21.4 What Is the Molecular Architecture of Photosynthetic Reaction Centers? 643 phyll (BChl). The e Ϫ is then transferred via the L bacteriopheophytin (BPheo) to Q A , which is also an L prosthetic group. The corresponding site on M is occupied by a loosely bound quinone, Q B , and electron transfer from Q A to Q B takes place. An interesting aspect of the system is that no electron transfer occurs through M, even though it has components apparently symmetric to and identical to the Le Ϫ transfer pathway. The reduced quinone formed at the Q B site is free to diffuse to a neighboring cytochrome bc 1 membrane complex, where its oxidation is coupled to H ϩ transloca- tion and, hence, ultimately to ATP synthesis. The use of light energy to drive ATP synthesis by the concerted action of these membrane proteins is called photophos- phorylation (Figure 21.15). Cytochrome c 2 , a periplasmic protein, serves to cycle electrons back to P870 ϩ via the four hemes of the reaction center cytochrome subunit. A specific tyrosine residue of L (Tyr 162 ) is situated between P870 and the closest cytochrome heme. This Tyr is the immediate e Ϫ donor to P870 ϩ and completes the light-driven elec- tron transfer cycle. The Molecular Architecture of PSII Resembles the R. viridis Reaction Center Architecture Type II photosystems of higher plants, green algae, and cyanobacteria contain more than 20 subunits and are considerably more complex than the R. viridis reaction center. The structure of PSII from the thermophilic cyanobacterium Light Outside Cytoplasm Cyt c 2 Cyt c 2 QH 2 Q B Q A LM H 2H + Fe 2H + 4H + 4H + 4H + Bacterial F 1 F 0 –ATP synthase + P i 2e – Q 2 H + Cyt b/c 1 ATP ADP ACTIVE FIGURE 21.15 Photophosphorylation. Photoexcitation of the R. viridis RC leads to reduction of a quinone, Q, to form QH 2 . Oxidation of QH 2 by the cytochrome bc 1 complex leads to H ϩ trans- location for ATP synthesis by the R. viridis F 1 F 0 –ATP synthase. Test yourself on the concepts in this figure at www.cengage.com/login. 644 Chapter 21 Photosynthesis Synechococcus elongatus has been revealed by X-ray crystallography, providing insight into PSII structures in general. Interestingly, both type II and type I photosystems show significant similarity to the R. viridis reaction center, thus establishing a strong evolutionary connection between reaction centers. S. elongatus PSII is a homodimeric structure. Each “monomer” has a mass of almost 350 kD and 23 different protein subunits, the 4 largest being the reaction center pair of subunits (D1 and D2) and two chlorophyll-containing inner antenna subunits (CP43 and CP47) that bracket D1 and D2 (Figure 21.16). Together, CP43 and CP47 have a total of 26 Chl a molecules, and exciton energy is collected and transferred from them to P680. Collectively, the protein subunits in a PSII “monomer” have at least 34 transmembrane ␣-helical segments, 22 of which are found in the D1-D2-CP43-CP47 “core” structure. D1 and D2 each have five membrane-spanning ␣-helices. Structurally and functionally, these two subunits are a direct counterpart of the L and M subunits of the R. viridis reaction center. P680 consists of a pair of Chl a molecules, with D1 and D2 each contributing one. D1 and D2 each have two other Chl a molecules, one near each P680 (Chl D1 and Chl D2 , re- spectively) and another that interacts with CP43/CP47 (Chl Z-D1 and Chl Z-D2 , respec- tively) (Figure 21.16). Two equivalents of pheophytin (Pheo) are located on D1 and D2. The tyrosine species D is Tyr 161 in the D1 amino acid sequence. Complexed to D2 is a tightly bound plastoquinone molecule, Q A . Electrons flow from P680* to Chl D1 and on to Pheo D1 . Pheo D1 then transfers the electron to Q A on D2, where it then moves to a second plastoquinone situated in the Q B site on D1 (Figure 21.16). Electron transfer from Q A and Q B is assisted by the iron atom located between them. Each plastoquinone that enters the Q B site accepts two electrons derived from water and two H ϩ from the stroma before it is released into the membrane as the hydroquinone PQH 2 . Thus, two photons are required to reduce each PQ that enters the Q B site. The stoichiometry of the overall reaction catalyzed by PSII is 2 H 2 O ϩ 2 PQ ϩ 4 h␷ ⎯⎯→ O 2 ϩ 2 PQH 2 . The (Mn) 4 complex is located on the Fe Phe D1 Phe D2 Chl D1 Chl D2 P D1 P D2 Y Z Y D Chl Z-D1 Chl Z-D2 D1 D2 (Mn) 4 CP43 CP47 Stroma Thylakoid lumen Q A Q B (a) (b) FIGURE 21.16 Molecular architecture of the Synechococ- cus elongatus PSII dimer. (a) The arrow shows the path of electron transfer from P680* to Chl D1 to Pheo D1 to Q A on D2 and then, via the Fe atom, to Q B on D1.The Tyr 161 residue of D1, symbolized by Y z , is situated between P680 and the (Mn) 4 cluster. (b) Structure of S. elongatus PSII (pdb id ϭ 1S5L). Chlorophylls of the reaction center and electron transfer path are shown in green; pheo- phytins, in blue.The OECs are shown in brick red. (Adapted from Barber, J., 2003.Photosystem II:The engine of life. Quarterly Review of Biophysics 36:71–89.) Go to CengageNOW and click CengageInteractive to explore the R. viridis reaction center, a complex scaffold for transduction of light energy. 21.4 What Is the Molecular Architecture of Photosynthetic Reaction Centers? 645 lumenal side of the thylakoid membrane. Thus, protons liberated from H 2 O mole- cules at the Mn site are deposited directly into the lumen. How Does PSII Generate O 2 from H 2 O? PSII catalyzes what is probably the most thermodynamically difficult reaction in na- ture, the light-driven oxidation of water to molecular oxygen. The protons and elec- trons released in this reaction are used to reduce NADP ϩ to NADPH and to estab- lish a proton gradient across the photosynthetic membrane that can be tapped to drive chemiosmotic ATP synthesis (see Figure 21.20). Accumulation of molecular oxygen in the atmosphere as a by-product of the photo-oxidation of water has trans- formed the planet since the evolutionary appearance of this reaction some 2 billion years ago in cyanobacteria. How does PSII evolve oxygen? The Structure of the Oxygen-Evolving Complex The architecture of the S. elon- gatus photosynthetic OEC reveals a large globular protein domain juxtaposed on the lumenal side of the D1 subunit of PSII (Figure 21.16). The active site of the OEC contains a cubelike metal cluster that consists of four manganese ions, one calcium ion, and five oxygen atoms bridging the Mn atoms, as shown in Figure 21.17. This metal cluster is held by Glu 189 , Asp 342 , His 332 , and His 337 of the PSII D1 subunit and Glu 354 of CP43. Chloride ion (Cl Ϫ ) is required for O 2 evolution, and Cl Ϫ is believed to be a Ca 2ϩ ligand. Note also that Tyr 161 (Y Z ) is situated near the metal cluster, ide- ally poised to serve in electron transfer between H 2 O and P680 ϩ . When four e Ϫ have been removed from the cluster (one from each Mn atom) through e Ϫ transfer to PSII via Tyr 161 , two H 2 O molecules provide the four e Ϫ needed to re-reduce the Mn atoms and O 2 is evolved. The four H ϩ released contribute to the proton gradient. The Molecular Architecture of PSI Resembles the R. viridis Reaction Center and PSII Architecture The structure of PSI from the cyanobacterium S. elongatus also has been solved by X-ray crystallography, completing our view of reaction center structure and confirming the fundamental similarities in organization that exist in these energy- transducing integral membrane proteins. Because of direct correlations with infor- mation about eukaryotic PSI, this cyanobacterial PSI provides a general model for all P700-dependent photosystems. S. elongatus PSI exists as a cloverleaf-shaped trimeric structure. Each “monomer” (356 kD) consists of 12 different protein subunits and 127 cofactors: 96 chlorophyll a molecules, 2 phylloquinones, 3 Fe 4 S 4 clusters, 22 carotenoids, and 4 lipids that are an intrinsic part of the protein complex (Figure 21.18). All of the electron-transferring prosthetic groups essential to PSI function are localized to just three polypeptides: PsaA, PsaB, and PsaC. PsaA and PsaB (83 kD each) compose the reaction center het- erodimer, a structural pattern now seen as universal in photosynthetic reaction centers. PsaA and PsaB each have 11 transmembrane ␣-helices, with the 5 most C-terminal ␣-helices of each serving as the scaffold for the reaction center photosynthetic electron- transfer apparatus. PsaC interacts with the stromal face of the PsaA–PsaB heterodimer. PsaC carries the two Fe 4 S 4 clusters, F A and F B , and interacts with PsaD. Together they provide a docking site for ferredoxin. The electron-transfer system of PSI consists of three pairs of chlorophyll molecules: P700 (a heterodimer of Chl a and an epimeric form, Chl aЈ) and two additional Chl a pairs (symbolized by A 0 ) that mediate e Ϫ trans- fer to the quinone acceptor. The S. elongatus quinone acceptor (A 1 ) is phylloquinone (also known as vitamin K 1 ). The Fx Fe 4 S 4 cluster bridges PsaA and PsaB; two of its four cysteine ligands come from PsaA, the other two from PsaB. Photochemistry begins with exciton absorption at P700, almost instantaneous electron transfer and charge separation (P700 ϩ ϺA 0 Ϫ ), followed by transfer of the electron from A 0 to A 1 and on to F X and then F A and F B , where it goes on to reduce a ferredoxin molecule at the “stromal” side of the membrane. The positive charge at P700 ϩ and the e Ϫ at F A /F B represent a charge separation across the membrane, an energized condition created His 332 Asp 170 Asp 342 CP43 Glu 354 Glu 333 Tyr 161 His 190 His 337 Gln 165 Glu 189 Mn A Mn B Mn C Mn D Ca Ala 344 (C-term) FIGURE 21.17 Structure of the PSII oxygen-evolving complex (OEC). Four Mn atoms (red, lettered A–D) and a Ca atom (green) form the water-splitting metal cluster of the OEC. Bridging O atoms are purple. (Adapted from Figure 4 in Yano, J.,et al., 2006.Where water is oxidized to dioxy- gen: Structure of the photosynthetic Mn 4 Ca cluster. Science 314:821–825.) 646 Chapter 21 Photosynthesis FeS A FeS B FeS X P700 A 1 O O O O A 0 Plastocyanin Plastocyanin docking Lumen PsaA PsaB PsaC PsaD Ferredoxin docking Ferredoxin PsaF (a) (b) FIGURE 21.18 The molecular architecture of PSI. (a) Subunit organization. (Adapted from Golbeck, J.H., 1992. Structure and function of photosystem I. Annual Review of Plant Physi- ology and Plant Molecular Biology 43:293–324; and Fromme, P., Jordan,P., and Krausse, N., 2001. Structure of photosystem I. Biochimica Biophysica Acta 1507:5–31.) (b) Molecular graphic of theoretical model for PSI. PsaA is orange; PgaB is magenta; PsaC is yellow.The iron- sulfur clusters are red (pdb id ϭ 1YO9). FIGURE 21.19 View of the plant PSI-LHC1 supercom- plex, from the stromal side of the thylakoid membrane. ChI molecules are shown in green and carotenoids and lipids in red.The 16 protein subunits are shown as ribbon diagrams in the background, with the positions of PsaG, PsaH, PsaK, and LHC1-4 subunits indicated. (Adapted from Figure 4 in Nelson, N.,and Yocum, C. F., Structure and function of photosystems I and II. Annual Review of Plant Biology 57: 521–566 (2006). Figure courtesy of the authors.) 21.5 What Is the Quantum Yield of Photosynthesis? 647 by light. Plastocyanin (or in cyanobacteria, a lumenal cytochrome designated cyto- chrome c 6 ) delivers an electron to fill the electron hole in P700 ϩ . How Do Green Plants Carry Out Photosynthesis? Do the higher plant photosystems follow the structural and functional pattern first revealed in the bacterial RC and recapitulated in the cyanobacterial PSI and PSII? With new structural information for the higher plant PSI (from Pisum sativum, garden pea) and PSII (from spinach), the fundamental organization pattern for photosystems seen earlier is confirmed for higher plants. Further, the structure of a plant membrane protein supercomplex consisting of the PSI reac- tion center and its light-harvesting antenna LHC1 (light-harvesting complex 1) has been described. This supercomplex exists as a “monomer” composed of 16 distinct protein subunits and about 200 prosthetic groups, including 167 chloro- phylls, 2 phylloquinones, and 3 Fe 4 S 4 clusters (Figure 21.19). The four LHC1 subunits form an arc around one side of the PSI RC. A second light-harvesting complex (LHC2) binds to another side. This plant PSI system, like all photosys- tems, is remarkably efficient, showing a quantum efficiency of nearly 1 (one elec- tron transferred per photon falling anywhere within the supercomplex). The many Chl and other light-harvesting molecules of the supercomplex form an in- tegrated network for highly efficient transfer of light energy into P700. 21.5 What Is the Quantum Yield of Photosynthesis? The quantum yield of photosynthesis can be defined as the amount of product formed per equivalent of light input. In terms of exciton delivery to reaction center Chl dimers and subsequent e Ϫ transfer, the quantum yield of photosynthesis typically approaches the theoretical limit of 1. The quantum yield of photosynthesis can also be expressed as the ratio of CO 2 fixed or O 2 evolved per photon absorbed. Interest- ingly, an overall stoichiometry of three H ϩ translocated into the thylakoid vesicle has been observed for each electron passing from H 2 O to NADP ϩ . Two photons per cen- ter would allow a pair of electrons to flow from H 2 O to NADP ϩ (see Figure 21.11), resulting in the formation of 1 NADPH and ᎏ 1 2 ᎏ O 2 . More appropriately, 4 h␷ per cen- ter (8 quanta total) would drive the evolution of 1 O 2 , the reduction of 2 NADP ϩ , and the translocation of 12 H ϩ . Current estimates suggest that 3 ATPs are formed for every 14 H ϩ translocated, so (12/14)3 ϭ 2.57 ATP would be synthesized from an in- put of 8 quanta. The energy of a photon depends on its wavelength, according to the equation E ϭ h␷ ϭ hc/␭, where E is energy, c is the speed of light, and ␭ is its wavelength. Ex- pressed in molar terms, an Einstein is the amount of energy in Avogadro’s number of photons: E ϭ Nhc/␭. Light of 700-nm wavelength is the longest-wavelength and the lowest-energy light acting in the eukaryotic photosystems. An Einstein of 700-nm light is equivalent in energy to approximately 170 kJ. Eight Einsteins of this light, 1360 kJ, theoretically generate 2 moles of NADPH, 2.57 moles of ATP, and 1 mole of O 2 . Calculation of the Photosynthetic Energy Requirements for Hexose Synthesis Depends on H ؉ /h␷ and ATP/H ؉ Ratios The fixation of carbon dioxide to form hexose, the dark reactions of photosynthesis, requires considerable energy. The overall stoichiometry of this process (see Equation 21.3) involves 12 NADPH and 18 ATPs. Thus, the ATP/NADPH ratio for CO 2 fixation is 1.5. To generate 12 equivalents of NADPH necessitates the consumption of 48 Ein- steins of light, minimally 170 kJ each. However, if the preceding ratio of 1.29 ATPs per NADPH is correct, only 15.5 or so ATPs would be produced for CO 2 fixation. To make up the deficit of 2.5 ATPs would require 35 H ϩ or about 12 more e Ϫ transferred from H 2 O to NADP ϩ (an additional 24 Einsteins). From 72 Einsteins, or 12,240 kJ, 1 mole 648 Chapter 21 Photosynthesis of hexose would be synthesized. The standard free energy change, ⌬G°Ј, for hexose formation from carbon dioxide and water (the exact reverse of cellular respiration) is ϩ2870 kJ/mol. Note that many assumptions underlie these calculations, including assumptions about the ATP/H ϩ ratio, the H ϩ /e Ϫ ratio, and ultimately, the relation- ship between quantum input and overall yields of NADPH and ATP. Also, cyclic pho- tophosphorylation (see later section titled Cyclic Photophosphorylation Generates ATP but Not NADPH or O 2 ) leads to ATP synthesis and may aid in making up the ATP deficit just mentioned. 21.6 How Does Light Drive the Synthesis of ATP? Light-driven ATP synthesis, or photophosphorylation, is a fundamental part of the photosynthetic process. The conversion of light energy to chemical energy results in electron-transfer reactions, which lead to the generation of reducing power (re- duced quinones or NADPH). Coupled with these electron transfers, protons are driven across the thylakoid membranes from the stromal side to the lumenal side. These proton translocations occur in a manner analogous to the proton transloca- tions accompanying mitochondrial electron transport that provide the driving force for oxidative phosphorylation (see Chapter 20). Figure 21.11 indicates that proton translocations can occur at a number of sites. For example, protons are produced in the thylakoid lumen upon photolysis of water by PSII. The oxidation–reduction events as electrons pass through the plastoquinone pool and the Q cycle are an- other source of proton translocations. The proton transfer accompanying NADP ϩ reduction also can be envisioned as protons being taken from the stromal side of the thylakoid vesicle. The current view is that three protons are translocated for each electron that flows from H 2 O to NADP ϩ . Because this electron transfer re- quires two photons, one falling at PSII and one at PSI, the overall yield is 1.5 pro- tons per quantum of light. The Mechanism of Photophosphorylation Is Chemiosmotic The thylakoid membrane is asymmetrically organized, or “sided,” like the mito- chondrial membrane. It also shares the property of being a barrier to the passive diffusion of H ϩ ions. Photosynthetic electron transport thus establishes an electro- chemical gradient, or proton-motive force, across the thylakoid membrane with the interior, or lumen, side accumulating H ϩ ions relative to the stroma of the chloro- plast. Like oxidative phosphorylation, the mechanism of photophosphorylation is chemiosmotic. A proton-motive force of approximately Ϫ250 mV is needed to achieve ATP syn- thesis. This proton-motive force, ⌬p, is composed of a membrane potential, ⌬␺, and a pH gradient, ⌬pH (see Chapter 20). The proton-motive force is defined as the free energy difference, ⌬G, divided by Ᏺ, Faraday’s constant: ⌬p ϭ ⌬G/Ᏺ ϭ ⌬␺ Ϫ (2.3 RT/Ᏺ)⌬pH (21.5) In chloroplasts, the value of ⌬␺ is typically Ϫ50 to Ϫ100 mV and the pH gradient is equivalent to about 3 pH units, so Ϫ(2.3 RT/Ᏺ)⌬pH ϭϪ170 mV. This situation con- trasts with the mitochondrial proton-motive force, where the membrane potential contributes relatively more to ⌬p than does the pH gradient. CF 1 CF 0 –ATP Synthase Is the Chloroplast Equivalent of the Mitochondrial F 1 F 0 –ATP Synthase The transduction of the electrochemical gradient into the chemical energy repre- sented by ATP is carried out by the chloroplast ATP synthase, which is highly analo- gous to the mitochondrial F 1 F 0 –ATP synthase. The chloroplast enzyme complex is called CF 1 CF 0 –ATP synthase, “C” symbolizing chloroplast. Like the mitochondrial complex, CF 1 CF 0 –ATP synthase is a heteromultimer of ␣-, ␤-, ␥-, ␦-, ⑀-, a-, b-, and 21.6 How Does Light Drive the Synthesis of ATP? 649 c-subunits (see Chapter 20), consisting of a knoblike structure some 9 nm in diame- ter (CF 1 ) attached to a stalked base (CF 0 ) embedded in the thylakoid membrane. The mechanism of action of CF 1 CF 0 –ATP synthase in coupling ATP synthesis to the col- lapse of the pH gradient is similar to that of the mitochondrial ATP synthase de- scribed in Chapter 20. However, higher plant CF 1 CF 0 –ATP synthase is believed to have 14 c-subunits in its F 0 rotor, implying that one turn of F 0 would require 14 H ϩ and lead to synthesis of 3 ATPs. The mechanism of photophosphorylation is summarized schematically in Figure 21.20. Photophosphorylation Can Occur in Either a Noncyclic or a Cyclic Mode Photosynthetic electron transport, which pumps H ϩ into the thylakoid lumen, can occur in two modes, both of which lead to the establishment of a transmembrane proton-motive force. Thus, both modes are coupled to ATP synthesis and are con- sidered alternative mechanisms of photophosphorylation, even though they are dis- tinguished by differences in their electron transfer pathways. The two modes are cyclic and noncyclic photophosphorylation. Noncyclic photophosphorylation has been the focus of our discussion and is represented by the scheme in Figure 21.20, where electrons activated by quanta at PSII and PSI flow from H 2 O to NADP ϩ , with concomitant establishment of the proton-motive force driving ATP synthesis. Note that in noncyclic photophosphorylation, O 2 is evolved and NADP ϩ is reduced. Cyclic Photophosphorylation Generates ATP but Not NADPH or O 2 In cyclic photophosphorylation, the “electron hole” in P700 ϩ created by electron loss from P700 is filled not by an electron derived from H 2 O via PSII but by a cyclic pathway in which the photoexcited electron returns ultimately to P700 ϩ . This path- way is schematically represented in Figure 21.11 by the dashed line connecting ferredoxin (Fd) and plastoquinone (PQ) within the membrane. This pathway di- verts the activated e Ϫ lost from PSI back through the PQ pool, the cytochrome b 6 f complex, and plastocyanin to re-reduce P700 ϩ (Figure 21.21). Lumen H 2 O PQ 2 H + 4.5 H + 4.5 H + 4 H + Cyt b 6 Cyt b 6 Cyt f O 2 + 2 H + Q QH 2 Plastocyanin docking CF 1 CF 0 – ATP synthase Stroma H + + NADP + FNR NADPH ADP + P i Cu + Cu 2+ PC PC Ferredoxin docking Ferredoxin Fd ATP FeS A FeS B FeS X P700 A 1 O O O O A 0 Fe Phe D1 Phe D2 Chl D1 Chl D2 P D1 P D2 Y Z Y D Chl Z-D1 Chl Z-D2 D1 D2 (Mn) 4 Q A Q B Photon 1 2 Photon FIGURE 21.20 The mechanism of photophosphorylation. Photosynthetic electron transport establishes a proton gradient that is tapped by the CF 1 CF 0 –ATP synthase to drive ATP synthesis. 650 Chapter 21 Photosynthesis Proton translocations accompany these cyclic electron transfer events so that ATP synthesis can be achieved. In cyclic photophosphorylation, ATP is the sole product of energy conversion. No NADPH is generated, and because PSII is not involved, no oxygen is evolved. Cyclic photophosphorylation theoretically yields 2 H ϩ per e Ϫ (2 H ϩ /h␷) from the operation of the cytochrome b 6 f complex. Thus, cyclic photo- phosphorylation provides a mechanism for overcoming the ATP deficit for CO 2 fix- ation (see the earlier section titled Calculation of the Photosynthetic Energy Re- quirements for Hexose Synthesis Depends on H ϩ /h␷ and ATP/H ϩ Ratios, page 647). Estimates indicate that cyclic photophosphorylation may contribute about 10% of total chloroplast ATP synthesis and thereby elevate the ATP/NADPH ratio. 21.7 How Is Carbon Dioxide Used to Make Organic Molecules? As we began this chapter, we saw that photosynthesis traditionally is equated with the process of CO 2 fixation, that is, the net synthesis of carbohydrate from CO 2 . Indeed, the capacity to perform net accumulation of carbohydrate from CO 2 distinguishes the phototrophic (and autotrophic) organisms from heterotrophs. Although animals possess enzymes capable of linking CO 2 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 CO 2 to acetyl-CoA to form malonyl- CoA (see Chapter 24). Nevertheless, this “fixed CO 2 ” is liberated in the very next re- action, so no net CO 2 incorporation occurs. Elucidation of the pathway of CO 2 fixation represents one of the earliest appli- cations of radioisotope tracers to the study of biology. In 1945, Melvin Calvin and his colleagues at the University of California, Berkeley, were investigating photo- synthetic CO 2 fixation in Chlorella. Using 14 CO 2 , they traced the incorporation of radioactive 14 C into organic products and found that the earliest labeled product was 3-phosphoglycerate (see Figure 17.14). Although this result suggested that the CO 2 acceptor was a two-carbon compound, further investigation revealed that, in ATP ADP H + H + Photon Plastocyanin docking Lumen Stroma + P i CF 1 CF 0 PC H + H + FeS A FeS B FeS X P700 A 1 O O O O A 0 PQ Cyt b 6 Cyt b 6 Cyt f FIGURE 21.21 The pathway of cyclic photophosphoryla- tion by PSI. (Adapted from Arnon, D. I., 1984.The discovery of photosynthetic phosphorylation. Trends in Biochemical Sciences 9:258–262.) 21.7 How Is Carbon Dioxide Used to Make Organic Molecules? 651 reality, 2 equivalents of 3-phosphoglycerate were formed following addition of CO 2 to a five-carbon (pentose) sugar: CO 2 ϩ 5-carbon acceptor⎯⎯→[6-carbon intermediate]⎯⎯→ two 3-phosphoglycerates Ribulose-1,5-Bisphosphate Is the CO 2 Acceptor in CO 2 Fixation The five-carbon CO 2 acceptor was identified as ribulose-1,5-bisphosphate (RuBP), and the enzyme catalyzing this key reaction of CO 2 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 CO 2 or, alternatively, O 2 with RuBP. Rubisco is found in the chloro- plast 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 iden- tical large subunits (55 kD) and eight small subunits (15 kD) (Figure 21.22). The large subunit is the catalytic unit of the enzyme. It binds both substrates (CO 2 and RuBP) and Mg 2ϩ (a divalent cation essential for enzymatic activity). The small subunit modu- lates the catalytic efficiency of the enzyme, increasing k cat more than 100-fold. The ru- bisco 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 ac- tive rubisco heteromultimers occurs within chloroplasts following transit of the small subunit polypeptide across the chloroplast membrane. 2-Carboxy-3-Keto-Arabinitol Is an Intermediate in the Ribulose-1,5-Bisphosphate Carboxylase Reaction The addition of CO 2 to ribulose-1,5-bisphosphate results in the formation of an enzyme-bound intermediate, 2-carboxy-3-keto-arabinitol (Figure 21.23, II). This in- termediate arises when CO 2 adds to the enediol intermediate generated from ribulose-1,5-bisphosphate. Hydrolysis of the C 2 OC 3 bond of the intermediate gen- erates two molecules of 3-phosphoglycerate. The CO 2 ends up as the carboxyl group of one of the two molecules. Ribulose-1,5-Bisphosphate Carboxylase Exists in Inactive and Active Forms Rubisco exists in three forms: an inactive form, designated E; a carbamylated, but in- active, form, designated EC; and an active form, ECM, which is carbamylated and has Mg 2ϩ at its active sites as well. Carbamylation of rubisco takes place by addition of CO 2 FIGURE 21.22 Molecular graphic of ribulose bisphos- phate carboxylase.The enzyme consists of 8 equivalents each of two subunits. Clusters of four small subunits (orange and red) are located at each end of the sym- metric octamer formed by the L subunits (light and dark green).The active sites are revealed in the ribbon dia- gram by bound ribulose-1,5-bisphosphate (yellow) (pdb id ϭ 1RXO). H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– H 2 COPO 3 2– 2 3 4 5 1 2 3 4 5 1 CO HCOH HCOH C COH HCOH CC HCOH C O HO III O O – O – CC HCOH C HO III O O – HO OH HCOH C 2 O O – H 2 O H + H + CO 2 FIGURE 21.23 The ribulose bisphosphate carboxylase reaction. Mg 2ϩ at the active site aids in stabilizing the 2,3-enediol transition state (I) for CO 2 addition and in facilitating the carbon–carbon bond cleavage that leads to product formation. 652 Chapter 21 Photosynthesis to its Lys 201 ⑀-NH 2 groups (to give ⑀ONHOCOO Ϫ derivatives). The CO 2 molecules used to carbamylate Lys residues do not become substrates. The carbamylation reac- tion occurs spontaneously at slightly alkaline pH (pH 8). Carbamylation of rubisco completes the formation of a binding site for the Mg 2ϩ that participates in the catalytic reaction. Once Mg 2ϩ binds to EC, rubisco achieves its active ECM form. Activated rubisco displays a K m for CO 2 of 10 to 20 ␮M. The relative abundance of CO 2 in the atmosphere is low, about 0.03%. The concentration of CO 2 dissolved in aqueous so- lutions equilibrated with air is about 10 ␮M. Substrate RuBP binds much more tightly to the inactive E form of rubisco (K D ϭ 20 nM) than to the active ECM form (K m for RuBP ϭ 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 Mg 2ϩ binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco. CO 2 Fixation into Carbohydrate Proceeds Via the Calvin–Benson Cycle The immediate product of CO 2 fixation, 3-phosphoglycerate, must undergo a series of transformations before the net synthesis of carbohydrate is realized. Among car- bohydrates, hexoses (particularly glucose) occupy center stage. Glucose is the build- ing 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 CO 2 fixation is amply justified. Also, sucrose (␣- D-glucopyranosyl-(1⎯→2)-␤-D-fructofuranoside) is the major carbon form translo- cated out of leaves to other plant tissues. In nonphotosynthetic tissues, sucrose is me- tabolized via glycolysis and the TCA cycle to produce ATP. 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 dis- coverers. The reaction series is indeed cyclic because not only must carbohydrate appear as an end product, but the five-carbon acceptor, RuBP, must be regenerated to provide for continual CO 2 fixation. Balanced equations that schematically repre- sent this situation are 6(1) ϩ 6(5)⎯⎯→12(3) 12(3)⎯⎯→1(6) ϩ 6(5) Net: 6(1) ⎯⎯→1(6) Each number in parentheses represents the number of carbon atoms in a com- pound, and the number preceding the parentheses indicates the stoichiometry of the reaction. Thus, 6(1), or 6 CO 2 , 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 Serve Three Metabolic Purposes The Calvin cycle enzymes serve three important ends: 1. They constitute the predominant CO 2 fixation pathway in nature. 2. They accomplish the reduction of 3-phosphoglycerate, the primary product of CO 2 fixation, to glyceraldehyde-3-phosphate so that carbohydrate synthesis be- comes feasible. 3. They catalyze reactions that transform three-carbon compounds into four-, five-, six-, and seven-carbon compounds. Most of the enzymes mediating the reactions of the Calvin cycle also participate in either glycolysis (see Chapter 18) or the pentose phosphate pathway (see Chap- ter 22). The aim of the Calvin scheme is to account for hexose formation from