Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

(From Knight,

22.8 ● Carbon Dioxide Fixation

As we began this chapter, we saw that photosynthesis traditionally is equated with the process of CO2 fixation, that is, the net synthesis of carbohydrate from CO2. Indeed, the capacity to perform net accumulation of carbohydrate from CO2 distinguishes the phototrophic (and autotrophic) organisms from heterotrophs. Although animals possess enzymes capable of linking CO2 to organic acceptors, they cannot achieve a net accumulation of organic material by these reactions. For example, fatty acid biosynthesis is primed by covalent attachment of CO2 to acetyl-CoA to form malonyl-CoA (Chapter 25). Nevertheless, this “fixed CO2” is liberated in the very next reaction, so no net CO2 incorporation occurs.

Elucidation of the pathway of CO2 fixation represents one of the earliest applications of radioisotope tracers to the study of biology. In 1945, Melvin Calvin and his colleagues at the University of California, Berkeley, were investigating photosynthetic CO2 fixation in Chlorella. Using 14CO2, they traced the incorporation of radioactive 14C into organic products and found that the earliest labeled product was 3-phosphoglycerate (see Figure 18.13). Although this result suggested that the CO2 acceptor was a two-carbon compound, further investigation revealed that, in reality, two equivalents of 3-phosphoglycerate were formed following addition of CO2 to a five-carbon (pentose) sugar:

CO2 5-carbon acceptor 88n [6-carbon intermediate] 88n

Two 3-phosphoglycerates

Ribulose-1,5-Bisphosphate Is the CO2 Acceptor in CO2 Fixation

The five-carbon CO2 acceptor was identified as ribulose-1,5-bisphosphate (RuBP), and the enzyme catalyzing this key reaction of CO2 fixation is ribulose bisphosphate carboxylase/oxygenase, or, in the jargon used by workers in this field, rubisco. The name ribulose bisphosphate carboxylase/oxygenase reflects the fact that rubisco catalyzes the reaction of either CO2 or, alternatively, O2 with RuBP. Rubisco is found in the chloroplast stroma. It is a very abundant enzyme, constituting more than 15% of the total chloroplast protein. Given the preponderance of plant material in the biosphere, rubisco is probably the world’s most abundant protein. Rubisco is large: in higher plants, rubisco is a 550-kD heteromultimeric ( 8 8) complex consisting of eight identical large subunits (55 kD) and eight small subunits (15 kD) (Figure 22.23). The large subunit is the catalytic unit of the enzyme. It binds both substrates (CO2 and RuBP) and Mg2 (a divalent cation essential for enzymatic activity). The small subunit modulates the activity of the enzyme, increasing kcat more than 100fold.2

The Ribulose-1,5-Bisphosphate Carboxylase Reaction

The addition of CO2 to ribulose-1,5-bisphosphate results in the formation of an enzyme-bound intermediate, 2-carboxy,3-keto-arabinitol (Figure 22.24). This intermediate arises when CO2 adds to the enediol intermediate gener-

2The rubisco large subunit is encoded by a gene within the chloroplast DNA, whereas the small subunit is encoded by a multigene family in the nuclear DNA. Assembly of active rubisco heteromultimers occurs within chloroplasts following transit of the small subunit polypeptide across the chloroplast membrane.

22.8 ● Carbon Dioxide Fixation

731

S monomers

L2 dimer

FIGURE 22.23 ● Schematic diagram of the subunit organization of ribulose bisphosphate carboxylase as revealed by X-ray crystallography. The enzyme consists of eight equivalents each of two types of subunits, large L (55 kD) and small S (15 kD). Clusters of four small subunits are located at each end of the symmetrical octamer formed by four L2 dimers.

S., Andersson, I., and Branden, C. I., 1990. Journal of Molecular Biology 215:113–160.)

732 Chapter 22 ●

Photosynthesis

1H2

COPO32–

H2

COPO23–

H2

COPO32–

1H2

COPO32–

O

O

2C

O

C

O–

CO2

HO

C

C

HO

2C

C

O–

O O–

3

O–

3

HCOH

COH

C

O

HO

C

OH

2

C

4

4

HCOH

H+

HCOH

HCOH

H2O

HCOH

H+

HCOH

5

5

H2COPO32–

H2COPO32–

H2COPO32–

H2COPO32–

H2COPO32–

I

II

III

FIGURE 22.24 ● The ribulose bisphosphate carboxylase reaction. Enzymatic abstraction of the C-3 proton of RuBP yields a 2,3-enediol intermediate (I), which is stereospecifically carboxylated at C-2 to create the six-carbon -keto acid intermediate (II) known as 2-carboxy,3- keto-arabinitol. Intermediate II is rapidly hydrated to give the gem-diol form (III). Deprotonation of the C-3 hydroxyl and cleavage yield two 3-phosphoglycerates. Mg2 at the active site aids in stabilizing the 2,3-enediol transition state for CO2 addition and in facilitating the carbon–carbon bond cleavage that leads to product formation. Note that CO2, not HCO3 (its hydrated form), is the true substrate.

ated from ribulose-1,5-bisphosphate. Hydrolysis of the C2–C3 bond of the intermediate generates two molecules of 3-phosphoglycerate. The CO2 ends up as the carboxyl group of one of the two molecules.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity

Rubisco exists in three forms: an inactive form designated E; a carbamylated, but inactive, form designated EC; and an active form, ECM, which is carbamy-

lated and has Mg2 at its active sites as well. Carbamylation of rubisco takes place by addition of CO2 to its Lys201 -NH2 groups (to give ONHOCOO

derivatives). The CO2 molecules used to carbamylate Lys residues do not become substrates. The carbamylation reaction is promoted by slightly alkaline pH (pH 8). Carbamylation of rubisco completes the formation of a binding site for the Mg2 that participates in the catalytic reaction. Once Mg2 binds to EC, rubisco achieves its active ECM form. Activated rubisco displays a Km for CO2 of 10 to 20 M.3

Substrate RuBP binds much more tightly to the inactive E form of rubisco (KD 20 nM) than to the active ECM form (Km for RuBP 20 M). Thus, RuBP is also a potent inhibitor of rubisco activity. Release of RuBP from the active site of rubisco is mediated by rubisco activase. Rubisco activase is a regulatory protein; it binds to E-form rubisco and, in an ATP-dependent reaction, promotes the release of RuBP. Rubisco then becomes activated by carbamylation and Mg2 binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco.

22.9 ● The Calvin–Benson Cycle

The immediate product of CO2 fixation, 3-phosphoglycerate, must undergo a series of transformations before the net synthesis of carbohydrate is realized. Among carbohydrates, hexoses (particularly glucose) occupy center stage. Glucose is the building block for both cellulose and starch synthesis. These plant polymers constitute the most abundant organic material in the living world, and thus, the central focus on glucose as the ultimate end product of CO2 fixation is amply justified. Also, sucrose ( -D-glucopyranosyl-(1 n 2)- -D- fructofuranoside) is the major carbon form translocated out of leaves to other plant tissues. In nonphotosynthetic tissues, sucrose is metabolized via glycolysis and the TCA cycle to produce ATP.

3The relative abundance of CO2 in the atmosphere is low, about 0.03%. The concentration of CO2 dissolved in aqueous solutions equilibrated with air is about 10 M.

22.9 ● The Calvin–Benson Cycle

733

The set of reactions that transforms 3-phosphoglycerate into hexose is named the Calvin–Benson cycle (often referred to simply as the Calvin cycle) for its discoverers. The reaction series is indeed cyclic because not only must carbohydrate appear as an end product, but the 5-carbon acceptor, RuBP, must be regenerated to provide for continual CO2 fixation. Balanced equations that schematically represent this situation are

6(1) 6(5) 88n		12(3)
12(3)	88n	1(6) 6(5)
Net : 6(1)	88n	1(6)

Each number in parentheses represents the number of carbon atoms in a compound, and the number preceding the parentheses indicates the stoichiometry of the reaction. Thus, 6(1), or 6 CO2, condense with 6(5) or 6 RuBP to give 12 3-phosphoglycerates. These 12(3)s are then rearranged in the Calvin cycle to form one hexose, 1(6), and regenerate the six 5-carbon (RuBP) acceptors.

The Enzymes of the Calvin Cycle

The Calvin cycle enzymes serve three important ends:

1.They constitute the predominant CO2 fixation pathway in nature.

2.They accomplish the reduction of 3-phosphoglycerate, the primary product of CO2 fixation, to glyceraldehyde-3-phosphate so that carbohydrate synthesis becomes feasible.

3.They catalyze reactions that transform 3-carbon compounds into 4-, 5-, 6-, and 7-carbon compounds.

Most of the enzymes mediating the reactions of the Calvin cycle also participate in either glycolysis (Chapter 19) or the pentose phosphate pathway (Chapter 23). The aim of the Calvin scheme is to account for hexose formation from 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 (22.3).

The Calvin cycle of reactions starts with ribulose bisphosphate carboxylase catalyzing formation of 3-phosphoglycerate from CO2 and RuBP and concludes with ribulose-5-phosphate kinase (also called phosphoribulose kinase), which forms RuBP (Figure 22.25 and Table 22.1). The carbon balance is given at the right side of the table. Several features of the reactions in Table 22.1 merit discussion. Note that the 18 equivalents of ATP consumed in hexose formation are expended in reactions 2 and 15: 12 to form 12 equivalents of 1,3-bisphospho- glycerate 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.

Balancing the Calvin Cycle Reactions To

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 3 hexoses in the form

6 ADP

6 ATP Phosphoribulose kinase

15

H2

COPO23–

H2

COPO23–

12 ATP 12 ADP

12

12 NADP+ + 12 P

6 CO2

2–

NADPH

C

O

1

HOCH

2

O

OPO3

3

COO–

C

CHO

HCOH

6

12

12

Ribulose

+

Phospho-

HCOH

Glyceraldehyde-

HCOH

HCOH

bisphos-

COO–

COPO32–

COPO32–

2–

phate

glycerate

H2

3-phosphate

H2

carboxylase

kinase

dehydrogenase

H2COPO3

HCOH

1,3-Bisphospho-

Glyceraldehyde-

Ribulose-1,5-bis-

glycerate

3-phosphate

6

H2COPO23–

phosphate

(BPG)

(G3P)

(RuBP)

Two 3-Phospho-

glycerates

(3PG)

12

4 5

H2

COH

Triose phosphate isomerase

C

O

H2

COPO23–

CHO

HCOH

C

O

HCOH

7

HCOH

H2COH

HCOH

H

COPO2–

Dihydroxyacetone

2

3

H2COPO23–

3 phosphate (DHAP)

Ribulose-

5

5-phosphate

Erythrose-4-

2

phosphate (E4P)

Aldolase

(Ru5P)

3

Aldolase

2

H2COPO32–

13 Phosphopentose

10

Glucose

C

O

epimerase

2–

2

H2

COPO3

4

8 Glucose-6-

4

HOCH

P

C

O

phosphatase

Phospho- 14

HCOH

HOCH

CHO

pentose

HCOH

isomerase

HCOH

HCOH

H2COPO32–

HCOH

HOCH

Fructose-1,6-

HCOH

bisphosphate (FBP)

HCOH

6

H2COPO23–

Fructose

HCOH

H2

COH

CHO

P

3

bisphosphatase

Sedoheptulose-1,7

bisphosphate (SBP)

H2COPO32–

C

O

HCOH

Glucose-6-

11

2

HOCH

H2

COH

phosphate

HCOH

P 2

Sedoheptulose

(G6P)

C

O

HCOH

HCOH

7

H2

COPO32–

H2

COPO32–

H2

COH

HOCH

Xylulose-5-

Ribose-5-

C

O

HCOH

Phospho-

1

phosphate (Xu5P)

phosphate (R5P)

HOCH

HCOH

glucoisomerase

H2

COPO32–

HCOH

Fructose-6-

HCOH

2

phosphate (F6P)

4

HCOH

2

9

H2COPO23–

2

Transketolase

Sedoheptulose-7

phosphate (S7P)

2

12

2

2

Transketolase

734

FIGURE 22.25

22.9 ● The Calvin–Benson Cycle

735

▲

● 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 22.1.

of fructose bisphosphate (Figure 22.25). (Recall that the G° for the aldolase reaction in the glycolytic direction is 23.9 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, distributed as two fructose- 6-phosphates, four 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 23). Reaction 11 is mediated by sedoheptulose-1,7-bis- phosphatase. This phosphatase is unique to plants; it generates sedoheptulose- 7-P, the seven-carbon sugar serving as the transketolase substrate. Likewise, phosphoribulose kinase carries out the unique plant function of providing RuBP from Ru-5-P (reaction 15). The net conversion accounts for the fixation of six equivalents of carbon dioxide into one hexose at the expense of 18 ATP and 12 NADPH.

Table 22.1

The Calvin Cycle Series of Reactions

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 is 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 CO2 6 H2O 6 RuBP 88n 12 3-PG	6(1) 6(5)	88n	12(3)
2.	3-Phosphoglycerate kinase: 12 3-PG 12 ATP 88n 12 1,3-BPG 12 ADP	12(3)	88n	12(3)
3.	NADP -glyceraldehyde-3-P dehydrogenase:
	12 1,3-BPG 12 NADPH 88n 12 NADP 12 G3P 12 Pi	12(3)	88n	12(3)
4.	Triose-P isomerase: 5 G3P 88n 5 DHAP	5(3)	88n	5(3)
5.	Aldolase: 3 G3P 3 DHAP 88n 3 FBP	3(3) 3(3)	88n	3(6)
6.	Fructose bisphosphatase: 3 FBP 3 H2O 88n 3 F6P 3 P1	3(6)	88n	3(6)
7.	Phosphoglucoisomerase: 1 F6P 88n 1 G6P	1(6)	88n	1(6)
8.	Glucose phosphatase: 1 G6P 1 H2O 88n 1 GLUCOSE 1 Pi	1(6)	88n	1(6)
	The remainder of the pathway involves regenerating six RuBP acceptors ( 30 C)
	from the leftover two F6P (12 C), four G3P (12 C), and two DHAP (6 C).
9.	Transketolase: 2 F6P 2 G3P 88n 2 Xu5P 2 E4P	2(6) 2(3)	88n	2(5) 2(4)
10.	Aldolase: 2 E4P 2 DHAP 88n 2 sedoheptulose-1,7-bisphosphate (SBP)	2(4) 2(3)	88n	2(7)
11.	Sedoheptulose bisphosphatase: 2 SBP 2 H2O 88n 2 S7P 2 Pi	2(7)	88n	2(7)
12.	Transketolase: 2 S7P 2 G3P 88n 2 Xu5P 2 R5P	2(7) 2(3)	88n	4(5)
13.	Phosphopentose epimerase: 4 Xu5P 88n 4 Ru5P	4(5)	88n	4(5)
14.	Phosphopentose isomerase: 2 R5P 88n 2 Ru5P	2(5)	88n	2(5)
15.	Phosphoribulose kinase: 6 Ru5P 6 ATP 88n 6 RuBP 6 ADP	6(5)	88n	6(5)
Net: 6 CO2 18 ATP 12 NADPH 12 H 12 H2O 88n			88n
	glucose 18 ADP 18 Pi 12 NADP	6(1)		1(6)

FIGURE 22.27

FIGURE 22.26

736 Chapter 22 ● Photosynthesis

ADP +

P

ATP

i r

is,

c t ic

a

ci

s

c

ly

d

c

y

o

y

l

cl

G

e

CO2 + H2O

Hexose

CO

x

n

2

fi atio

+

ADP +

P

ATP

Light

● Light regulation of CO2 fixation prevents a substrate cycle between cellular respiration and hexose synthesis by CO2 fixation. Because plants possess mitochondria and are capable of deriving energy from hexose catabolism (glycolysis and the citric acid cycle), regulation of photosynthetic CO2 fixation by light activation controls the net flux of carbon between these opposing routes.

8		Stroma
pH		Thylakoid
6
		space
dark	light	dark

● Light-induced pH changes in chloroplast compartments. Illumination of chloroplasts leads to proton pumping and pH changes in the chloroplast, such that the pH within the thylakoid space falls and the pH of the stroma rises. These pH changes modulate the activity of key Calvin cycle enzymes.

22.10 ● Regulation of Carbon Dioxide Fixation

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 CO2 by glycolysis and the citric acid cycle in one direction, and CO2 to carbohydrate by the CO2 fixation pathway in the opposite direction, is thwarted through regulation of the Calvin cycle (Figure 22.26). In this regulation, the activities of key Calvin cycle enzymes are coordinated with the output of photosynthesis. In effect, these enzymes respond indirectly to light activation. Thus, when light energy is available to generate ATP and NADPH for CO2 fixation, the Calvin cycle proceeds. In the dark, when ATP and NADPH cannot be produced by photosynthesis, fixation of CO2 ceases. The light-induced changes in the chloroplast which regulate key Calvin cycle enzymes include (1) changes in stromal pH, (2) generation of reducing power, and (3) Mg2 efflux from the thylakoid lumen.

Light-Induced pH Changes in Chloroplast Compartments

As discussed in Section 22.7, illumination of chloroplasts leads to light-driven pumping of protons into the thylakoid lumen, which causes pH changes in both the stroma and the thylakoid lumen (Figure 22.27). The stromal pH rises, typically to pH 8. Because rubisco and rubisco activase are more active at pH 8, CO2 fixation is activated as stromal 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 lightinduced pH increase in the stroma.

Light-Induced Generation of Reducing Power

Illumination of chloroplasts initiates photosynthetic electron transport, which generates reducing power in the form of reduced ferredoxin and NADPH. Several enzymes of CO2 fixation, notably fructose-1,6-bisphosphatase, sedoheptulose- 1,7-bisphosphatase, and ribulose-5-phosphate kinase, are activated upon reduction of specific Cys-Cys disulfide bonds to cysteine sulfhydryls. The reduced form of thioredoxin mediates this reaction. 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). Thioredoxin serves as the hydrogen carrier between NADPH or Fdred and enzymes regulated by light (Figure 22.28).

FIGURE 22.28 ● The pathway for light regulation of Calvin cycle enzymes. Light-generated reducing power (Fdred reduced ferredoxin) provides e for reduction of thioredoxin (T) by FTR (ferredoxin–thioredoxin reductase). Several Calvin cycle enzymes have pairs of Cys residues that are involved in the disulfidesulfhydryl transition between an inactive (OSOSO) form and an active (OSH HSO) form, as shown here. These enzymes include fructose-1,6-bisphosphatase (residues Cys174 and Cys179), NADP -malate dehydrogenase (residues Cys10 and Cys15), and ribulose-5-P kinase

(residues Cys16 and Cys55).

Light-Induced Mg2 Efflux from Thylakoid Vesicles

When light-driven proton pumping across the thylakoid membrane occurs, a concomitant efflux of Mg2 ions from vesicles into the stroma is observed. This efflux of Mg2 somewhat counteracts the charge accumulation due to H

Fdox	SH	S	Active	SH
	FTR	T
	SH	S	enzyme	SH
PSI
Fdred	S	SH	Inactive	S
	FTR	T
			enzyme
	S	SH		S

22.11 ● The Ribulose Bisphosphate Oxygenase Reaction: Photorespiration

737

influx and is one reason why the membrane potential change in response to proton pumping is less in chloroplasts than in mitochondria (Eq. 22.5). Both ribulose bisphosphate carboxylase and fructose-1,6-bisphosphatase are Mg2 -activated enzymes, and Mg2 flux into the stroma as a result of light-driven proton pumping stimulates the CO2 fixation pathway at these key steps. Activity measurements have indicated that fructose bisphosphatase may be the rate-limiting step in the Calvin cycle. The recurring theme of fructose 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.

22.11 ● The Ribulose Bisphosphate Oxygenase

Reaction: Photorespiration

As indicated, ribulose bisphosphate carboxylase/oxygenase catalyzes an alternative reaction in which O2 replaces CO2 as the substrate added to RuBP (Figure 22.29a). The ribulose-1,5-bisphosphate oxygenase reaction diminishes plant

(a) H2

COPO32–

C

O

Ribulose

–

O

bisphosphate

O

H2

COPO32–

HCOH

+

O2

C

+

carboxylase/

C

–

HCOH

oxygenase

HCOH

O

O

H2

COPO32–

H2

COPO32–

3-Phospho-

Phospho-

glycerate

glycolate

(b)

H2

COH

ADP

ATP

Phosphoglycolate

HCOH

phosphatase

C

–

O

O

Glycerate

P

NAD+

H

O

H2O2

O2

C

H2COH

NADH

C

C

+ H+

Glycolate

–

–

O

oxygenase

O

O

O

Glyoxylate

Glycolate

Serine

Transamination

Hydroxypyruvate

H2COH

H2

COH

C

O

+

+

HC

C

CH2

NH3

NH3

–O

2 Glycine

+

CO2

O

C

C

–

O

–

O

O

O

Glycine

Serine

FIGURE 22.29 ● The oxygenase reaction of rubisco. (a) The reaction of ribulose bisphosphate carboxylase with O2 in the presence of ribulose bisphosphate leads to wasteful cleavage of RuBP to yield 3-phosphoglycerate and phosphoglycolate. (b) Conversion of phosphoglycolate to glycine. In mitochondria, two glycines from photorespiration are converted into one serine plus CO2. This step is the source of the CO2 evolved in photorespiration. Transamination of glyoxylate to glycine by the product serine yields hydroxypyruvate; reduction of hydroxypyruvate yields glycerate, which can

be phosphorylated to 3-phosphoglycerate. 3-Phosphoglycerate can fuel resynthesis of ribulose bisphosphate by the Calvin cycle (Figure 22.25).

738 Chapter 22 ● Photosynthesis

productivity because it leads to loss of RuBP, the essential CO2 acceptor. The Km for O2 in this oxygenase reaction is about 200 M. Given the relative abundance of CO2 and O2 in the atmosphere and their relative Km values in these rubisco-mediated reactions, the ratio of carboxylase to oxygenase activity in vivo is about 3 or 4 to 1.

The products of ribulose bisphosphate oxygenase activity are 3-phospho- glycerate and phosphoglycolate. Dephosphorylation and oxidation convert phosphoglycolate to glyoxylate, the -keto acid of glycine (Figure 22.29b). Transamination yields glycine. Other fates of phosphoglycolate are also possible, including oxidation to CO2, 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 O2 and release of CO2, 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.

22.12 ● The C-4 Pathway of CO2 Fixation

Tropical grasses are less susceptible to the effects of photorespiration, as noted earlier. Studies employing 14CO2 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 and Slack, two Australian biochemists, first discovered this C-4 product of CO2 fixation, and the C-4 pathway of CO2 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 CO2 fixation scheme. Instead, it functions as a CO2 delivery system, carrying carbon dioxide 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 CO2 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 [O2]. The C-4 compounds serving as CO2 transporters are malate or aspartate.

Compartmentation of these reactions to prevent photorespiration involves the interaction of two cell types, mesophyll cells and bundle sheath cells. The mesophyll cells take up CO2 at the leaf surface, where O2 is abundant, and use it to carboxylate phosphoenolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 22.30). 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.4 The 4-C CO2 carrier (malate or aspartate) then is transported to the bundle sheath cells, where it is decarboxylated to yield CO2 and a 3-C product. The CO2 is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another CO2 (Figure 22.30). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of CO2 uptake (C3 plants).

4A number of different biochemical subtypes of C4 plants are known. They differ in whether OAA or malate is the CO2 carrier to the bundle sheath cell and in the nature of the reaction by which the CO2 carrier is decarboxylated to regenerate a 3-C product. In all cases, the 3-C product is returned to the mesophyll cell and reconverted to PEP.

FIGURE 22.30

		CO2
Mesophyll
cell		PEP	Oxaloacetate
AMP +
	P P	P	NADPH
ATP	+ P		NADP+
		Pyruvate	Malate
			Bundle
		NADPH	sheath
	Pyruvate		NADP+ Malate cell

CO2 Ribulose-

1,5-bisphosphate

Calvin cycle

Glucose

2 3-Phospho- glycerate

22.12 ● The C-4 Pathway of CO2 Fixation

739

● Essential features of the compartmentation and biochemistry of the Hatch–Slack pathway of carbon dioxide uptake in C4 plants. Carbon dioxide is fixed into organic linkage by PEP carboxylase of mesophyll cells, forming OAA. Either malate (the reduced form of OAA) or aspartate (the aminated form) serves as the carrier transporting CO2 to the bundle sheath cells. Within the bundle sheath cells, CO2 is liberated by decarboxylation of malate or aspartate; the C-3 product is returned to the mesophyll cell. Formation of PEP by pyruvate Pi dikinase reinitiates the cycle. The CO2 liberated in the bundle sheath cell is used to synthesize hexose by the conventional rubisco–Calvin cycle series of reactions.

Intercellular Transport of Each CO2 via a

C-4 Intermediate Costs 2 ATP

The transport of each CO2 requires the expenditure of two high-energy phosphate bonds. The energy of these bonds is expended in the phosphorylation of pyruvate to PEP (phosphoenolpyruvate) by the plant enzyme pyruvate-Pi dikinase; the products are PEP, AMP, and pyrophosphate (PPi). 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 Pi. The reaction mechanism involves an enzyme phosphohistidine intermediate. The -phosphate of ATP is transferred to Pi, whereas formation of E-His-P occurs by addition of the -phosphate from ATP:

EOHis AMP OP OP Pi 88n EOHisOP AMP P Pi EOHisOP pyruvate 88n PEP EOHis

Net: ATP pyruvate Pi 88n AMP PEP PPi

Pyruvate-Pi dikinase is regulated by reversible phosphorylation of a threonine residue, the nonphosphorylated form being active. Interestingly, ADP is the phosphate donor in this interconvertible regulation. Despite the added metabolic expense of two phosphodiester bonds for each equivalent of carbon dioxide taken up, CO2 fixation is more efficient in C4 plants, provided that light intensities and temperatures are both high. (As temperature rises, photorespiration in C3 plants rises and efficiency of CO2 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. Approximately 8% of the incident light energy on a sugarcane field appears as chemical energy in the form of CO2 fixed into carbohydrate. This efficiency compares dramatically with the estimated pho-

740 Chapter 22 ● Photosynthesis

tosynthetic efficiency of 0.2% for uncultivated plant areas. Research on photorespiration is actively pursued in hopes of enhancing the 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.

22.13 ● Crassulacean Acid Metabolism

In contrast to C4 plants, which have separated CO2 uptake and fixation into distinct cells in order to minimize photorespiration, succulent plants native to semiarid and tropical environments separate CO2 uptake and fixation in time. Carbon dioxide (as well as O2) enters the leaf through microscopic pores known as stomata, and water vapor escapes from plants via these same openings. In nonsucculent plants, the stomata are open during the day, when light can drive photosynthetic CO2 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 H2O in their arid habitats would doom them. Instead, these plants open their stomata to take up CO2 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 carboxylase; OAA is then reduced to malate by malate dehydrogenase and stored within vacuoles until morning. During the day, the malate is released from the vacuoles and decarboxylated to yield CO2 and a 3-C product. The CO2 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 metabolism, and plants capable of it are called CAM plants.

PROBLEMS

1.In photosystem I, P700 in its ground state has an ° 0.4 V. Excitation of P700 by a photon of 700-nm light alters the ° of P700* to 0.6 V. What is the efficiency of energy capture in this

light reaction of P700?

2.What is the ° for the light-generated primary oxidant of photosystem II if the light-induced oxidation of water (which leads to O2 evolution) proceeds with a G° of 25 kJ/mol?

3.Assuming that the concentrations of ATP, ADP, and Pi in chloroplasts are 3 mM, 0.1 mM, and 10 mM, respectively, what is the G for ATP synthesis under these conditions? Photosynthetic electron transport establishes the proton-motive force driving photophosphorylation. What redox potential difference is necessary to achieve ATP synthesis under the foregoing conditions, assuming an electron pair is transferred per molecule of ATP generated?

4.14C-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, ribose-5-phosphate. In which carbon atoms will radioactivity be found?

5.Write a balanced equation for the synthesis of a glucose molecule from ribulose-1,5-bisphosphate and CO2 that involves the first three reactions of the Calvin cycle and subsequent conversion of the two glyceraldehyde-3-P molecules into glucose.

6.If noncyclic photosynthetic electron transport leads to the translocation of 3 H /e and cyclic photosynthetic electron transport leads to the translocation of 2 H /e , what is the relative pho-

tosynthetic efficiency of ATP synthesis (expressed as the number of photons absorbed per ATP synthesized) for noncyclic versus

cyclic photophosphorylation? (Assume that the CF1CF0 ATP synthase yields 1 ATP/3 H .)

7.The photosynthetic CO2 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?

8.The overall equation for photosynthetic CO2 fixation is

6 CO2 6 H2O 88n C6H12O6 6 O2

All the O atoms evolved as O2 come from water; none comes from carbon dioxide. But 12 O atoms are evolved as 6 O2, and only 6 O atoms appear as 6 H2O in the equation. Also, 6 CO2 have 12 O atoms, yet there are only 6 O atoms in C6H12O6. How can you account for these discrepancies? (Hint: Consider the partial reactions of photosynthesis: ATP synthesis, NADP+ reduction, photolysis of water, and the overall reaction for hexose synthesis in the Calvin–Benson cycle.)