Stage 2 begins as stromal 3-phosphoglycerate kinase catalyzes the transfer of a phosphoryl group from ATP to 3-phosphoglycerate, yielding 1,3-bisphosphoglycerate. Next, NADPH donates electrons in a reduction catalyzed by the chloroplast-specific isozyme of glyceraldehyde 3-phosphate dehydrogenase, producing glyceraldehyde 3-phosphate and $P_{i}$ $upper P Subscript i$ . The high concentrations of NADPH and ATP in the chloroplast stroma allow this thermodynamically unfavorable pair of reactions to proceed in the direction of glyceraldehyde 3-phosphate formation. Triose phosphate isomerase then interconverts glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, producing the two substrates for aldolase, which condenses them into fructose 1,6-bisphosphate. Thus far, the process has employed the same enzymes we saw in glycolysis, but operating in the reverse direction.

Most of the triose phosphate and fructose 1,6-bisphosphate produced by photosynthesis is used to regenerate ribulose 1,5-bisphosphate, the essential starting material for photosynthesis. Any excess triose phosphate is either converted to starch in the chloroplast and stored for later use or immediately exported to the cytosol and converted to sucrose for transport to growing regions of the plant.

Stage 3: Regeneration of Ribulose 1,5-Bisphosphate from Triose Phosphates

For the continuous flow of ${CO}_{2}$ $CO Subscript 2$ into carbohydrate, ribulose 1,5-bisphosphate must be constantly regenerated. This is accomplished in a series of mostly reversible reactions (Fig. 20-31) that, together with stages 1 and 2, constitute the reductive pentose phosphate pathway summarized in Figure 20-26. Three exergonic reactions, shown with blue arrows in Figure 20-31, make the whole process irreversible. These are the reactions catalyzed by fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and ribulose 5-phosphate kinase.

A figure shows the third stage of C O 2 assimilation that interconvert triose phosphate and pentose phosphates. — FIGURE 20-31 Third stage of ${CO}_{2}$ $bold CO Subscript bold 2$ assimilation. This schematic diagram shows the interconversions of triose phosphates and pentose phosphates. Red dots represent the number of carbons in each compound. Compounds that appear more than once are highlighted. The starting materials are glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Reactions catalyzed by aldolase ( and ) and transketolase ( and ) produce pentose phosphates that are converted to ribulose 1,5-bisphosphate — ribose 5-phosphate by ribose 5-phosphate isomerase and xylulose 5-phosphate by ribulose 5-phosphate epimerase. Ribulose 5-phosphate is phosphorylated , regenerating ribulose 1,5-bisphosphate. The reactions with blue arrows are exergonic and make the whole process irreversible: fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and ribulose 5-phosphate kinase.

FIGURE 20-31 Third stage of ${CO}_{2}$ $bold CO Subscript bold 2$ assimilation. This schematic diagram shows the interconversions of triose phosphates and pentose phosphates. Red dots represent the number of carbons in each compound. Compounds that appear more than once are highlighted. The starting materials are glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Reactions catalyzed by aldolase ( and ) and transketolase ( and ) produce pentose phosphates that are converted to ribulose 1,5-bisphosphate — ribose 5-phosphate by ribose 5-phosphate isomerase and xylulose 5-phosphate by ribulose 5-phosphate epimerase. Ribulose 5-phosphate is phosphorylated , regenerating ribulose 1,5-bisphosphate. The reactions with blue arrows are exergonic and make the whole process irreversible: fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and ribulose 5-phosphate kinase.

Glyceraldehyde 3-phosphate is shown at the upper left in a blue box above a chain of three red spheres. Dihydroxyacetone phosphate is shown to its right in a purple box above a chain of three red spheres. A curved double-headed arrow labeled 1 and aldolase points from up from fructose 1,6-bisphosphate and splits to point to glyceraldehyde 3-phosphate and dihydroxyacetone. Fructose 1,6-bisphosphate is shown as a five-carbon chain of red spheres. An arrow labeled 2 and fructose 1,6-bisphosphatase points down to fructose 6-phosphate, shown as a chain of six red spheres. This arrow has an arrow curving off to show the loss of P subscript i end subscript. To the right, glyceraldehyde 3-phosphate is shown in a blue box above a chain of three red spheres. Two double-headed arrows that meet in the middle, where they are labeled 3 and transketolase, point from erythrose 4-phosphate and xylulose 5-phosphate below to fructose 6-phospahte and glyceraldehyde 3-phosphate above. Erythrose 4-phosphate is shown as a chain of four spheres. Dihydroxyacetone phosphate is shown to its left in a purple box above a chain of three red spheres. A curved double-headed arrow labeled 4 and aldolase points from sedoheptulose 1,7-bisphosphate below to split to point to dihydroxyacetone phosphate and erythrose 4-phospahte above. Sedoheptulose 1,7-bisphosphate is shown as a chain of seven red spheres. An arrow labeled 5 and sedoheptulose 1,7-bisphosphate points down to sedoheptulose 7-phosphate. The arrow is accompanied by a curved arrow showing the loss of P subscript i end subscript. Glyceraldehyde 3-phosphate is shown to the left in a blue box above a chain of three red spheres. A double-headed arrow labeled 6 and transketolase points from ribose 5-phosphate and an orange box labeled xylulose 5-phosphate below to glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate above. Ribose 5-phosphate is shown as a chain of five red spheres and xylulose 5-phosphate is shown as a chain of five red spheres. Upward- and downward-pointing arrows labeled 7 and ribose 5-phosphate isomerase show a reversible reaction that yields a yellow box labeled ribulose 5-phosphate, shown as a chain of 5 red spheres. An arrow labeled 9 and ribulose 5-phosphate kinase points right to a green box labeled ribulose 1,5-bisphosphate above a chain of five spheres. The arrow is accompanied by a curved arrow showing the addition of an orange oval of A T P and loss of A D P. Xylulose 5-phosphate above has diagonal forward and reverse arrows to the upper right labeled 8 and ribulose 5-phosphate epimerase. This yields a yellow box labeled ribulose 5-phosphate above a chain of five red spheres from which a blue arrow accompanied by a curved arrow showing the addition of an orange oval labeled A T P and loss of A D P is labeled 9 yields an green box labeled ribulose 1,5-bisphosphate, shown as a chain of five red spheres. Xylulose 5-phosphate above, produced by reaction 3, has upward- and downward-pointing arrows labeled 8 and ribulose 5-phosphate epimerase below showing that is also produces ribulose 5-phosphate that can undergo reaction 9 to produce ribulose 1,5-bisphosphate.

Synthesis of Each Triose Phosphate from ${CO}_{2}$ $bold upper C upper O bold 2$ Requires Six NADPH and Nine ATP

The net result of three turns of the Calvin cycle is the conversion of three molecules of ${CO}_{2}$ $CO Subscript 2$ and one molecule of phosphate to a molecule of triose phosphate. The stoichiometry of the overall path from ${CO}_{2}$ $CO Subscript 2$ to triose phosphate, with regeneration of ribulose 1,5-bisphosphate, is shown in Figure 20-32.

A figure shows the stoichiometry of C O 2 assimilation in the Calvin cycle. — FIGURE 20-32 Stoichiometry of ${CO}_{2}$ $bold upper C upper O bold 2$ assimilation in the Calvin cycle. For every three ${CO}_{2}$ $CO Subscript 2$ molecules fixed, one molecule of triose phosphate (glyceraldehyde 3-phosphate) is produced and nine ATP and six NADPH are consumed.

A cycle of reactions is shown. Each molecule is shown with a number in a yellow box to its left. At the top, a yellow box labeled 3 C O 2 plus H 2 O has a blue arrow down to show that it adds at the 12 o’clock position to an arrow from 3 ribulose 1,5-bisphosphate at the ten o’clock position to 6 3-phosphoglycerate at the two o’clock position. An arrow points to 6 1,3-bisphosphoglycerate at the three o’clock position accompanied by a curved arrow showing the addition of 6 orange ovals labeled A T P and loss of 6 A D P. An arrow points to a box at the five-o’clock position labeled 6 glyceraldehyde 3-phosphate with upward- and downward-pointing arrows to dihydroxyacetone phosphate below. This arrow is accompanied by a curved arrow showing the addition of 6 orange ovals labeled N A D P H plus 6 H plus and the loss of 6 N A D P plus followed by a branching curved arrow showing the low of 6 P i. A blue arrow points clockwise and then points down at the six o’clock position to a yellow box labeled 1 glyceraldehyde 3-phosphate. Another blue arrow continues the cycle from the six o’clcok position to the seven o’clock position, where there is a box labeled 5 glyceraldehyde 3-phosphate with upward- and downward-pointing arrows to dihydroxyacetone phosphate below. A series of three arrows points upward to 3 ribulose 5-phosphate at the nine o’clock position. Between the first and second arrows, an arrow curves away to show the loss of 2 P subscript i end subscript. An arrow points up to 3 ribulose 1,5-bisphosphate at the 11 o’clock position accompanied by a curved arrow showing the addition of three orange ovals labeled A T P and the loss of 3 A D P. All data are approximate.

One molecule of glyceraldehyde 3-phosphate is the net product of the ${CO}_{2}$ $CO Subscript 2$ -assimilation pathway. The other five triose phosphate molecules (15 carbons) are rearranged in steps to of Figure 20-31 to form three molecules of ribulose 1,5-bisphosphate (15 carbons). The last step in this conversion requires one ATP per ribulose 1,5-bisphosphate, or a total of three ATP. Thus, in summary, for every molecule of triose phosphate produced by photosynthetic ${CO}_{2}$ $CO Subscript 2$ assimilation, six NADPH and nine ATP are required.

NADPH and ATP are produced in the light-dependent reactions of photosynthesis in about the same ratio (2:3) as they are consumed in the Calvin cycle. Nine ATP molecules are converted to ADP and phosphate in the generation of a molecule of triose phosphate; eight of the phosphates are released as $P_{i}$ $upper P Subscript i$ and combined with eight ADP to regenerate ATP. The ninth phosphate is incorporated into the triose phosphate itself. To convert the ninth ADP to ATP, a molecule of $P_{i}$ $upper P Subscript i$ must be imported from the cytosol, as we shall see.

In the dark, the production of ATP and NADPH by photophosphorylation and the incorporation of ${CO}_{2}$ $CO Subscript 2$ into triose phosphate (once referred to as the dark reactions) cease. The “dark reactions” of photosynthesis were so named to distinguish them from the primary light-driven reactions of electron transfer to ${NADP}^{+}$ $NADP Superscript plus$ and synthesis of ATP. They do not, in fact, occur at significant rates in the dark and are thus more appropriately called the ${CO}_{2}$ $CO Subscript 2$ -assimilation reactions. Later in this section we describe the regulatory mechanisms that turn ${CO}_{2}$ $CO Subscript 2$ assimilation on in the light and turn it off in the dark.

The chloroplast stroma contains all the enzymes necessary to convert the triose phosphates produced by ${CO}_{2}$ $CO Subscript 2$ assimilation (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate) to starch, which is temporarily stored in the chloroplast as insoluble granules. Aldolase condenses the triose phosphates to fructose 1,6-bisphosphate; fructose 1,6-bisphosphatase produces fructose 6-phosphate; phosphohexose isomerase yields glucose 6-phosphate; and phosphoglucomutase produces glucose 1-phosphate, the starting material for starch synthesis (see Section 20.6).

All the reactions of the Calvin cycle except those catalyzed by rubisco, sedoheptulose 1,7-bisphosphatase, and ribulose 5-phosphate kinase also take place in animal tissues. Lacking these three enzymes, animals cannot carry out significant conversion of ${CO}_{2}$ $CO Subscript 2$ to glucose.

A Transport System Exports Triose Phosphates from the Chloroplast and Imports Phosphate

The inner chloroplast membrane is impermeable to most phosphorylated compounds, including fructose 6-phosphate, glucose 6-phosphate, and fructose 1,6-bisphosphate. It does, however, have a specific antiporter that catalyzes the one-for-one exchange of $P_{i}$ $upper P Subscript i$ with a triose phosphate, either dihydroxyacetone phosphate or 3-phosphoglycerate (Fig. 20-33). This antiporter simultaneously moves $P_{i}$ $upper P Subscript i$ into the chloroplast, where it is used in photophosphorylation, and moves triose phosphate into the cytosol, where it can be used to synthesize sucrose, the form in which the fixed carbon is transported to distant plant tissues.

A figure shows the P subscript i end subscript phosphate antiport system of the inner chloroplast membrane. — FIGURE 20-33 The $P_{i} - triose$ $bold upper P Subscript bold i Baseline en-dash bold t r i o s e$ phosphate antiport system of the chloroplast inner membrane. This transporter facilitates the exchange of cytosolic $P_{i}$ $upper P Subscript i$ for stromal dihydroxyacetone phosphate. The products of photosynthetic ${CO}_{2}$ $CO Subscript 2$ assimilation are thus moved into the cytosol, where they serve as a starting point for sucrose biosynthesis, and $P_{i}$ $upper P Subscript i$ required for photophosphorylation is moved into the stroma. This same antiporter can transport 3-phosphoglycerate, and it acts indirectly in the export of ATP and reducing equivalents (see Fig. 20-34).

FIGURE 20-33 The $P_{i} - triose$ $bold upper P Subscript bold i Baseline en-dash bold t r i o s e$ phosphate antiport system of the chloroplast inner membrane. This transporter facilitates the exchange of cytosolic $P_{i}$ $upper P Subscript i$ for stromal dihydroxyacetone phosphate. The products of photosynthetic ${CO}_{2}$ $CO Subscript 2$ assimilation are thus moved into the cytosol, where they serve as a starting point for sucrose biosynthesis, and $P_{i}$ $upper P Subscript i$ required for photophosphorylation is moved into the stroma. This same antiporter can transport 3-phosphoglycerate, and it acts indirectly in the export of ATP and reducing equivalents (see Fig. 20-34).

A cycle of reactions is shown on the left. A wavy yellowish-orange arrow points from light to the word photosynthesis above a curved arrow from 9 A D P plus 9 P I at the upper right to 9 A T P at the upper left. An arrow points down to 9 A T P, then another curved arrow completes the bottom of the circle and extends up to 9 A D P plus 8 P subscript i end subscript plus P subscript i end subscript. A horizontal arrow runs across the bottom of the bottom curved arrow, meeting it at the six o’clock position, and the top of the Calvin cycle meets the horizontal arrow at the same point. The Calvin cycle is shown as two clockwise arrows. The horizontal arrow points to dihydroxyacetone phosphate. The cycle of reactions at the upper left is completed by an arrow from 9 A D P plus 8 P subscript I end subscript plus P subscript i end subscript to 9 A D P plus 9 P subscript i end subscript. A horizontal membrane labeled chloroplast inner membrane is shown to the right. The region on the left is labeled stroma and the region on the right is labeled cytosol. A channel labeled P subscript i end subscript-triose phosphate antiporter provides a horizontal passage across the membrane. An arrow points from dihydroxyacetone in the stroma through the channel to dihydroxyacetone in the cytosol, from which a series of three arrows points right to sucrose. The third arrow has a curved branch to show the loss of P subscript i end subscript, from which a horizontal arrow points left to P subscript i in the cytosol near the entrance to the channel, from which a horizontal arrow points right to P subscript i at the end of the equation 9 A D P plus 8 P subscript i end subscript plus P subscript i end subscript at the lower right of the cycle shown in the stroma.

Sucrose synthesis in the cytosol and starch synthesis in the chloroplast are the major pathways by which the excess triose phosphate from photosynthesis is harvested. Sucrose synthesis (described later) releases four $P_{i}$ $upper P Subscript i$ molecules from the four triose phosphates required to make sucrose. For every molecule of triose phosphate removed from the chloroplast, one $P_{i}$ $upper P Subscript i$ is transported into the chloroplast, providing the ninth $P_{i}$ $upper P Subscript i$ mentioned above, to be used in regenerating ATP. If this exchange were blocked, triose phosphate synthesis would quickly deplete the available $P_{i}$ $upper P Subscript i$ in the chloroplast, slowing ATP synthesis and suppressing assimilation of ${CO}_{2}$ $CO Subscript 2$ into starch.

The $P_{i}$ $upper P Subscript i$ –triose phosphate antiport system serves one additional function. ATP and reducing power are needed in the cytosol for a variety of synthetic and energy-requiring reactions. These requirements are met to an as-yet-undetermined degree by mitochondria, but a second potential source of energy is the ATP and NADPH generated in the chloroplast stroma during the light-dependent reactions. However, neither ATP nor NADPH can cross the chloroplast membrane. The $P_{i}$ $upper P Subscript i$ –triose phosphate antiport system has the indirect effect of moving ATP equivalents and reducing equivalents from the chloroplast to the cytosol (Fig. 20-34). Dihydroxyacetone phosphate formed in the stroma is transported to the cytosol, where it is converted by glycolytic enzymes to 3-phosphoglycerate, generating ATP and NADH. 3-Phosphoglycerate reenters the chloroplast, completing the cycle.

A figure shows the role of the P subscript i end subscript-triose phosphate antiporter in the transport of A T P and reducing equivalents. — FIGURE 20-34 Role of the $P_{i} - triose$ $bold upper P Subscript bold i Baseline en-dash bold t r i o s e$ phosphate antiporter in the transport of ATP and reducing equivalents. Dihydroxyacetone phosphate leaves the chloroplast and is converted to glyceraldehyde 3-phosphate in the cytosol. The cytosolic glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase reactions then produce NADH, ATP, and 3-phosphoglycerate. The latter reenters the chloroplast and is reduced to dihydroxyacetone phosphate, completing a cycle that effectively moves ATP and reducing equivalents (NAD(P)H) from chloroplast to cytosol.

A cycle of reactions is shown with a horizontal membrane labeled chloroplast inner membrane across the center. The left side is labeled stroma and the right side is labeled cytosol. Two P subscript i end subscript phosphate antiporter channels are present in the membrane, providing horizontal passages between the stroma and the cytosol. An arrow points from P subscript i end subscript in the stroma through the top channel to P subscript i end subscript in the cytosol. 3-phosphoglycerate in the cytosol has an arrow pointing through the same top channel to 3-phosphoglycerate in the stroma, from which an arrow curves down to 1,3-bisphosphoglycerate at the ten o’clock position. This arrow is accompanied by a curved arrow showing the addition of an orange oval of A T P and loss of A D P. An arrow points down to glyceraldehyde 3-phosphate at the seven o’clock position accompanied by an arrow showing the addition of an orange oval labeled N A D P H plus H plus and loss of N A D plus. Forward and reverse arrows point to dihydroxyacetone just to the left of a channel at the six o’clock position. A horizontal arrow points from dihydroxyacetone in the stroma through the channel to dihydroxyacetone in the cytosol. An arrow points from P subscript i end subscript through the same channel to P subscript i end subscript in the stroma. Forward and reverse arrows labeled triose phosphate isomerase point to from dihydroxyacetone just to the right of the bottom channel at the six o’clock position to glyceraldehyde 3-phosphate at the four o’clock position. An arrow labeled glyceraldehyde 3-phosphate, accompanied by a curved arrow showing the addition of N A D plus and loss of an orange oval labeled N A D H and N plus, points to 1,3-bisphosphoglycerate at the two o’clock position. From this point, an arrow labeled phosphoglycerate kinase accompanied by a curved arrow showing the addition of a D P and loss of an orange oval labeled A D P yields 3-phosphoglycerate. All data are approximate.

Four Enzymes of the Calvin Cycle Are Indirectly Activated by Light

The reductive assimilation of ${CO}_{2}$ $CO Subscript 2$ requires a lot of ATP and NADPH, and their stromal concentrations increase when chloroplasts are illuminated (Fig. 20-35). The light-induced transport of protons across the thylakoid membrane also increases the stromal pH from about 7 to about 8, and it is accompanied by a flow of ${Mg}^{2 +}$ $Mg Superscript 2 plus$ from the thylakoid compartment into the stroma, raising the $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ from 1 to 3 mm to 3 to 6 mm. Several stromal enzymes have evolved to take advantage of these light-induced conditions, which signal the availability of ATP and NADPH: the enzymes are more active in an alkaline environment and at high $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ . For example, activation of rubisco by formation of carbamoyllysine is faster at alkaline pH, and high stromal $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ favors formation of the enzyme’s active ${Mg}^{2 +}$ $Mg Superscript 2 plus$ complex. Fructose 1,6-bisphosphatase requires ${Mg}^{2 +}$ $Mg Superscript 2 plus$ and is very dependent on pH (Fig. 20-36); its activity increases more than 100-fold when pH and $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ rise during chloroplast illumination.

A figure shows sources of A T P and N A D P H in a chloroplast and how their production affects p H and magnesium concentration. — FIGURE 20-35 Source of ATP and NADPH. ATP and NADPH produced by the light-dependent reactions are essential substrates for the reduction of ${CO}_{2}$ $CO Subscript 2$ . The photosynthetic reactions that produce ATP and NADPH are accompanied by movement of protons (red) from the stroma into the thylakoid, creating alkaline conditions in the stroma. Magnesium ions pass from the thylakoid into the stroma, increasing the stromal $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ .

A curved membrane is shown across the top with the light green region below labeled stroma. A horizontal darker green bar branches into two horizontal bars. The top bar is labeled thylakoid. Wavy yellowish-orange arrows point from light to the top and bottom bars. At the right end of the bottom bar, M g 2 plus is shown with an arrow pointing out to the stroma. Red highlighted H plus is shown beneath the bottom bar with a red arrow pointing into the thylakoid beneath text reading, photosynthetic electron transfer. A cycle of reactions is shown below and labeled the C O 2-assimilation cycle. An arrow curves away at the three o’clock position to show the loss of P subscript i. An arrow curves away at the six o’clock position to show the loss of glyceraldehyde 3-phosphate. A curved line shows the addition of C O 2 at the nine o’clock position. At the eleven o’clock position, the cycle meets a curved arrow that points up to A D P plus P subscript i end subscript, from which an arrow curves to an orange oval labeled A T P and then another arrow curves back to complete the cycle. At the one o’clock position, the cycle meets a curved arrow from an orange oval labeled N A D P H next to plus H plus and curves up to N A D P plus before curving back to N A D P H plus H plus. All data are approximate.

A graph plots F B P ase-1 activity against concentration of magnesium chloride at three p H values to show factors influencing the activity of chloroplast fructose 1,6-bisphosphatase. — FIGURE 20-36 Activation of chloroplast fructose 1,6-bisphosphatase. Reduced fructose 1,6-bisphosphatase (FBPase-1) is activated by light and by the combination of high pH and high $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ in the stroma, both of which are results of illumination. [Information from B. Halliwell, *Chloroplast Metabolism: The Structure and Function of Chloroplasts in Green Leaf Cells*, p. 97, Clarendon Press, 1984.]

FIGURE 20-36 Activation of chloroplast fructose 1,6-bisphosphatase. Reduced fructose 1,6-bisphosphatase (FBPase-1) is activated by light and by the combination of high pH and high $[{Mg}^{2 +}]$ $left-bracket Mg Superscript 2 plus Baseline right-bracket$ in the stroma, both of which are results of illumination. [Information from B. Halliwell, *Chloroplast Metabolism: The Structure and Function of Chloroplasts in Green Leaf Cells*, p. 97, Clarendon Press, 1984.]

Four Calvin cycle enzymes are subject to a special type of regulation by light. Ribulose 5-phosphate kinase, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and glyceraldehyde 3-phosphate dehydrogenase are activated by light-driven reduction of disulfide bonds between two Cys residues critical to their catalytic activities. When these Cys residues are disulfide-bonded (oxidized), the enzymes are inactive; this is the normal situation in the dark. With illumination, electrons flow from photosystem I to ferredoxin (Fig. 20-12), which passes electrons to a small, soluble, disulfide-containing protein called thioredoxin (Fig. 20-37), in a reaction catalyzed by ferredoxin: thioredoxin reductase. Reduced thioredoxin donates electrons for the reduction of the disulfide bonds of the light-activated enzymes, and these reductive cleavage reactions are accompanied by conformational changes that increase enzyme activities. At nightfall, the Cys residues in the four enzymes are reoxidized to their disulfide forms, the enzymes are inactivated, and ATP is not expended in ${CO}_{2}$ $CO Subscript 2$ assimilation. Instead, starch synthesized and stored during the daytime is degraded to fuel glycolysis and oxidative phosphorylation at night.

A figure shows light activation of several enzymes of the Calvin cycle, which is mediated by thioredoxin. — FIGURE 20-37 Light activation of several enzymes of the Calvin cycle. The light activation is mediated by thioredoxin, a small, disulfide-containing protein. In the light, thioredoxin is reduced by electrons moving from photosystem I through ferredoxin (Fd) (blue arrows), then thioredoxin reduces critical disulfide bonds in each of the enzymes sedoheptulose 1,7-bisphosphatase, fructose 1,6-bisphosphatase, ribulose 5-phosphate kinase, and glyceraldehyde 3-phosphate dehydrogenase, activating these enzymes. In the dark, the OSH groups undergo reoxidation to disulfides, inactivating the four enzymes.

A wavy yellowish-orange arrow point from light to photosystem Roman numeral 1 at the center of an arrow curving up from F d subscript ox end subscript at the bottom to F e subscript red end subscript at the top. A similar arrow curves down to show the reverse reaction, but meets a curved downward arrow to its right. The top half of the downward arrow from F d subscript red end subscript to F d subscript ox end subscript is blue until it meets the arrow to its right, at which point the arrow becomes gray and the curved arrow to its right becomes blue. This right-hand curved arrow, labeled ferredoxin-thioredoxin reductase, runs from a gray oval labeled thioredoxin at the top with two bonds to S atoms above and a bond connecting these two S together, down to an orange oval labeled thioredoxin below that has two bonds to S H below. An arrow curves up from this orange oval labeled thioredoxin to the gray oval labeled thioredoxin above and meets an upward arrow to its right. The bottom half of the arrow is blue, but it becomes gray and the right-hand arrow becomes blue after they meet. The right-hand arrow curves up from a gray oval labeled enzyme (inactive) with bonds to two S atoms below that are bonded to each other Where the two curved arrows meet to an orange oval labeled enzyme (active) that has bonds to two S H above. An arrow curves down from the orange oval labeled enzyme (active to the gray oval labeled enzyme (inactive) and the top half is blue until it branches off to O 2 (in dark), then the bottom half is gray.

Glucose 6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway, is also regulated by this light-driven reduction mechanism, but in the opposite sense. During the day, when photosynthesis produces plenty of NADPH, this enzyme is not needed for NADPH production. Reduction of a critical disulfide bond by electrons from ferredoxin inactivates the enzyme.

SUMMARY 20.4 Carbon-Assimilation Reactions

Photosynthesis in eukaryotes takes place in chloroplasts. In the ${CO}_{2}$ $CO Subscript 2$ -assimilating reactions (the Calvin cycle), ATP and NADPH are used to reduce ${CO}_{2}$ $CO Subscript 2$ to triose phosphates. These reactions occur in three stages: the fixation reaction itself, catalyzed by rubisco; reduction of the resulting 3-phosphoglycerate to glyceraldehyde 3-phosphate; and regeneration of ribulose 1,5-bisphosphate from triose phosphates.
Stromal enzymes rearrange the carbon skeletons of triose phosphates to generate intermediates of three, four, five, six, and seven carbons, eventually yielding pentose phosphates. The pentose phosphates are converted to ribulose 5-phosphate, which is phosphorylated to ribulose 1,5-bisphosphate to complete the Calvin cycle.
The cost of fixing three ${CO}_{2}$ $CO Subscript 2$ into one triose phosphate is nine ATP and six NADPH, which are provided by the light-dependent reactions of photosynthesis.
An antiporter in the inner chloroplast membrane exchanges $P_{i}$ $upper P Subscript i$ in the cytosol for 3-phosphoglycerate or dihydroxyacetone phosphate molecules produced by ${CO}_{2}$ $CO Subscript 2$ assimilation in the stroma. Oxidation of dihydroxyacetone phosphate in the cytosol generates ATP and NADH, thus moving ATP and reducing equivalents from the chloroplast to the cytosol.
Four enzymes of the Calvin cycle are activated indirectly by light and are inactive in the dark, so that hexose synthesis does not compete with glycolysis — which is required to provide energy in the dark.