Photosynthesis and Carbohydrate Synthesis in Plants

CHAPTER 20 PHOTOSYNTHESIS AND CARBOHYDRATE SYNTHESIS IN PLANTS As we examine this process, these principles will emerge: The capture of solar energy by photosynthetic organisms and its conversion to the chemical energy of reduced organic compounds is the ultimate source of nearly all biological energy and organic nutrients for all of the nonphotosynthetic organisms, including humans. It is arguably the most important biochemical process in the biosphere. Photosynthetic organisms use tightly organized light- harvesting complexes to absorb sunlight and capture its energy in chemical form: a separation of positive and negative charge leading to electron flow. The energy from an absorbed photon moves from one antenna chlorophyll to another and another until it arrives at the reaction center where it promotes the photochemical reaction that sends electrons through a series of electron carriers. The light-driven flow of electrons through specialized protein carriers is coupled to ATP synthesis. A strong reducing agent (NADPH) is also produced, and simultaneously, water is oxidized to O2, which is released into the atmosphere. Evolution yielded a universal mechanism for coupling ATP synthesis to the flow of electrons. A proton gradient created by electron flow is used to energize the ATP- synthesizing enzyme in microorganisms, animals, and plants. The ATP and NADPH produced in the light-dependent reactions of photosynthesis provide the energy and the reducing power to convert atmospheric CO2 into simple organic compounds. High concentrations of ATP and NADPH allow the chloroplast to carry out redox reactions that are thermodynamically unfavorable. Photosynthesis encompasses two processes: the light-dependent reactions, in which sunlight provides the energy for the synthesis of ATP and NADPH, and the CO2-assimilation reactions, in which ATP and NADPH are used to reduce CO2 to form triose phosphates via a set of reactions known as the Calvin cycle (Fig. 20-1). We heterotrophs are alive because the enormous energy of sunlight has been captured and tamed by autotrophs by photosynthesis and made available to us as fuel, vitamins, and building blocks. How do they do it? FIGURE 20-1 Assimilation of CO2 provides all of the carbon a plant needs. The light- driven synthesis of ATP and NADPH provides energy and reducing power for the fixation of CO2 into trioses in the Calvin cycle. All of the carbon-containing compounds of the plant cell are synthesized from this fixation of CO2. All vascular plants, as well as algae and cyanobacteria, carry out the same basic process of photosynthesis, but some are more amenable to study than others. Algae and cyanobacteria have been extensively studied because of the relative ease of culturing and manipulating them in the laboratory. Spinach is a vascular plant commonly used for studies of photosynthesis because of the ease of obtaining large amounts of material; and for genetic approaches, the small plant Arabidopsis thaliana is a favorite. What we say here about photosynthesis is essentially true of photosynthesis in all of these organisms. Aer looking at photosynthesis, we will discuss the conversion of trioses produced in the Calvin cycle to sucrose (for sugar transport) and starch (for energy storage) (see Fig. 20-1). This conversion is accomplished by mechanisms analogous to those used by animal cells to make glycogen. We also describe the synthesis of the cellulose of plant cell walls. Finally, we consider how carbohydrate metabolism is integrated within a plant cell and throughout the plant. Although strikingly different on the surface, the processes of photophosphorylation in the chloroplast and oxidative phosphorylation in the mitochondrion are closely similar at the molecular level, and the mechanism for ATP synthesis is virtually identical: a proton gradient drives rotary catalysis by a remarkable ATP synthase. 20.1 Light Absorption Photophosphorylation (ATP synthesis driven by light) resembles oxidative phosphorylation in that electron flow through a series of membrane carriers is coupled to proton pumping, producing the proton motive force that powers ATP formation. The processes are compared in Figure 20-2. In oxidative phosphorylation, the electron donor is NADH and the ultimate electron acceptor is O2, forming H2O. In photophosphorylation, electrons flow in the opposite direction: H2O is the electron donor and NADPH is formed. How is this endergonic process possible?

FIGURE 20-2 The chemiosmotic mechanism for ATP synthesis in chloroplasts and mitochondria. (a) Movement of electrons through a chain of membrane-bound carriers in the chloroplast membrane is driven by the energy of photons absorbed by the green pigment chlorophyll. Electron flow leads to the movement of protons and positive charge across the membrane, creating an electrochemical potential. This electrochemical potential drives ATP synthesis by the membrane-bound ATP synthase, which is fundamentally similar in structure and mechanism to (b) the mitochondrial machinery for oxidative phosphorylation of mitochondria. In mitochondria, the force that moves electrons through the complexes is a large difference in the reduction potentials of electron donor and acceptor. In both systems, the energy made available by electron transfer is captured as a transmembrane proton gradient, which drives ATP synthesis by an ATP synthase. Water is a poor donor of electrons; its standard reduction potential is 0.816 V, compared with −0.320 V for NADH, a good electron donor. Photosynthesis requires the input of energy in the form of light to create a good electron donor and a good electron acceptor. Electrons flow from the electron donor through a series of membrane-bound carriers, including cytochromes, quinones, and iron-sulfur proteins, while protons are pumped across a membrane to create an electrochemical potential. Electron transfer and proton pumping are catalyzed by a membrane complex that is homologous in structure and function to Complex III of mitochondria. The electrochemical potential so produced is the driving force for ATP synthesis from ADP and Pi, catalyzed by a membrane-bound ATP synthase complex closely similar to that of mitochondria and bacteria. Chloroplasts Are the Site of Light- Driven Electron Flow and Photosynthesis in Plants In photosynthetic eukaryotic cells, both the light-dependent and the CO2-assimilation reactions take place in chloroplasts (Fig. 20-3), organelles that are variable in shape and generally a few micrometers in diameter. Like mitochondria, chloroplasts are surrounded by two membranes: an outer membrane that is permeable to small molecules and ions, and an impermeable inner membrane that bears specific transporters for a variety of ions and metabolites. The space enclosed by the inner membrane is called the stroma in chloroplasts and is analogous to the mitochondrial matrix; it is an aqueous phase containing most of the soluble enzymes required for the CO2-assimilation reactions. Throughout the stroma is a highly organized set of topologically continuous internal membranes, forming a single compartment or lumen. This complex membrane system forms flattened sacks called thylakoids. Granal thylakoids are disk-like pouches arranged in stacks; they are connected by stromal thylakoids, which are flatter and spiral around a stack of grana. The thylakoid membranes provide a large area for the machinery of photophosphorylation — the photosynthetic pigments and enzyme complexes that carry out the light-dependent reactions and ATP synthesis. Traffic across these membranes is also mediated by specific transporters. FIGURE 20-3 Chloroplast structure. (a) Schematic diagram. (b) Colorized electron micrograph at high magnification, showing the highly organized thylakoid membrane system. In 1937, Robert Hill found that when leaf extracts containing chloroplasts were illuminated, they (1) evolved O2 and (2) reduced a nonbiological electron acceptor added to the medium, according to the Hill reaction 2H2O + 2A  light −−→  2AH2+ O2 where A is an artificial electron acceptor, or Hill reagent. One Hill reagent, the dye 2,6-dichlorophenolindophenol, is blue when oxidized (A) and colorless when reduced (AH2), making the reaction easy to follow. When a leaf extract supplemented with the dye was illuminated, the blue dye became colorless and O2 was evolved. In the dark, no O2 evolution or dye reduction took place. This was the first evidence that absorbed light energy causes electrons to flow from some electron donor (now known to be H2O) to an electron acceptor. Moreover, Hill found that CO2 was neither required nor reduced to a stable form under these conditions; O2 production could be dissociated from CO2 reduction. Several years later, Severo Ochoa showed that NADP+ is the biological electron acceptor in chloroplasts, according to the equation 2H2O + 2NADP+  light −−→  2NADPH + 2H+ + O2 To understand this photochemical process, we must first consider the more general topic of the effects of light absorption on molecular structure. Visible light is electromagnetic radiation of wavelengths 400 to 700 nm, a small part of the electromagnetic spectrum (Fig. 20-4), ranging from violet to red. The energy of a single photon (a quantum of light) is greater at the violet end of the spectrum than at the red end; shorter wavelength (and higher frequency) corresponds to higher energy. The energy, E, in a single photon of visible light is given by the Planck equation: E = hν= hc/λ where h is Planck’s constant (6.626× 10−34 J ∙s), ν is the frequency of the light in cycles/s, c is the speed of light (3.00× 108 m/s), and λ is the wavelength of the light in meters. The energy of a photon of visible light ranges from 150 kJ/einstein for red light to ∼300 kJ/einstein for violet light. FIGURE 20-4 Electromagnetic radiation. The spectrum of electromagnetic radiation, and the energy of photons in the visible range. One einstein is 6.022× 1023 photons. WORKED EXAMPLE 20-1 Energy of a Photon The light used by vascular plants for photosynthesis has a wavelength of about 700 nm. Calculate the energy in a “mole” of photons (an einstein) of light of this wavelength, and compare this with the energy needed to synthesize a mole of ATP. SOLUTION: The energy in a single photon is given by the Planck equation. At a wavelength of 700× 10−9 m, the energy of a photon is E = hc/λ = = 2.84× 10−19 J An einstein of light is Avogadro’s number of photons (6.022× 1023); thus the energy of one einstein of photons at 700 nm is given by (2.84× 10−19 J /photon)(6.022 × 1023 photons/einstein) = 17.1× 104 J /einstein = 171 kJ /einstein So, a “mole” of photons of red light has about five times the energy needed to produce a mole of ATP from ADP and Pi (30.5 kJ/mol). When a photon is absorbed, an electron in the absorbing molecule (chromophore) is lied to a higher energy level. This is an all-or-nothing event: to be absorbed, the photon must contain a quantity of energy, called a quantum, that exactly matches the energy of the electronic transition. A molecule that has absorbed a photon is in an excited state, which is generally unstable. An electron lied into a higher-energy orbital usually returns rapidly to its lower-energy orbital; that is, the excited molecule decays to the stable ground state, giving up the absorbed quantum as light [(6.626× 10−34 J ∙s)(3.00× 108 m/s)] (7.00× 10−7 m) or heat or using it to do chemical work. Light emission accompanying decay of excited molecules, fluorescence, is always at a longer wavelength (lower energy) than that of the absorbed light (see Box 12-1). An alternative mode of decay, central to photosynthesis, involves direct transfer of excitation energy from an excited molecule to a neighboring molecule. Just as the photon is a quantum of light energy, so the exciton is a quantum of energy passed from an excited molecule to another molecule in a process called exciton transfer. Chlorophylls Absorb Light Energy for Photosynthesis The most important light-absorbing pigments in the thylakoid membranes are the chlorophylls, green pigments with polycyclic, planar structures resembling the protoporphyrin of hemoglobin, except that M g2+, not Fe2+, occupies the central position (Fig. 20- 5a; compare to Fig. 5-1). The four inward-oriented nitrogen atoms of chlorophyll are coordinated with the M g2+. All chlorophylls have a long phytol side chain, esterified to a carboxyl-group substituent in ring IV, and chlorophylls also have a fih five- membered ring not present in heme. FIGURE 20-5 Primary and secondary photopigments. (a) Chlorophylls a and b and bacteriochlorophyll are the primary gatherers of light energy. (b) β -Carotene (a carotenoid) and (c) lutein (a xanthophyll) are accessory pigments in plants. (d) Phycoerythrobilin and phycocyanobilin (phycobilins) are accessory pigments in cyanobacteria and red algae. The conjugated bond systems in these molecules (alternating single and double bonds, shaded) have delocalized electrons that are easily excited by photons with the wavelengths of visible light. The heterocyclic five-ring system that surrounds the M g2+ has an extended polyene structure, with alternating single and double bonds. Such polyenes characteristically show strong absorption in the visible region of the spectrum (Fig. 20-6); the chlorophylls have unusually high molar extinction coefficients (see Box 3-1) and are therefore particularly well-suited for absorbing visible light during photosynthesis. FIGURE 20-6 Absorption of visible light by photopigments. Plants are green because their pigments absorb light from the red and blue regions of the spectrum, leaving primarily green light to be reflected. Compare the absorption spectra of the pigments with the spectrum of sunlight reaching the earth’s surface; the combination of chlorophylls (a and b) and accessory pigments enables plants to harvest most of the energy available in sunlight. The relative amounts of chlorophylls and accessory pigments are characteristic of a particular plant species. Variation in the proportions of these pigments is responsible for the range of colors of photosynthetic organisms, from the deep blue-green of spruce needles, to the greener green of maple leaves, to the red, brown, or purple color of some species of multicellular algae and the leaves of some foliage plants favored by gardeners. Chloroplasts always contain both chlorophyll a and chlorophyll b (Fig. 20-5a). Although both are green, their absorption spectra are sufficiently different (Fig. 20-6) that they complement each other’s range of light absorption in the visible region. Most plants contain about twice as much chlorophyll a as chlorophyll b. The chlorophyll in cyanobacteria differs only slightly from those of plants. In addition to chlorophylls, thylakoid membranes of plants contain secondary light-absorbing pigments, or accessory pigments, called carotenoids. Carotenoids may be yellow, red, or purple. The two most prominent in plant leaves are β -carotene, a red-orange isoprenoid, and the yellow carotenoid lutein (Fig. 20- 5b, c). Cyanobacteria and red algae use the accessory pigments phycocyanobilin and phycoerythrobilin (Fig. 20-5d). Accessory pigments absorb light at wavelengths not absorbed by the chlorophylls (Fig. 20-6) and thus are supplementary light receptors. They also protect downstream components from a highly reactive form of oxygen (singlet oxygen) that is formed when intense light exceeds the system’s capacity to accept electrons. Experimental determination of the effectiveness of light of different colors in promoting photosynthesis yields an action spectrum (Fig. 20-7), oen useful in identifying the pigment primarily responsible for a biological effect of light. By capturing light in a region of the spectrum not used by other organisms, a photosynthetic organism can claim a unique ecological niche.

FIGURE 20-7 Two ways to determine the action spectrum for photosynthesis. (a) Results of a classic experiment performed by T. W. Engelmann in 1882 to determine the wavelength of light that is most effective in supporting photosynthesis. Engelmann placed cells of a filamentous photosynthetic alga on a microscope slide and illuminated them with light from a prism, so that one part of the algal filament received mainly blue light, another part yellow, another red. To determine which cells carried out photosynthesis most actively, Engelmann also placed on the microscope slide bacteria known to migrate toward regions of high O2 concentration. A er a period of illumination, the distribution of bacteria showed highest O2 levels (produced by photosynthesis) in the regions illuminated with violet and red light. (b) Results of a similar experiment that used modern techniques (an oxygen electrode) for the measurement of O2 production. An action spectrum, as shown here, describes the relative rate of photosynthesis for illumination with a constant number of photons of different wavelengths. An action spectrum is useful because, by comparison with absorption spectra (such as those in Fig. 20-6), it suggests which pigments can channel energy into photosynthesis. Chlorophylls Funnel Absorbed Energy to Reaction Centers by Exciton Transfer The light-absorbing pigments of thylakoid or bacterial membranes are arranged in functional arrays called photosystems. In spinach chloroplasts, for example, each photosystem contains about 200 chlorophyll and 50 carotenoid molecules. All the pigment molecules in a photosystem can absorb photons, but only one pair of chlorophyll molecules associated with the photochemical reaction center is specialized to transduce light into chemical energy. The other pigment molecules in a photosystem serve as antenna molecules. They absorb light energy and transmit it rapidly and efficiently to the reaction center (Fig. 20-8). Some chlorophylls are part of a core complex around the reaction center. Others form light-harvesting complexes (LHCs) around the periphery of the core complex. Chlorophyll and other pigments are always associated with specific binding proteins, which fix the chromophores in relation to each other, to other protein complexes, and to the membrane. For example, each monomer of the trimeric light-harvesting complex LHCII (Fig. 20-9) contains seven molecules of chlorophyll a, five of chlorophyll b, and two of lutein. FIGURE 20-8 Organization of photosystems in the thylakoid membrane. Photosystems are tightly packed in the thylakoid membrane, with several hundred antenna chlorophylls and accessory pigments surrounding each reaction center. Absorption of a photon by any of the antenna chlorophylls leads to excitation of the reaction center by exciton transfer (red arrow). FIGURE 20-9 The light-harvesting complex LHCII of the pea. The functional unit is a trimer, with 36 chlorophyll and 6 lutein molecules. Shown here is a monomer, viewed in the plane of the membrane, with its three transmembrane α -helical segments, seven chlorophyll a molecules (light green), five chlorophyll b molecules (dark green), and two molecules of lutein (yellow), which form an internal cross-brace. [Data from PDB ID 2BHW, J. Standfuss et al., EMBO J. 24:919, 2005.] The chlorophyll molecules in light-harvesting complexes and other chlorophyll-binding proteins have light-absorption properties that are subtly different from those of free chlorophyll. When isolated chlorophyll molecules are excited by light, the absorbed energy is quickly released as fluorescence and heat; but when chlorophyll in intact leaves is excited by visible light (Fig. 20-10, step ), very little fluorescence is observed. Instead, the excited antenna chlorophyll transfers energy directly to a neighboring chlorophyll molecule, which becomes excited as the first molecule returns to its ground state (step ). This transfer of energy, exciton transfer, extends to a third, fourth, or subsequent neighbor, until one of a “special pair” of chlorophyll a molecules at the photochemical reaction center is excited (step ). The special pair of chlorophyll molecules, oen designated (Chl)2, are held close enough to each other to share bonding orbitals, and to react as a single compound when excited. In this excited chlorophyll pair, an electron is promoted to a higher-energy orbital. This electron then passes to a nearby electron acceptor that is part of the photosynthetic electron-transfer chain, leaving the reaction-center chlorophyll pair with a missing electron (an “electron hole,” denoted by + in Fig. 20-10) (step ). The electron acceptor acquires a negative charge in this transaction. The electron lost by the reaction-center chlorophyll pair is replaced by an electron from a neighboring electron-donor molecule (step ), which thereby becomes positively charged.

In this way, excitation by light causes electric charge separation and initiates an oxidation-reduction chain.

FIGURE 20-10 Exciton and electron transfer. This generalized scheme shows conversion of the energy of an absorbed photon into separation of charges at the reaction center. Note that step may repeat between successive antenna molecules until the exciton reaches the special pair of chlorophylls in the reaction center. An asterisk (*) denotes the excited state of a molecule. SUMMARY 20.1 Light Absorption Photosynthesis takes place in plant chloroplasts, structures enclosed in double membranes and filled with an elaborate system of thylakoid membranes containing the photosynthetic machinery. Chlorophyll molecules and other light-absorbing pigments are associated with proteins in light-harvesting complexes arrayed around photochemical reaction centers. The proteins are embedded in thylakoid membranes. The many chlorophyll molecules that surround the reaction center serve as antennas for light. When they absorb light, they pass its energy (exciton) to the reaction center. There the energy is used to create a charge separation that initiates electron flow through a series of oxidation-reduction reactions. 20.2 Photochemical Reaction Centers Studies on a variety of bacteria that carry out photosynthesis have been helpful in understanding the mechanisms of photosynthesis in cyanobacteria, algae and vascular plants. Photosynthetic bacteria have relatively simple phototransduction machinery, with one of two general types of photosystems. Both systems send electrons through a cytochrome complex that pumps protons, producing the electrochemical gradient that drives ATP synthesis. Photosynthetic Bacteria Have Two Types of Reaction Center The type II photosystem in purple bacteria consists of three basic modules (Fig. 20-11a): a single P870 reaction center; a cytochrome bc1 electron-transfer complex similar to Complex III of the mitochondrial electron-transfer chain; and an ATP synthase, also similar to that of mitochondria. Illumination lis an electron in the reaction center to its excited state (P870*), from which it passes through pheophytin (chlorophyll a lacking its central M g2+) and a quinone to the cytochrome bc1 complex. Aer passing through the bc1 complex, electrons flow through cytochrome c2 back to the reaction center, restoring its preillumination state and completing one cycle. This light-driven cyclic electron transfer provides the energy for proton pumping by the cytochrome bc1 complex. Powered by the resulting proton gradient, ATP synthase produces ATP, exactly as in mitochondria. The type I photosystem in green sulfur bacteria involves the same three modules as in purple bacteria, but the process differs in several respects and includes additional enzymatic reactions (Fig. 20-11b). Excitation by light causes an electron to move from the excited reaction center to the cytochrome bc1 complex via a quinone carrier. Electron transfer through this complex powers proton transport and creates the proton-motive force used for ATP synthesis, just as in purple bacteria and in mitochondria. However, in contrast to the cyclic electron transfer path in purple bacteria, some electrons follow a linear electron transfer path from the reaction center to the soluble iron-sulfur protein ferredoxin (see Fig. 19-5), which then passes electrons via ferredoxin: NAD+ reductase to NAD+, producing NADH. The electrons taken from the reaction center to reduce NAD+ are replaced by the oxidation of H2S to elemental S in the reaction that defines the green sulfur bacteria. This oxidation of H2S by bacteria is chemically analogous to the oxidation of H2O by oxygenic plants. Note that the path of electrons in the purple bacteria is cyclic; the path in the green sulfur bacteria can be either cyclic or linear, leading to NAD+ and producing NADH. FIGURE 20-11 Functional Modules of Photosynthetic Machinery in Purple Bacteria and Green Sulfur Bacteria. The position on the vertical scale of each electron carrier reflects its standard reduction potential. (a) In purple bacteria, light energy excites an electron in the reaction center P870. The electron passes through pheophytin (Pheo), a quinone (Q), and the cytochrome bc1 complex, then through cytochrome c2 and thus back to the reaction center. Electron transfer through the cytochrome bc1 complex causes proton pumping, creating an electrochemical potential that powers ATP synthesis. (b) Green sulfur bacteria have two routes for electrons driven by excitation of P840. A cyclic electron transfer route that goes through a quinone to the cytochrome bc1 complex and back to the reaction center via cytochrome c553 causes proton pumping. A linear electron transfer route that goes from the reaction center through the iron-sulfur protein ferredoxin (Fd) reduces NAD+ to NADH in a reaction catalyzed by ferredoxin: NAD+ reductase. In Vascular Plants, Two Reaction Centers Act in Tandem The photosynthetic apparatus of cyanobacteria, algae, and vascular plants is more complex than the one-center bacterial systems, and it most likely evolved through the combination of two simpler bacterial photosystems. The Z scheme diagram in Figure 20-12 outlines the path of electron flow between the two photosystems and the energy relationships in the light-dependent reactions. (The Z scheme takes its name from the zigzag pattern of the pathways in the diagram.) The thylakoid membranes of chloroplasts have two different kinds of photosystems, each with its own type of photochemical reaction center and set of antenna molecules. The two systems have distinct and complementary functions. Photosystem II (PSII) is a pheophytin-quinone type of system (like the single photosystem of purple bacteria) containing roughly equal amounts of chlorophylls a and b. Excitation of the P680 special pair in its reaction center drives electrons through the cytochrome b6f complex discussed below, with concomitant pumping of protons across the thylakoid membrane and ATP synthesis. Photosystem I (PSI) is structurally and functionally related to the photosynthetic machinery of green sulfur bacteria. It has a P700 reaction center and a high ratio of chlorophyll a to chlorophyll b. The excited P700 passes electrons through a linear chain of carriers to ferredoxin, then to NADP+, producing NADPH. An alternative pathway for electrons is cyclic: instead of following the linear path that leads to NADP+ reduction, electrons pass to plastoquinone (PQ) through a membrane- embedded protein complex, cytochrome b6f (again, with the movement of protons into the chloroplast lumen). The thylakoid membranes of a single spinach chloroplast have many hundreds of each kind of photosystem. FIGURE 20-12 Integration of photosystems I and II in chloroplasts. This “Z scheme” shows the pathway of linear electron transfer from H2O (lower le ) to NADP+ (far right). The position on the vertical scale of each electron carrier reflects its standard reduction potential. To raise the energy of electrons derived from H2O to the energy level required to reduce NADP+ to NADPH, each electron must be “li ed” twice (heavy arrows) by photons absorbed in PSII and PSI. One photon is required per electron in each photosystem. A er excitation, the high-energy electrons flow “downhill” through the carrier chains as shown. Protons move across the thylakoid membrane during the water-splitting reaction and during electron transfer through the cytochrome b6f complex, producing the proton gradient that is essential to ATP formation. An alternative path of electrons is cyclic electron transfer, in which electrons move from ferredoxin back to the plastoquinone and cytochrome b6f complex, instead of reducing NADP+ to NADPH. The cyclic pathway produces more ATP and less NADPH than the linear pathway. These two photosystems in plants act in tandem to catalyze the light-driven movement of electrons from H2O to NADP+. The electron carriers include large, integral protein complexes (PSI, PSII, and the proton-pumping complex cytochrome b6f); quinones that are lipid-soluble and move through the membrane between the protein complexes; and two soluble proteins, plastocyanin (analogous to cytochrome c in mitochondria) and ferredoxin. To replace the electrons that move from PSII through PSI to NADP+, H2O is oxidized, producing O2 (Fig. 20-12, bottom le). All O2-evolving photosynthetic cells — those of plants, algae, and cyanobacteria — contain both PSI and PSII. The Z scheme thus describes the complete route by which electrons flow from H2O to NADP+, according to the equation 2H2O + 2NADP+ + 8 photons→ O2+ 2NADPH + 2H+ For every two photons absorbed (one by each photosystem), one electron is transferred from H2O to NADP+. To form one molecule of O2, which requires transfer of four electrons from two H2O to two NADP+, a total of eight photons must be absorbed, four by each photosystem. Having seen the overall process, we’ll now look at how the structure of the photosystems informs our understanding of the electrochemistry. Photosystem II PSII is dimeric (Fig. 20-13). Each monomer is a huge complex of 19 proteins, including the accessory proteins CP47 and CP43, and the core complex of P680 reaction-center proteins D1 and D2; 2 chlorophyll-binding proteins; and associated chromophores, including carotenoids, a nonheme iron, and the critically important inorganic cofactor, M n4CaO5. Of the proteins in PSII, 16 have transmembrane segments, but 3 are peripheral proteins on the lumenal side that stabilize the M n4CaO5 cofactor. Surrounding PSII are additional chlorophyll-binding proteins and light-harvesting complexes. When a photon is absorbed by any of these antenna molecules, the resulting exciton moves very rapidly from one to another of the antenna chlorophylls until it reaches the reaction center and excites P680, the special pair of chlorophyll a molecules (Chl a)2, to initiate the photochemistry. FIGURE 20-13 Structure of photosystem II of the cyanobacterium Thermosynechococcus vulcanus. The enormous complex, visualized by x- ray crystallography, is a dimer; each monomer has its own reaction center. Chlorophyll-binding proteins CP43 and CP47 form the core antenna, directly associated with the PSII reaction-center proteins D1 and D2. Each PSII monomer contains 35 chlorophylls, 2 pheophytins, 11 β -carotenes, 2 plastoquinones, and 1 each of b-type cytochrome, c-type cytochrome, and nonheme iron. Water is oxidized to form O2 at the oxygen-evolving center (M n4CaO5). [Data from PDB ID 3WU2, Y. Umena et al., Nature 473:55, 2011.] Excitation of P680 in PSII (Fig. 20-14) produces P680*, an excellent electron donor that, within picoseconds, transfers an electron to pheophytin, giving it a negative charge (∙Pheo−). With the loss of its electron, P680* is transformed into a radical cation, P680+. ∙Pheo− very rapidly passes its extra electron to a protein- bound plastoquinone, PQA, which in turn passes its electron to another, more loosely bound plastoquinone, PQB. When PQB has acquired two electrons in two such transfers from PQA and two protons from the solvent water, it is in its fully reduced quinol form, PQBH2. The overall reaction initiated by light in PSII is 4 P680+ 4H+ + 2 PQB + 4 photons→ 4 P680+ + 2 PQBH2 (20-1) Eventually, the electrons in PQBH2 pass through the cytochrome b6f complex (see Fig. 20-12). The electron initially removed from P680 is replaced with an electron obtained from the oxidation of water, as described below. FIGURE 20-14 Electron transfer through photosystem II of the cyanobacterium Synechococcus elongatus. The monomeric form of the core complex shown here has two major transmembrane proteins, D1 and D2, each with its set of electron carriers. Although the two subunits are nearly symmetrical, electron transfer occurs through only one of the two branches of electron carriers: that on the right (in D1). The arrows show the path of electron transfer from the M n4CaO5 ion cofactor of the oxygen- evolving complex to plastoquinone PQB. The photochemical events occur in the sequence indicated by the step numbers. The role of the Tyr residues and the detailed structure of the M n4CaO5 cofactor are discussed below (see Fig. 20-20b). Photosystem I PSI and its antenna molecules are part of a supramolecular complex composed of at least 16 proteins, including 4 chlorophyll-binding proteins arranged around the periphery of the reaction center (Fig. 20-15). The complex also includes 35 carotenoids of several types, three 4Fe-4S clusters, and two phylloquinones. FIGURE 20-15 Structure of photosystem I in the cyanobacterium Synechococcus elongatus. PSI is a symmetric trimer, viewed here (a) in the plane of the thylakoid membrane and (b) from the stroma ( side of the membrane). (c) One of the three core complexes in PSI, displayed as the protein without its ligands and (d) the ligands alone. Note the four peripheral light-harvesting complexes (LHC) and the many chlorophyll molecules surrounding the reaction center. (e) Close-up view of the reaction center without the surrounding chlorophylls, showing the chlorophyll special pair, phylloquinones, and Fe-S centers. [Data from PDB ID 1JBO, P. Jordan et al., Nature 411:909, 2001; PDB ID 4RKU, Y. Mazor et al.] The photochemical events that follow the excitation of PSI at the reaction-center P700 (Fig. 20-16) are formally similar to those occurring in PSII. The excited reaction-center P700* loses an electron to an acceptor, designated A0 (a chlorophyll a molecule, functionally homologous to the pheophytin of PSII), creating A− 0 and P700+. Again, excitation results in charge separation at the photochemical reaction center. P700+ is a strong oxidizing agent, which quickly acquires an electron from plastocyanin, a soluble Cu-containing electron-transfer protein. A− 0 is an exceptionally strong reducing agent that passes its electron through a chain of carriers that leads to NADP+ (Fig. 20-12, right side). Phylloquinone (QK) accepts the electron and passes it to an iron- sulfur protein through three Fe-S centers in PSI. From here, the electron moves to ferredoxin (Fd). Recall that ferredoxin contains a 2Fe-2S center (see Fig. 19-5) that undergoes one-electron oxidation and reduction reactions. The fourth electron carrier in the chain is the flavoprotein ferredoxin:NADP+ reductase, which transfers electrons from reduced ferredoxin (Fdred) to NADP+: 2Fdred + 2H+ + NADP+ → 2Fdox+ NADPH + H+ FIGURE 20-16 The path of electrons through PSI. The path of electrons (blue arrows) through PSI, viewed in the plane of the membrane. When the reaction-center P700, the special pair of chlorophylls, is excited by a photon or an exciton, its reduction potential is dramatically reduced, making it a good electron donor. P700 then passes an electron through a nearby chlorophyll (referred to as A0) to phylloquinone (QK). Reduced QK is reoxidized as it passes two electrons, one at a time, to an Fe-S center (FX) near the side of the membrane. From FX, electrons move through two more Fe-S centers (FA and FB) to ferredoxin in the stroma. Ferredoxin then donates electrons to NADP+ (not shown), reducing it to NADPH, one of the forms in which the energy of photons is trapped in chloroplasts. The Cytochrome b6f Complex Links Photosystems II and I, Conserving the Energy of Electron Transfer Electrons temporarily held in plastoquinol as a result of the excitation of P680 in PSII are carried to P700 of PSI via the cytochrome b6f complex and the soluble protein plastocyanin (see Fig. 20-12, center). With a structure and role analogous with that of Complex III in mitochondria, the cytochrome b6f complex (Fig. 20-17) contains a b-type cytochrome with two heme groups (designated bH and bL), a Rieske iron-sulfur protein (M r 20,000), and cytochrome f (named for the Latin frons, “leaf”). Electrons flow through the cytochrome b6f complex from PQBH2 to cytochrome f, then to plastocyanin, and finally to P700+, thereby reducing it.

FIGURE 20-17 Electron and proton flow through the cytochrome b6f complex. (a) In addition to the hemes of cytochrome b (heme bH and bL; also called heme bN and bP, respectively, because of their proximity to the and sides of the bilayer) and cytochrome f (heme f), there is a fourth heme (heme x) near heme bH; also present is a β-carotene of unknown function. Two sites bind plastoquinone: the PQH2 site near the side of the bilayer, and the PQ site near the side. The Fe-S center of the Rieske protein lies just outside the bilayer on the side, and the heme f site is on a protein domain that extends well into the thylakoid lumen. The electron path is shown for just one of the monomers, but both sets of carriers in the dimer carry electrons to plastocyanin. (b) Plastoquinol (PQH2), formed in PSII, is oxidized by the cytochrome b6f complex in a series of steps like those of the Q cycle in Complex III of mitochondria (see Fig. 19-11). One electron from PQH2 passes to the Fe-S center of the Rieske protein, the other to heme bL of cytochrome b6. The net effect is passage of electrons from PQH2 to the soluble protein plastocyanin, which carries them to PSI. [Data from PDB ID 1VF5, G. Kurisu et al., Science 302:1009, 2003; PDB ID 2Q5B, Y. S. Bukhman- DeRuyter et al.] Like Complex III of mitochondria, cytochrome b6f conveys electrons from a reduced quinone — a mobile, lipid-soluble carrier of two electrons (Q in mitochondria, PQB in chloroplasts; P for plastoquinone) — to a water-soluble protein that carries one electron (cytochrome c in mitochondria, plastocyanin in chloroplasts) (Fig 20-17a). As in mitochondria, the function of this complex involves a Q cycle (Fig. 20-17b; see Fig. 19-11) in which electrons pass, one at a time, from PQBH2 to cytochrome b6. This cycle results in the pumping of protons across the membrane, from the stromal compartment to the thylakoid lumen. Up to four protons enter the lumen for each pair of electrons that passes through the cytochrome b6f complex. The result is production of a proton gradient across the thylakoid membrane as electrons pass from PSII to PSI. Because the volume of the flattened thylakoid lumen is small, the influx of a small number of protons has a relatively large effect on lumenal pH. The measured difference in pH between the stroma (pH 8) and the thylakoid lumen (pH 5) represents a 1,000-fold difference in proton concentration — a powerful driving force for ATP synthesis. Cyclic Electron Transfer Allows Variation in the Ratio of ATP/NADPH Synthesized Cyclic electron flow between PSI and cytochrome b6f increases the production of ATP relative to NADPH. The linear path of electrons from water, through PSII, cytochrome b6f, and PSI to NADP+ produces both a proton gradient, which is used to drive ATP synthesis, and NADPH, which is used in reductive biosynthetic processes (see Fig. 20-12). Some fraction of electrons passing from P700* to ferredoxin do not continue to NADP+, but cycle back through plastoquinone and the cytochrome b6f complex to plastocyanin. Plastocyanin then donates electrons to P700. In this way, electrons are repeatedly recycled through the cytochrome b6f complex and the reaction center of PSI, each electron propelled around the cycle by the energy of one photon. Cyclic electron flow is not accompanied by net formation of NADPH or evolution of O2. However, it is accompanied by proton pumping by the cytochrome b6f complex and by phosphorylation of ADP to ATP, referred to as cyclic photophosphorylation. The overall equation for cyclic electron flow and photophosphorylation is simply ADP + Pi  light −−→  AT P + H2O By regulating the partitioning of electrons between NADP+ reduction and cyclic photophosphorylation, a plant adjusts the ratio of ATP to NADPH produced in the light- dependent reactions to match its needs for these products in the CO2-assimilation reactions and other biosynthetic processes. As we shall see in Section 20.4, the CO2-assimilation reactions require ATP and NADPH in the ratio 3:2. This regulation of electron-transfer pathways is part of a short-term adaptation to changes in light color (wavelength) and quantity (intensity). State Transitions Change the Distribution of LHCII between the Two Photosystems Photosynthetic organisms are exposed to light of highly variable intensity and wavelength in the course of a day or a season, and, although they can alter their growth patterns somewhat, they cannot uproot themselves and move to optimize their light exposure. Instead, cellular mechanisms have evolved that allow plants to accommodate changing light conditions. The energy needed to excite PSI (P700) is less (light of longer wavelength, lower energy) than the energy needed to excite PSII (P680). If PSI and PSII were physically contiguous, excitons originating in the antenna system of PSII would migrate to the reaction center of PSI, leaving PSII chronically underexcited and thus interfering with the operation of the two-center system. This imbalance in the supply of excitons is prevented by physically separating the two photosystems in the thylakoid membrane (Fig. 20-18). PSII is located almost exclusively in the tightly appressed membrane stacks of granal thylakoids; its associated light-harvesting complex (LHCII) mediates the tight association of adjacent membranes in the grana. PSI and the ATP synthase complex are located almost exclusively in the nonappressed membranes of the stromal thylakoids, where they have access to the contents of the stroma, including ADP and NADP+. The cytochrome b6f complex is present primarily in the granal thylakoids. FIGURE 20-18 Localization of PSI and PSII in thylakoid membranes. (a) Structures of the complexes and soluble proteins of the photosynthetic apparatus of a vascular plant or alga, drawn to the same scale. The bovine ATP synthase is shown. (b) Light-harvesting complex LHCII and ATP synthase are located both in appressed regions of the thylakoid membrane (granal thylakoids, in which several membranes are in contact) and in nonappressed regions (stromal thylakoids), and have ready access to ADP and NADP+ in the stroma. PSII is present almost exclusively in the appressed granal regions, and PSI almost exclusively in nonappressed stromal regions. LHCII is the “adhesive” that holds appressed thylakoid membranes together (see Fig. 20-19). [(a) Data from PSII: PDB ID 3WU2, Y. Umena et al., Nature 473:55, 2011; cyt b6fcomplex: PDB ID 2E74, E. Yamashita et al., J. Mol. Biol. 370:39, 2007; plastocyanin: PDB ID 1AG6, Y. Xue et al., Protein Sci. 7:2099, 1998; PSI: PDB ID 4RKU, Y. Mazor et al.; ferredoxin: PDB ID 1A70, C. Binda et al., Acta Crystallogr. D Biol. Crystallogr. 54:1353, 1998; ferredoxin:NADP reductase: PDB ID 1QG0, Z. Deng et al., Nat. Struct. Biol. 6:847, 1999; ATP synthase: PDB ID 5ARA, A. Zhou et al., eLife 4:e10180, 2015.] The association of LHCII with PSI and PSII depends on light intensity and wavelength, which can change in the short term and lead to state transitions in the chloroplast. In state 1, LHCII, PSII, and PSI are poised to maximize the capture of light energy. A critical Thr residue in LHCII is unphosphorylated, and LHCII associates with PSII. Under conditions of intense or blue light, which favor absorption by PSII, that photosystem reduces plastoquinone to plastoquinol (PQH2) faster than PSI can oxidize it. The resulting accumulation of PQH2 activates a protein kinase that triggers the transition to state 2 by phosphorylating a Thr residue on LHCII (Fig. 20-19). Phosphorylation weakens the interaction of LHCII with the appressed membrane and with PSII; some LHCII dissociates and moves to the stromal thylakoids. Here it captures photons (excitons) for PSI, speeding the oxidation of PQH2 and reversing the imbalance between electron flow in PSI and PSII. In less intense light (in the shade, with more red light), PSI oxidizes PQH2 faster than PSII can make it, and the resulting increase in [PQ] triggers dephosphorylation of LHCII, reversing the effect of phosphorylation. The state transition in LHCII localization and the transition from cyclic to linear electron transfer are coordinately regulated: the path of electrons is primarily linear in state 1 and primarily cyclic in state 2. FIGURE 20-19 Electron transfer in PSI and PSII is balanced through state transitions. In granal thylakoids, a hydrophobic domain of LHCII in one membrane inserts into the neighboring membrane and closely appresses the two (state 1). Accumulation of plastoquinol (not shown) stimulates a protein kinase that phosphorylates a Thr residue in the hydrophobic domain of LHCII, which reduces its affinity for the neighboring membrane and converts appressed granal thylakoids to nonappressed stromal thylakoids (state 2). A specific protein phosphatase reverses this regulatory phosphorylation when the [PQ]/[PQH2] ratio increases. When light is so intense that the combined activity of PSII and PSI cannot synthesize ATP and NADPH fast enough to keep up with the supply of photons, carotenoids in LHCII absorb the excitons and very rapidly quench the excited chlorophyll before it can create damaging reactive oxygen species (ROS). The trigger for switching from an efficient light-harvesting state to an energy- dissipating state is the lowering of pH in the lumenal space, but the detailed mechanism for this transition is not yet known. Water Is Split at the Oxygen-Evolving Center The ultimate source of the electrons passed to NADPH in plant (oxygenic) photosynthesis is water. Having given up an electron to pheophytin, P680+ (of PSII) must acquire an electron to return to its ground state in preparation for capture of another photon. In principle, the required electron might come from any number of organic or inorganic compounds. Photosynthetic bacteria use a variety of electron donors for this purpose — acetate, succinate, malate, or sulfide — depending on what is available in a particular ecological niche. About 2.5 billion years ago, evolution of primitive photosynthetic bacteria (progenitors of the modern cyanobacteria) produced a photosystem capable of taking electrons from a donor that is always available: water. Two water molecules are oxidized, yielding four electrons, four protons, and molecular oxygen: 2H2O → 4H+ + 4e− + O2 A single photon of visible light does not have enough energy to break the bonds in water; four photons are required in this photolytic cleavage reaction. The four electrons abstracted from water do not pass directly to P680+, which can accept only one electron at a time. Instead, a remarkable molecular device, the oxygen-evolving center, passes four electrons one at a time to P680+ (Fig. 20-20a). The immediate electron donor to P680+ is a Tyr residue (sometimes designated Z or T yrZ) in subunit D1 of the PSII reaction center. The Tyr residue loses both a proton and an electron, generating the electrically neutral Tyr free radical, ∙T yr: 4 P680+ + 4 T yr→ 4 P680+ 4 ∙T yr (20-2) The Tyr radical regains its missing electron and proton by oxidizing a cofactor of four manganese ions and one calcium ion in the oxygen-evolving center. With each single-electron transfer, the M n4CaO5 cofactor becomes more oxidized; four single- electron transfers, each corresponding to the absorption of one photon, produce a charge of 4+ on the M n4CaO5 cofactor (Fig. 20-20a): 4 ∙T yr+ [M n4CaO5]0→ 4 T yr+ [M n4CaO5]4+ (20-3) In this state, the M n4CaO5 cofactor can take four electrons from a pair of water molecules, releasing four H+ and O2: [M n4CaO5]4+ + 2H2O → [M n4CaO5]0+ 4H+ + O2 (20-4) Because the four protons produced in this reaction are released into the thylakoid lumen, the oxygen-evolving center acts as a proton pump, driven by electron transfer. We saw in Equation 20-1 that the overall reaction initiated by light in PSII is 4 P680+ 4H+ + 2 PQB + 4 photons→ 4 P680+ + 2 PQBH2 The sum of Equations 20-1 through 20-4 is 2H2O + 2PQB + 4 photons→ O2+ 2 PQBH2 (20-5) FIGURE 20-20 Water-splitting activity of the oxygen-evolving center. (a) The process that produces a four-electron oxidizing agent — a multinuclear center with four Mn ions, one Ca ion, and five oxygen atoms — in the oxygen-evolving center of PSII. The sequential absorption of four photons (excitons), each absorption causing the loss of one electron from the M n4CaO5 cofactor, produces an oxidizing agent that can remove four electrons from two molecules of water, producing O2. The electrons lost from the M n4CaO5 cofactor pass one at a time to an oxidized Tyr residue in a PSII protein, then to P680+. (b) The chair-shaped metallic center of the oxygen-evolving center. Tyr161, known to participate in the oxidation of water, is seen hydrogen-bonded to a network of water molecules, including several directly in contact with the M n4CaO5 cofactor. This is the site of one of the most important reactions in the biosphere. [(b) Data from PDB ID 3WU2, Y. Umena et al., Nature 473:55, 2011.] The oxygen-evolving cofactor takes the shape of a chair (Fig. 20-20b). The seat and legs of the chair are made up of three Mn ions, one Ca ion, and four O atoms; the fourth Mn and another O form the back of the chair. Four water molecules are also seen in the crystal structure, two associated with one of the Mn ions, the other two with the Ca ion. It may be one (or more) of these water molecules that undergoes oxidation to produce O2. This metal cofactor is associated with several peripheral membrane proteins on the lumenal side of the thylakoid membrane that are believed to stabilize the cofactor. The Tyr residue designated Z, through which electrons move between water and the PSII reaction center, is connected with a network of hydrogen-bonded water molecules that includes the four associated with the M n4CaO5 cofactor. The detailed mechanism of water oxidation by the M n4CaO5 cofactor is not known but is under intense investigation. The reaction is central to life on Earth and may involve novel bioinorganic chemistry. Determination of the structure of the polymetallic center has inspired several reasonable and testable hypotheses. Stay tuned. SUMMARY 20.2 Photochemical Reaction Centers Bacteria have a single photochemical reaction center. Purple bacteria have a type II photosystem where electrons from an excited special pair of chlorophyll molecules (P870*) flow through pheophytin, quinones, and a proton-pumping cytochrome complex, back to the special pair of chlorophylls. Green sulfur bacteria have a type I photosystem that can send electrons through a similar cyclic path or through a linear path that reduces NAD+ to NADH. In cyanobacteria, algae, and plants, two different reaction centers are arranged in tandem. In the reaction center of PSII, when the special pair of chlorophylls (P680) is excited by light, it passes electrons to plastoquinone, and the electrons lost from P680 are replaced by electrons from H2O. PSI passes electrons from the excited special pair (P700*) in its reaction center through a series of carriers to ferredoxin, which then reduces NADP+ to NADPH. Electron flow from either photosystem through the cytochrome b6f complex drives protons across the thylakoid membrane, creating a proton-motive force that provides the energy for ATP synthesis by an ATP synthase. Linear electron transfer through the photosystems produces NADPH and ATP. Cyclic electron transfer produces only ATP and allows variability in the proportions of NADPH and ATP formed. The distribution of PSI and PSII between the granal and stromal thylakoids can change and is indirectly controlled by light intensity, optimizing the distribution of excitons between PSI and PSII for efficient energy capture. The oxygen-evolving center, which contains a M n4CaO5 cofactor, uses energy from light to split water, producing O2. For each O2 formed at the oxygen-evolving center, four protons are pumped into the thylakoid lumen, contributing to the proton motive force. 20.3 Evolution of a Universal Mechanism for ATP Synthesis The combined activities of the two plant photosystems move electrons from water to NAD P+, conserving some of the energy of absorbed light as NADPH (Fig. 20-12). Simultaneously, protons are pumped across the thylakoid membrane and energy is conserved as an electrochemical potential. We turn now to the process by which this proton gradient drives the synthesis of ATP, the other energy-conserving product of the light-dependent reactions. A Proton Gradient Couples Electron Flow and Phosphorylation Although the energy source and electron carriers in photophosphorylation in chloroplasts differ from those of oxidative phosphorylation in mitochondria, they use essentially the same mechanism to capture the energy of the proton gradient. Electron-transferring molecules in the chain of carriers connecting PSII and PSI are oriented asymmetrically in the thylakoid membrane, so photoinduced electron flow results in the net movement of protons across the membrane, from the stromal side to the thylakoid lumen (Fig. 20-21). FIGURE 20-21 Proton and electron circuits during photophosphorylation. In the linear electron pathway (blue arrows), electrons move from H2O through PSII, through the intermediate chain of carriers of the cytochrome b6f complex, through PSI, and finally to NAD P+. In the cyclic pathway, electrons move from PSI back to plastoquinone and cytochrome b6f. Protons (red arrows) are pumped into the thylakoid lumen by the flow of electrons through cytochrome b6f, and they reenter the stroma through proton channels formed by CFo of ATP synthase. The CF1 subunit catalyzes synthesis of ATP. The Approximate Stoichiometry of Photophosphorylation Has Been Established As electrons move from water to NAD P+ in chloroplasts, about 12 protons move from the stroma into the thylakoid lumen per 4 electrons passed (that is, per O2 formed). Of these protons, 4 are moved by the oxygen-evolving center, and up to 8 are moved by the cytochrome b6f complex. The measurable result is a 1,000- fold difference in H+ concentration across the thylakoid membrane (ΔpH = 3). Recall that the energy stored in a proton gradient (the electrochemical potential) has two components: a proton concentration difference (ΔpH) and an electrical potential (Δψ) due to charge separation. In chloroplasts, ΔpH is the dominant component; counterion movement apparently dissipates most of the electrical potential. In illuminated chloroplasts, the energy stored in the proton gradient per mole of protons is ΔG = 2.3RT ΔpH + ZF Δψ=−17 kJ /mol so the movement of 12 mol of protons across the thylakoid membrane represents conservation of about 200 kJ of energy — enough energy to drive the synthesis of several moles of ATP (ΔG′°= 30.5 kJ /mol). Experimental measurements yield values of about 3 ATP per O2 produced. At least 8 photons must be absorbed to drive 4 electrons from 2 H2O to 2 NADPH (one photon per electron at each reaction center). The energy in 8 photons of visible light is more than enough for the synthesis of three molecules of ATP. ATP synthesis is not the only energy-conserving reaction of photosynthesis in plants; the NADPH formed in the final electron transfer is also energetically rich. The overall equation for this linear photophosphorylation is (20-6) The ATP Synthase Structure and Mechanism Are Nearly Universal The enzyme responsible for ATP synthesis in chloroplasts is a large complex with two functional components, CFo and CF1 (C denoting its location in chloroplasts). CFo is a transmembrane proton pore composed of several integral membrane proteins and is homologous to mitochondrial Fo. CF1 is a peripheral membrane protein complex very similar in subunit composition, structure, and function to mitochondrial F1. Electron microscopy of sectioned chloroplasts shows ATP synthase complexes as projections on the outside (stromal, or N) surface of thylakoid membranes; these complexes correspond to the ATP synthase complexes that project on the inside (matrix, or N) surface of the inner mitochondrial membrane. Thus, the relationship between the orientation of the ATP synthase and the direction of proton pumping is the same in chloroplasts and mitochondria. In both cases, the F1 portion of ATP synthase is located on the more alkaline (N) side of the membrane through 2H2O + 8 photons+ 2NAD P+ +∼3AD P+ +∼3Pi→ O2+∼3AT P which protons flow down their concentration gradient; the direction of proton flow relative to F1 is the same in both cases: P to N (Fig. 20-22). FIGURE 20-22 Orientation of ATP synthase is fixed relative to the proton gradient. Superficially, the direction of proton pumping in chloroplasts may seem to be opposite to that in mitochondria and bacteria. In mitochondria and bacteria, protons are pumped out of the organelle or cell, and F1 is on the inside of the membrane; in chloroplasts, protons are pumped into the thylakoid lumen, and CF1 is on the outside of the thylakoid membrane. However, exactly the same mechanism of energy conversion (from proton gradient to ATP) occurs in all three cases. ATP is synthesized in the matrix of mitochondria, the stroma of chloroplasts, and the cytosol of bacteria. The mechanism of chloroplast ATP synthase is essentially identical to that of its mitochondrial analog; ADP and Pi readily condense to form ATP on the enzyme surface, and the release of this enzyme-bound ATP requires a proton-motive force. Rotational catalysis sequentially engages each of the three β subunits of the ATP synthase in ATP synthesis, ATP release, and AD P + Pi binding (see Figs. 19-26 and 19-27). The appearance of oxygenic photosynthesis on Earth about 2.5 billion years ago was a crucial event in the evolution of the biosphere. Before that, Earth’s atmosphere was composed of methane, CO2, and N2. The planet was essentially devoid of molecular oxygen and lacked the ozone layer that protects organisms from solar UV radiation. Oxygenic photosynthesis made available a nearly limitless supply of reducing agent (H2O) to drive the production of organic compounds by reductive biosynthetic reactions. And mechanisms evolved that allowed organisms to use O2 as a terminal electron acceptor in highly energetic electron transfers from organic substrates, employing the energy of oxidation to support metabolism. The complex photosynthetic apparatus of a modern vascular plant is the culmination of a series of evolutionary events, the most recent of which was the acquisition by eukaryotic cells of a cyanobacterial endosymbiont. The chloroplasts of modern organisms share several properties with mitochondria and originated by the same mechanism that gave rise to mitochondria: endosymbiosis. Like mitochondria, chloroplasts contain their own DNA and protein-synthesizing machinery. Some of the polypeptides of chloroplast proteins are encoded by chloroplast genes and synthesized in the chloroplast; others are encoded by nuclear genes, synthesized outside the chloroplast, and imported (Chapter 27). When plant cells grow and divide, chloroplasts give rise to new chloroplasts by division, during which their DNA is replicated and divided between daughter chloroplasts. The machinery and mechanisms for light capture, electron flow, and ATP synthesis in modern cyanobacteria are similar in many respects to those in plant chloroplasts. These observations led to the now widely accepted hypothesis that the evolutionary progenitors of modern plant cells were primitive eukaryotes that engulfed photosynthetic cyanobacteria and established stable endosymbiotic relationships with them (see Fig. 1-37). At least half of the photosynthetic activity on Earth now occurs in microorganisms — algae, other photosynthetic eukaryotes, and photosynthetic bacteria. Cyanobacteria have PSII and PSI in tandem, and the PSII has an associated oxygen-evolving activity resembling that of plants. However, the other groups of photosynthetic bacteria have single reaction centers and do not split H2O or produce O2. Many are obligate anaerobes and cannot tolerate O2; they must use some compound other than H2O as an electron donor. Some photosynthetic bacteria use inorganic compounds as electron (and hydrogen) donors. For example, green sulfur bacteria use hydrogen sulfide: 2H2S + CO2  light −−→  (CH2O)+ H2O + 2S These bacteria, instead of producing molecular O2, form elemental sulfur as the oxidation product of H2S. (They further oxidize the S to SO2− 4 .) Other photosynthetic bacteria use organic compounds such as lactate as electron donors: The fundamental similarity of photosynthesis in plants and bacteria, despite the differences in the electron donors they employ, becomes more obvious when the equation of photosynthesis is written in the more general form 2H2D + CO2  light −−→  (CH2O)+ H2O + 2D in which H2D is an electron (and hydrogen) donor and D is its oxidized form. H2D may be water, hydrogen sulfide, lactate, or some other organic compound, depending on the species. Most likely, the bacteria that first developed photosynthetic ability used H2S as their electron source. Modern cyanobacteria can synthesize ATP by oxidative phosphorylation or by photophosphorylation, although they have neither mitochondria nor chloroplasts. The enzymatic machinery for both processes is in a highly convoluted plasma membrane (Fig. 20-23). Three protein components function in both processes, giving evidence that the processes have a common evolutionary origin (Fig. 20-24). First, the proton-pumping 2 Lactate+ CO2  light −−→  (CH2O)+ H2O + 2 pyruvate cytochrome b6f complex carries electrons from plastoquinone to cytochrome c6 in photosynthesis, and also carries electrons from ubiquinone to cytochrome c6 in oxidative phosphorylation — the role played by cytochrome bc1 in mitochondria. Second, cytochrome c6, homologous to mitochondrial cytochrome c, carries electrons from Complex III to Complex IV in cyanobacteria; it can also carry electrons from the cytochrome b6f complex to PSI — a role performed in plants by plastocyanin. We therefore see the functional homology between the cyanobacterial cytochrome b6f complex and the mitochondrial cytochrome bc1 complex, and between cyanobacterial cytochrome c6 and plant plastocyanin. The third conserved component is the ATP synthase, which functions in oxidative phosphorylation and photophosphorylation in cyanobacteria, and in the mitochondria and chloroplasts of photosynthetic eukaryotes. The structure and remarkable mechanism of this enzyme have been strongly conserved throughout evolution. FIGURE 20-23 The photosynthetic membranes of a cyanobacterium. In these thin sections of a cyanobacterium, viewed with a transmission electron microscope, the multiple layers of the internal membranes are seen to fill half the total volume of the cell. The extensive membrane system serves the same role as the thylakoid membranes of vascular plants, providing a large surface area containing all of the photosynthetic machinery. (Bar= 100 nm.) [S. R. Miller et al. Discovery of a free-living chlorophyll d-producing cyanobacterium with a hybrid proteobacterial/cyanobacterial small-subunit rRNA gene. Proc. Natl. Acad. Sci. USA 102:850, 2005, Fig. 2. © 2005 National Academy of Sciences.] FIGURE 20-24 Dual roles of cytochrome b6f and cytochrome c6 in cyanobacteria reflect evolutionary origins. Cyanobacteria use cytochrome b6f, cytochrome c6, and plastoquinone for both oxidative phosphorylation and photophosphorylation. (a) In photophosphorylation, electrons flow (blue arrows) from water to NAD P+. (b) In oxidative phosphorylation, electrons flow from NADH to O2. Both processes are accompanied by proton movement across the membrane, accomplished by a Q cycle. SUMMARY 20.3 Evolution of a Universal Mechanism for ATP Synthesis In plants, both the water-splitting reaction and electron flow through the cytochrome b6f complex are accompanied by proton pumping across the thylakoid membrane. The proton-motive force thus created drives ATP synthesis by a CFoCF1 complex similar to the mitochondrial FoF1 complex in both structure and catalytic mechanism. Direct measurements show that eight photons drive the production of one O2 from oxidation of two H2O, making three ATP molecules. About 2.5 billion years ago, cyanobacteria appeared on Earth. They had acquired two photosystems — one of the type now found in purple bacteria, the other of the type found in green sulfur bacteria — that operated in tandem, and a water-splitting activity that released oxygen into the atmosphere. Many photosynthetic microorganisms obtain electrons for photosynthesis not from water but from donors such as H2S, forming an oxidized product such as elemental sulfur (not oxygen). Chloroplasts, like mitochondria, evolved from bacteria living as endosymbionts in early eukaryotic cells. The ATP synthases of bacteria, cyanobacteria, mitochondria, and chloroplasts share a common evolutionary precursor and a common enzymatic mechanism. 20.4 CO2-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 CO2 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. FIGURE 20-25 Products of photosynthesis. Green plants contain in their chloroplasts the enzymatic machinery that catalyzes the conversion of CO2 to simple (reduced) organic compounds, a process called CO2 assimilation. This process has also been called CO2 fixation, but we reserve this term for the specific reaction in which CO2 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 oen 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 CO2 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 M g2+ 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 CO2 into biomolecules (Fig. 20-26) is the CO2-fixation reaction: condensation of CO2 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 CO2 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 CO2 to triose and hexose phosphates. FIGURE 20-26 The three stages of CO2 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 CO2 are fixed for the net synthesis of one molecule of glyceraldehyde 3-phosphate. Fructose 6-phosphate is a key intermediate in stage 3 of CO2 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 CO2, 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 CO2 into 3- Phosphoglycerate An important clue to the nature of the CO2-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 (14CO2) 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 14C 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 C3 plants, in contrast to the C4 plants described below. Most plant species — 80% to 90% — are C3, including most trees, wheat, oats, rice, beans, peas, and spinach. The enzyme that catalyzes incorporation of CO2 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 CO2 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 CO2 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 CO2. It has a complex form I structure (Fig. 20-27a), with eight identical large catalytic subunits (Mr 53,000; encoded in the chloroplast genome), and eight identical small subunits (Mr 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 CO2 are fixed per second per molecule of rubisco at 25 °C. To achieve high rates of CO2 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. 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 Mr> 500,000. A transition-state analog, 2-carboxyarabinitol bisphosphate (yellow), is shown bound to each of the eight substrate-binding sites. M g2+ 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.] Central to the proposed mechanism for plant rubisco is a carbamoylated Lys side chain with a bound M g2+ ion. The M g2+ 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. FIGURE 20-28 Central role of M g2+ in the active site of rubisco. M g2+ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on Lys201; two in the carboxyl groups of Glu204 and Asp203; two at C-2 and C-3 of the substrate, ribulose 1,5-bisphosphate; and one in the other substrate, CO2. A water molecule occupies the CO2- binding site in the crystal structure. In this figure, a CO2 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.] MECHANISM FIGURE 20-29 First stage of CO2 assimilation: rubisco’s carboxylase activity. The CO2-fixation reaction is catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase. The overall reaction accomplishes the combination of one CO2 and one ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate, one of which contains the carbon atom from CO2 (red). Additional proton transfers (not shown), involving Lys201, Lys175, and His294, occur in several of these steps. As the catalyst for the first step of photosynthetic CO2 assimilation, rubisco is a prime target for regulation. The enzyme is inactive until carbamoylated on the ε -amino group of Lys201 (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 Lys201 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 CO2; this is followed by M g2+ binding, which activates the rubisco. FIGURE 20-30 Role of rubisco activase in carbamoylation of Lys201 of 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 Pi. 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 CO2 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. FIGURE 20-31 Third stage of CO2 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. Synthesis of Each Triose Phosphate from CO2 Requires Six NADPH and Nine ATP The net result of three turns of the Calvin cycle is the conversion of three molecules of CO2 and one molecule of phosphate to a molecule of triose phosphate. The stoichiometry of the overall path from CO2 to triose phosphate, with regeneration of ribulose 1,5-bisphosphate, is shown in Figure 20- 32. FIGURE 20-32 Stoichiometry of CO2 assimilation in the Calvin cycle. For every three CO2 molecules fixed, one molecule of triose phosphate (glyceraldehyde 3-phosphate) is produced and nine ATP and six NADPH are consumed. One molecule of glyceraldehyde 3-phosphate is the net product of the CO2-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 CO2 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 Pi 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 Pi 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 CO2 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+ and synthesis of ATP. They do not, in fact, occur at significant rates in the dark and are thus more appropriately called the CO2-assimilation reactions. Later in this section we describe the regulatory mechanisms that turn CO2 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 CO2 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 CO2 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 Pi with a triose phosphate, either dihydroxyacetone phosphate or 3-phosphoglycerate (Fig. 20-33). This antiporter simultaneously moves Pi 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. FIGURE 20-33 The Pi–triose phosphate antiport system of the chloroplast inner membrane. This transporter facilitates the exchange of cytosolic Pi for stromal dihydroxyacetone phosphate. The products of photosynthetic CO2 assimilation are thus moved into the cytosol, where they serve as a starting point for sucrose biosynthesis, and Pi 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). 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 Pi molecules from the four triose phosphates required to make sucrose. For every molecule of triose phosphate removed from the chloroplast, one Pi is transported into the chloroplast, providing the ninth Pi mentioned above, to be used in regenerating ATP. If this exchange were blocked, triose phosphate synthesis would quickly deplete the available Pi in the chloroplast, slowing ATP synthesis and suppressing assimilation of CO2 into starch. The Pi–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 Pi–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. FIGURE 20-34 Role of the Pi–triose 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. Four Enzymes of the Calvin Cycle Are Indirectly Activated by Light The reductive assimilation of CO2 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 M g2+ from the thylakoid compartment into the stroma, raising the [M g2+] 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 [M g2+]. For example, activation of rubisco by formation of carbamoyllysine is faster at alkaline pH, and high stromal [M g2+] favors formation of the enzyme’s active M g2+ complex. Fructose 1,6-bisphosphatase requires M g2+ and is very dependent on pH (Fig. 20-36); its activity increases more than 100-fold when pH and [M g2+] rise during chloroplast illumination. FIGURE 20-35 Source of ATP and NADPH. ATP and NADPH produced by the light-dependent reactions are essential substrates for the reduction of CO2. 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 [M g2+]. 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 [M g2+] 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 CO2 assimilation. Instead, starch synthesized and stored during the daytime is degraded to fuel glycolysis and oxidative phosphorylation at night. 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. 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 CO2-assimilating reactions (the Calvin cycle), ATP and NADPH are used to reduce CO2 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 CO2 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 Pi in the cytosol for 3-phosphoglycerate or dihydroxyacetone phosphate molecules produced by CO2 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. 20.5 Photorespiration and the C4 and CAM Pathways As we have seen, photosynthetic cells produce O2 (by the splitting of H2O) during the light-driven reactions and use CO2 during the light- independent processes, so the net gaseous change during photosynthesis is the uptake of CO2 and release of O2: CO2+ H2O → O2+ (CH2O) In the dark, plants also carry out mitochondrial respiration, the oxidation of substrates to CO2 and the conversion of O2 to H2O. And there is another process in plants that, like mitochondrial respiration, consumes O2 and produces CO2 and, like photosynthesis, is driven by light. This process, photorespiration, is a costly side reaction of photosynthesis, a result of the lack of specificity of the enzyme rubisco. In this section we describe this side reaction and the strategies plants use to minimize its metabolic consequences. Photorespiration Results from Rubisco’s Oxygenase Activity Rubisco is not absolutely specific for CO2 as a substrate. Molecular oxygen (O2) competes with CO2 at the active site, and about once in every three or four turnovers, rubisco catalyzes the condensation of O2 with ribulose 1,5-bisphosphate to form 3- phosphoglycerate and 2-phosphoglycolate (Fig. 20-38), a metabolically unneeded product. This is the oxygenase activity referred to in the full name of rubisco: ribulose 1,5-bisphosphate carboxylase/oxygenase. The reaction with O2 results in no fixation of CO2 and is presumably a net liability to the cell; salvaging the carbons from 2-phosphoglycolate (by the pathway outlined below) consumes significant amounts of cellular energy and releases some previously fixed CO2.

FIGURE 20-38 Oxygenase activity of rubisco. Rubisco can incorporate O2 rather than CO2 into ribulose 1,5-bisphosphate. The unstable intermediate thus formed splits into 2-phosphoglycolate (recycled as described in Fig. 20-39) and 3- phosphoglycerate, which can reenter the Calvin cycle. Phosphoglycolate Is Salvaged in a Costly Set of Reactions in C3 Plants The glycolate pathway converts two molecules of 2-phosphoglycolate to a molecule of serine (three carbons) and a molecule of CO2 (Fig. 20-39). In the chloroplast, a phosphatase converts 2-phosphoglycolate to glycolate, which is exported to the peroxisome. There, glycolate is oxidized by molecular oxygen, and the resulting aldehyde (glyoxylate) undergoes transamination to glycine. The hydrogen peroxide formed as a side product of glycolate oxidation is rendered harmless by peroxidases in the peroxisome. Glycine passes from the peroxisome to the mitochondrial matrix, where it undergoes oxidative decarboxylation by the glycine decarboxylase complex, an enzyme similar in structure and mechanism to two mitochondrial complexes we have already encountered: the pyruvate dehydrogenase complex and the α -ketoglutarate dehydrogenase complex (Chapter 16). The glycine decarboxylase complex oxidizes glycine to CO2 and NH3, with the concomitant reduction of NAD + to NADH and transfer of the remaining carbon from glycine to the cofactor tetrahydrofolate. The one-carbon unit carried on tetrahydrofolate is then transferred to a second glycine by serine hydroxymethyltransferase, producing serine. The net reaction catalyzed by the glycine decarboxylase complex and serine hydroxymethyltransferase is 2 Glycine+ NAD + + H2O → serine+ CO2+ NH3+ NADH + H+ The serine is converted to hydroxypyruvate, then to glycerate, and finally to 3-phosphoglycerate, which is used to regenerate ribulose 1,5-bisphosphate, completing the long, expensive cycle (Fig. 20-39).

FIGURE 20-39 Glycolate pathway. This pathway, which salvages 2- phosphoglycolate (shaded light red) by converting it to serine and, eventually, to 3-phosphoglycerate, involves three cellular compartments. Glycolate formed by dephosphorylation of 2-phosphoglycolate in chloroplasts is oxidized to glyoxylate and transaminated to glycine in peroxisomes. In mitochondria, two glycine molecules condense to form serine and CO2, released in photorespiration. This reaction is catalyzed by glycine decarboxylase, an enzyme present at very high levels in the mitochondria of C3 plants. The serine is converted to hydroxypyruvate and then to glycerate in peroxisomes; glycerate reenters the chloroplasts to be phosphorylated, rejoining the Calvin cycle. Oxygen is consumed at two steps during photorespiration. In bright sunlight, the carbon flux through the glycolate salvage pathway can be very high, producing about five times more CO2 than is typically produced by all the oxidations of the citric acid cycle. To generate this large flux, mitochondria contain prodigious amounts of the glycine decarboxylase complex: the four proteins of the complex make up half of all the protein in the mitochondrial matrix in the leaves of pea and spinach plants. In nonphotosynthetic parts of a plant, such as potato tubers, mitochondria have very low concentrations of the glycine decarboxylase complex. The practical effects of this inefficiency are large and costly. The average yield of soybeans and wheat in the United States is reduced by an estimated 36% and 20%, respectively, by the necessity of recycling glycolate from photorespiration. The combined activity of the rubisco oxygenase and the glycolate salvage pathway consumes O2 and produces CO2 — hence the name photorespiration. Unlike mitochondrial respiration, photorespiration does not conserve energy and actually inhibits net biomass formation. This inefficiency has led to evolutionary adaptations in the CO2-assimilation processes, particularly in plants that have evolved in warm climates. The apparent inefficiency of rubisco, and its effect in limiting biomass production, has inspired efforts to genetically engineer a “better” rubisco, but this goal is not, as yet, within reach (Box 20-1). BOX 20-1 Will Genetic Engineering of Photosynthetic Organisms Increase Their Efficiency? Three pressing world problems have prompted serious attention to the possibility of engineering plants to be more efficient in converting sunlight into biomass: the greenhouse effect of increasing levels of atmospheric CO2 on climate change, the finite supply of oil for generating energy, and the need for more and better food for the world’s growing population. The concentration of CO2 in the earth’s atmosphere has risen steadily over the past 50 years (Fig. 1), a combined effect of the use of fossil fuels for energy and the clearing and burning of tropical forests to allow use of the land for agriculture. As atmospheric CO2 increases, the atmosphere absorbs more heat radiated from the earth’s surface and reradiates more heat toward the surface of the planet (and in all other directions). Retention of heat raises the temperature at the surface of the earth; this is the greenhouse effect. One way to limit the increase in atmospheric CO2 would be to engineer plants or microorganisms with a greater capacity for sequestering CO2. FIGURE 1 The concentration of CO2 in the atmosphere measured at the Mauna Loa Observatory in Hawaii. [Data from the National Oceanic and Atmospheric Administration and the Scripps Institution of Oceanography CO2 Program.] The estimated amount of total carbon in all terrestrial systems (atmosphere, soil, biomass) is about 3,200 gigatons (GT), or 3,200 billion metric tons. The atmosphere contains another 760 GT of CO2. The flux of carbon through these terrestrial reservoirs (Fig. 2) is largely due to the photosynthetic activities of plants and the degradative activities of microorganisms. Plants fix some 123 GT of carbon annually, then immediately release about half of that to the atmosphere as they respire. Much of the remainder is gradually released to the atmosphere by microbial action on dead plant materials, but biomass is sequestered in woody plants and trees for decades or centuries. Anthropogenic carbon flux, the amount of CO2 released into the atmosphere by human activities, is 9 GT per year — small compared with total biomass, but enough to tip the balance toward increased CO2 in the atmosphere. Estimates indicate that the forests of North America sequester 0.7 GT of carbon annually, which represents about a tenth of the annual global production of CO2 from fossil fuels. Clearly, preservation of forests and reforestation are effective ways to limit the flow of CO2 back into the atmosphere. FIGURE 2 The terrestrial carbon cycle. Carbon stocks (boxed text) are shown as gigatons (GT), and fluxes (arrows) are shown in GT per year. Animal biomass is negligible here — less than 0.5 GT. [Information from C. Jansson et al., BioScience 60:683, 2010, Fig. 1.] A second approach to limiting the increase of atmospheric CO2, while also addressing the need to replace dwindling fossil fuels, is to use renewable biomass as a source of ethanol to replace fossil fuels in internal combustion engines. This reduces the unidirectional movement of carbon from fossil fuels into the atmospheric pool of CO2, replacing it with the cyclic flow of CO2 from ethanol to CO2 and back to biomass. When maize, wheat, or switchgrass is fermented to ethanol for fuel, every increase in biomass production brought about by more efficient photosynthesis should result in a corresponding decrease in the use of fossil fuels. Finally, engineering of food crops to yield more food per acre of land, or per hour of work, could improve human nutrition worldwide. In principle, these goals might be accomplished by developing a rubisco that didn’t also catalyze the wasteful reaction with O2, or by increasing the turnover number for rubisco, or by increasing the level of rubisco or other enzymes in the pathway for CO2 fixation. Rubisco, as we have noted, is an unusually inefficient enzyme, with a turnover number of 3 s−1 at 25 °C; most enzymes have turnover numbers orders of magnitude larger. It also catalyzes the wasteful reaction with oxygen, which further reduces its efficiency in fixing CO2 and producing biomass. If rubisco could be genetically engineered to turn over faster or to be more selective for CO2 relative to O2, would the effect be greater photosynthetic production of biomass and thus greater sequestration of CO2, greater production of nonfossil fuel, and improved nutrition? The traditional view of metabolic pathways held that one step in any pathway was the slowest and therefore the limiting factor in material flow through the pathway. However, efforts to engineer cells or organisms to produce more of the “limiting” enzyme in a pathway have o en given discouraging results; the organisms o en show little or no change in the flux through that pathway. The Calvin cycle is an instructive case in point. Increasing the amount of rubisco in plant cells through genetic engineering has little or no effect on the rate of CO2 conversion into carbohydrate. Similarly, changes in the levels of enzymes known to be regulated by light and therefore suspected of playing key roles in the regulation of the CO2- assimilation pathway (fructose 1,6-bisphosphatase, 3-phosphoglycerate kinase, and glyceraldehyde 3-phosphate dehydrogenase) also produce little or no significant improvement in photosynthetic rate. This should probably not be surprising; in the living organism, pathways can be limited by more than one enzymatic step, because every change in one step results in compensating changes in other steps. Metabolic control analysis is the science of measuring, understanding, and eventually altering the factors that govern the overall flux through a pathway. Its application will be essential to the success of engineering plants for higher efficiency or greater yield. In C4 Plants, CO2 Fixation and Rubisco Activity Are Spatially Separated In many plants that grow in the tropics (and in temperate-zone crop plants native to the tropics, such as maize, sugarcane, and sorghum) a mechanism has evolved to circumvent the problem of wasteful photorespiration. The step in which CO2 is fixed into a three-carbon product, 3-phosphoglycerate, is preceded by several steps, one of which is temporary fixation of CO2 into oxaloacetate, a four-carbon compound. Plants that use this process are referred to as C4 plants, and the assimilation process is known as the C4 pathway, by comparison to the C3 pathway in which CO2 is first fixed in the three- carbon compound 3-phosphoglycerate. The C4 plants, which typically grow at high light intensity and high temperatures, have several important characteristics: high photosynthetic rates, high growth rates, low photorespiration rates, low rates of water loss, and a specialized leaf structure. Photosynthesis in the leaves of C4 plants involves two cell types: mesophyll and bundle-sheath cells (Fig. 20-40a).

FIGURE 20-40 CO2 assimilation in C4 plants. The C4 pathway, involving mesophyll cells and bundle-sheath cells, predominates in plants of tropical origin. (a) Electron micrograph showing chloroplasts of adjacent mesophyll and bundle-sheath cells. The bundle-sheath cell contains starch granules. Plasmodesmata connecting the two cells are visible. (b) The C4 pathway of CO2 assimilation, which occurs through a four-carbon intermediate. The fixation of CO2 into the four-carbon oxaloacetate occurs in the cytosol of leaf mesophyll cells. The reaction is catalyzed by phosphoenolpyruvate (PEP) carboxylase, for which the substrate is HCO−3, not CO2. The oxaloacetate thus formed is either reduced to malate at the expense of NADPH (as shown in Fig. 20-40b) or converted to aspartate by transamination: Oxaloacetate+ α-amino acid→ L-aspartate+ α-keto acid The malate or aspartate formed in the mesophyll cells then passes into neighboring bundle-sheath cells through plasmodesmata, protein-lined channels that connect two plant cells and provide a path for movement of metabolites and even small proteins between cells. In the bundle-sheath cells, malate is oxidized and decarboxylated to yield pyruvate and CO2 by the action of malic enzyme, reducing NADP+. In plants that use aspartate as the CO2 carrier, aspartate arriving in bundle-sheath cells is transaminated to form oxaloacetate and reduced to malate, then the CO2 is released by malic enzyme or PEP carboxykinase. Labeling experiments show that the free CO2 released in the bundle-sheath cells is the same CO2 molecule originally fixed into oxaloacetate in the mesophyll cells. This CO2 is now fixed again, this time by rubisco, in exactly the same reaction that occurs in C3 plants: incorporation of CO2 into C-1 of 3- phosphoglycerate. The pyruvate formed by decarboxylation of malate in bundle-sheath cells is transferred back to the mesophyll cells, where it is converted to PEP by an unusual enzymatic reaction catalyzed by pyruvate phosphate dikinase (Fig. 20-40b). This enzyme is called a dikinase because two different molecules are simultaneously phosphorylated by one molecule of ATP: pyruvate to PEP, and phosphate to pyrophosphate. The pyrophosphate is subsequently hydrolyzed to phosphate, so two high-energy phosphate groups of ATP are used in regenerating PEP. The PEP is now ready to receive another molecule of CO2 in the mesophyll cell. The PEP carboxylase of mesophyll cells has a high affinity for HCO−3 (which is favored relative to CO2 in aqueous solution) and can fix CO2 more efficiently than can rubisco. Unlike rubisco, it does not use O2 as an alternative substrate, so there is no competition between CO2 and O2. The PEP carboxylase reaction, then, serves to fix and concentrate CO2 in the form of malate. Release of CO2 from malate in the bundle-sheath cells yields a sufficiently high local concentration of CO2 for rubisco to function near its maximal rate, and for suppression of the enzyme’s oxygenase activity. Once CO2 is fixed into 3-phosphoglycerate in the bundle-sheath cells, the other reactions of the Calvin cycle take place exactly as described earlier. Thus in C4 plants, mesophyll cells carry out CO2 assimilation by the C4 pathway and bundle-sheath cells synthesize starch and sucrose by the C3 pathway. Three enzymes of the C4 pathway are regulated by light, becoming more active in daylight. Malate dehydrogenase is activated by the thioredoxin-dependent reduction mechanism shown in Figure 20-37; PEP carboxylase is activated by phosphorylation of a Ser residue; and pyruvate phosphate dikinase is activated by dephosphorylation. The pathway of CO2 assimilation has a greater energy cost in C4 plants than in C3 plants. For each molecule of CO2 assimilated in the C4 pathway, a molecule of PEP must be regenerated at the expense of two phosphoanhydride bonds in ATP. Thus C4 plants need five ATP molecules to assimilate one molecule of CO2, whereas C3 plants need only three (nine per triose phosphate). As the temperature increases (and the affinity of rubisco for CO2 decreases, as noted above), a point is reached, at about 28 to 30 °C, at which the gain in efficiency from the elimination of photorespiration more than compensates for this energetic cost. C4 plants (crabgrass, for example) outgrow most C3 plants during the summer, as any experienced gardener can attest. In CAM Plants, CO2 Capture and Rubisco Action Are Temporally Separated Succulent plants such as cactus and pineapple, which are native to very hot, very dry environments, have another variation on photosynthetic CO2 fixation, which reduces loss of water vapor through the pores (stomata) by which CO2 and O2 must enter leaf tissue. Instead of separating the initial trapping of CO2 and its fixation by rubisco across space (as do the C4 plants), they separate these two events over time. At night, when the air is cooler and moister, the stomata open to allow entry of CO2, which is then fixed into oxaloacetate by PEP carboxylase. The oxaloacetate is reduced to malate and stored in the vacuoles, to protect cytosolic and plastid enzymes from the low pH produced by malic acid dissociation. During the day the stomata close, preventing the water loss that would result from high daytime temperatures, and the CO2 trapped overnight in malate is released as CO2 by the NADP-linked malic enzyme. This CO2 is now assimilated by the action of rubisco and the Calvin cycle enzymes. Because this method of CO2 fixation was first discovered in stonecrops, perennial flowering plants of the family Crassulaceae, it is called crassulacean acid metabolism, and the plants are called CAM plants. Table 20-1 compares characteristics of C3, C4, and CAM plants. TABLE 20-1 Comparison of C3, C4, and CAM Plants C3 Plants C4 Plants CAM Plants Examples Spinach, pea, rice, wheat, beans, most trees Maize (corn), sugarcane, crabgrass Cactus, prickly pear, orchid, pineapple Most efficient environment 15 to 25 °C Hot and dry; 30 to 47 °C Extremely dry; 35 °C Path of CO2 fixation C3 photosynthesis only Sequential C4 and C3 cycles spatially separated: C4 in mesophyll cells followed by C3 in bundle-sheath cells C3 and C4 cycles, separated spatially and temporally Cell type Mesophyll cells C4 in mesophyll cells, C3 and C4 in the involved C3 in bundle-sheath cells same mesophyll cells Light conditions Light Light C3 in light; C4 in dark Initial CO2 acceptor Ribulose 1,5- bisphosphate Phosphoenolpyruvate Ribulose 1,5- bisphosphate in light; phosphoenolpyruvate in dark CO2-fixing enzyme Rubisco PEP carboxylase, then rubisco Rubisco in light; PEP carboxylase at night First stable product of CO2 fixation 3- Phosphoglycerate Oxaloacetate in C4 cycle 3-Phosphoglycerate in light; oxaloacetate in dark Energy needed for complete reduction of one molecule of CO2 3 ATP, 2 NADPH 5 ATP, 2 NADPH 6.5 ATP, 2 NADPH Photorespiration Present Absent or suppressed Absent or suppressed SUMMARY 20.5 Photorespiration and the C4 and CAM Pathways Rubisco is not completely specific for CO2 as its substrate; it can also use O2, producing 2-phosphoglycolate, which must be disposed of in an oxygen-dependent pathway. The result is increased consumption of O2 — photorespiration. The 2-phosphoglycolate is converted to glyoxylate, to glycine, and then to serine in a pathway that involves enzymes in the chloroplast stroma, peroxisomes, and mitochondria. In C4 plants, the CO2-assimilation pathway minimizes photorespiration: CO2 is first fixed in mesophyll cells into a four- carbon compound, which passes into bundle-sheath cells and releases CO2 in high concentrations. The released CO2 is fixed by rubisco, and the remaining reactions of the Calvin cycle occur as in C3 plants. In CAM plants, CO2 is fixed into malate in the dark and stored in vacuoles until daylight, when the stomata are closed (minimizing water loss), and the stored malate serves as a source of CO2 for rubisco. 20.6 Biosynthesis of Starch, Sucrose, and Cellulose During active photosynthesis in bright light, a plant leaf produces more carbohydrate (as triose phosphates) than it needs for generating energy or synthesizing precursors. The excess is converted to sucrose and transported to other parts of the plant, to be used as fuel or stored. In most plants, starch is the main storage form of carbohydrate, but in a few plants, such as sugar beet and sugarcane, sucrose is the primary storage form. The synthesis of sucrose and starch occurs in different cellular compartments (cytosol and plastids, respectively), and these processes are coordinated by a variety of regulatory mechanisms that respond to changes in light level and photosynthetic rate. The synthesis of sucrose and starch is important to the plant but also to humans: starch provides more than 80% of human dietary calories worldwide. ADP-Glucose Is the Substrate for Starch Synthesis in Plant Plastids and for Glycogen Synthesis in Bacteria Starch, like glycogen, is a high molecular weight polymer of D- glucose in (α1→ 4) linkage. It is synthesized in chloroplasts for temporary storage as one of the stable end products of photosynthesis, and for long-term storage it is synthesized in amyloplasts of the nonphotosynthetic parts of plants: seeds, roots, and tubers (underground stems). The mechanism of glucose activation in starch synthesis is similar to that in glycogen synthesis, described in Chapter 15. An activated sugar nucleotide, in this case ADP-glucose, is formed by condensation of glucose 1-phosphate with ATP in a reaction made essentially irreversible by the presence in plastids of inorganic pyrophosphatase. Starch synthase then transfers glucose residues from ADP-glucose to preexisting starch molecules. The monomeric units are almost certainly added to the nonreducing end of the growing polymer, as they are in glycogen synthesis. The amylose of starch is unbranched, but amylopectin has numerous (α1→ 6)-linked branches (see Fig. 7-13). Chloroplasts contain a branching enzyme, similar to the glycogen-branching enzyme that introduces the (α1→ 6) branches of amylopectin. Taking into account the hydrolysis by inorganic pyrophosphatase of the PPi produced during ADP-glucose synthesis, the overall reaction for starch formation from glucose 1-phosphate is Starchn + glucose 1-phosphate + AT P → starchn+1+ AD P + 2Pi ΔG′°=−50 kJ /mol Starch synthesis is regulated at the level of ADP-glucose formation, as discussed below. UDP-Glucose Is the Substrate for Sucrose Synthesis in the Cytosol of Leaf Cells Most of the triose phosphate generated by CO2 fixation in plants is converted to sucrose (Fig. 20-41) or starch. In the course of evolution, sucrose may have been selected as the transport form of carbon because of its unusual linkage between the anomeric C- 1 of glucose and the anomeric C-2 of fructose. This bond is not hydrolyzed by amylases or other common carbohydrate-cleaving enzymes, and the unavailability of the sucrose molecule’s anomeric carbons prevents it from reacting nonenzymatically (as does glucose) with amino acids and proteins. FIGURE 20-41 Sucrose synthesis. Sucrose is synthesized from UDP-glucose and fructose 6-phosphate, which are synthesized from triose phosphates in the plant cell cytosol. The sucrose 6-phosphate synthase of most plant species is allosterically regulated by glucose 6-phosphate and Pi. Sucrose is synthesized in the cytosol, beginning with dihydroxyacetone phosphate and glyceraldehyde 3-phosphate exported from the chloroplast. Aer condensation of two triose phosphates to form fructose 1,6-bisphosphate (catalyzed by aldolase), hydrolysis by fructose 1,6-bisphosphatase yields fructose 6-phosphate. Sucrose 6-phosphate synthase then catalyzes the reaction of fructose 6-phosphate with UDP-glucose to form sucrose 6-phosphate (Fig. 20-41). Finally, sucrose 6- phosphate phosphatase removes the phosphate group, making sucrose available for export to other tissues. The reaction catalyzed by sucrose 6-phosphate synthase is a low-energy process (ΔG′°=−5.7 kJ /mol), but the hydrolysis of sucrose 6- phosphate to sucrose is sufficiently exergonic (ΔG′°=−16.5 kJ /mol) to make the overall synthesis of sucrose thermodynamically favorable. Sucrose synthesis is regulated and closely coordinated with starch synthesis, as we shall see. One remarkable difference between the cells of plants and animals is the absence in the plant cell cytosol of the enzyme inorganic pyrophosphatase, which catalyzes the reaction PPi+ H2O → 2Pi ΔG′°=−19.2 kJ /mol For many biosynthetic reactions that liberate PPi, pyrophosphatase activity makes the process more favorable energetically, tending to make these reactions irreversible. In plants, this enzyme is present in plastids but absent from the cytosol. As a result, the cytosol of leaf cells contains a substantial concentration of PPi — enough (~0.3 mM) to make reactions such as that catalyzed by UDP-glucose pyrophosphorylase (see Fig. 15- 7) readily reversible. Conversion of Triose Phosphates to Sucrose and Starch Is Tightly Regulated Triose phosphates produced by the Calvin cycle in bright sunlight, as we have noted, may be stored temporarily in the chloroplast as starch, or converted to sucrose and exported to nonphotosynthetic parts of the plant, or both. The balance between the two processes is tightly regulated, and both must be coordinated with the rate of CO2 fixation. Five-sixths of the triose phosphate formed in the Calvin cycle must be recycled to ribulose 1,5-bisphosphate (Fig. 20-32); if more than one-sixth of the triose phosphate is drawn out of the cycle to make sucrose and starch, the cycle will slow or stop. However, insufficient conversion of triose phosphate to starch or sucrose would tie up phosphate, leaving a chloroplast deficient in Pi, which is also essential for operation of the Calvin cycle. The flow of triose phosphates into sucrose is regulated by the activity of fructose 1,6-bisphosphatase (FBPase-1) and the enzyme that effectively reverses its action, PPi-dependent phosphofructokinase (PP-PFK-1). These enzymes are therefore critical points for determining the fate of triose phosphates produced by photosynthesis. Both enzymes are regulated by fructose 2,6-bisphosphate (F26BP), which inhibits FBPase-1 and stimulates PP-PFK-1. In vascular plants, the concentration of F26BP varies inversely with the rate of photosynthesis (Fig. 20- 42). Phosphofructokinase-2, responsible for F26BP synthesis, is inhibited by dihydroxyacetone phosphate or 3-phosphoglycerate and is stimulated by fructose 6-phosphate and Pi. During active photosynthesis, dihydroxyacetone phosphate is produced and Pi is consumed, resulting in inhibition of PFK-2 and lowered concentrations of F26BP. This favors greater flux of triose phosphate into fructose 6-phosphate formation and sucrose synthesis. With this regulatory system, sucrose synthesis occurs when the level of triose phosphate produced by the Calvin cycle exceeds that needed to maintain operation of the cycle. FIGURE 20-42 Fructose 2,6-bisphosphate as regulator of sucrose synthesis. The concentration of the allosteric regulator fructose 2,6- bisphosphate in plant cells is regulated by the products of photosynthetic CO2 assimilation and by Pi. Dihydroxyacetone phosphate and 3- phosphoglycerate produced by CO2 assimilation inhibit phosphofructokinase-2 (PFK-2), the enzyme that synthesizes the regulator; Pi stimulates PFK-2. The concentration of the regulator is therefore inversely proportional to the rate of photosynthesis. In the dark, the concentration of fructose 2,6-bisphosphate increases and stimulates the glycolytic enzyme PPi-dependent phosphofructokinase-1 (PP-PFK-1), while inhibiting the gluconeogenic enzyme fructose 1,6-bisphosphatase (FBPase- 1). When photosynthesis is active (in the light), the concentration of the regulator drops and the synthesis of fructose 6-phosphate and sucrose is favored. Sucrose synthesis is also regulated at the level of sucrose 6- phosphate synthase, which is allosterically activated by glucose 6- phosphate and inhibited by Pi. This enzyme is further regulated by phosphorylation and dephosphorylation; a protein kinase phosphorylates the enzyme on a specific Ser residue, making it less active, and a phosphatase reverses this inactivation by removing the phosphate (Fig. 20-43). Inhibition of the kinase by glucose 6-phosphate, and of the phosphatase by Pi, enhances the effects of these two compounds on sucrose synthesis. When hexose phosphates are abundant, sucrose 6-phosphate synthase is activated by glucose 6-phosphate; when Pi is elevated (as when photosynthesis is slow), sucrose synthesis is slowed. During active photosynthesis, triose phosphates are converted to fructose 6- phosphate, which is rapidly equilibrated with glucose 6- phosphate by phosphohexose isomerase. Because the equilibrium lies far toward glucose 6-phosphate, as soon as fructose 6- phosphate accumulates, the level of glucose 6-phosphate rises and sucrose synthesis is stimulated. FIGURE 20-43 Regulation of sucrose phosphate synthase by phosphorylation. A protein kinase (SPS kinase) specific for sucrose phosphate synthase (SPS) phosphorylates a Ser residue in SPS, inactivating it; a specific phosphatase (SPS phosphatase) reverses this inhibition. The kinase is inhibited allosterically by glucose 6-phosphate, which also activates SPS allosterically. The phosphatase is inhibited by Pi, which also inhibits SPS directly. Thus, when the concentration of glucose 6-phosphate is high as a result of active photosynthesis, SPS is activated and produces sucrose phosphate. A high Pi concentration, which occurs when photosynthetic conversion of ADP to ATP is slow, inhibits sucrose phosphate synthesis. The key regulatory enzyme in starch synthesis is ADP-glucose pyrophosphorylase (Fig. 20-44); it is activated by 3- phosphoglycerate, which accumulates during active photosynthesis, and inhibited by Pi, which accumulates when light-driven condensation of ADP and Pi slows. When sucrose synthesis slows, 3-phosphoglycerate formed by CO2 fixation accumulates, activating this enzyme and stimulating the synthesis of starch. FIGURE 20-44 Regulation of ADP-glucose pyrophosphorylase by 3- phosphoglycerate and Pi. This enzyme, which produces the precursor for starch synthesis, is rate-limiting in starch production. The enzyme is stimulated allosterically by 3-phosphoglycerate (3-PGA) and inhibited by Pi; in effect, the ratio [3-PG A]/[Pi], which rises with increasing rates of photosynthesis, controls starch synthesis at this step. The Glyoxylate Cycle and Gluconeogenesis Produce Glucose in Germinating Seeds Many plants store lipids (oils) and proteins in their seeds, to be used as sources of energy and as biosynthetic precursors during germination, before photosynthetic capacity has developed. These stored components are converted to carbohydrates by the combined action of several pathways. Glucogenic amino acids (see Table 14-4) derived from the breakdown of stored seed proteins are transaminated and oxidized to succinyl-CoA, pyruvate, oxaloacetate, fumarate, and α -ketoglutarate (Chapter 18) — all good starting materials for gluconeogenesis. Active gluconeogenesis in germinating seeds provides glucose for the synthesis of sucrose, polysaccharides, and many metabolites derived from hexoses. In plant seedlings, sucrose provides much of the chemical energy needed for initial growth. Triacylglycerols stored in seeds also provide fuel for the germinating plants. They are hydrolyzed to free fatty acids, which undergo β oxidation to acetyl-CoA in specialized peroxisomes called glyoxysomes that develop during seed germination (see Fig. 17-14). The acetyl-CoA formed from seed oils enters the glyoxylate cycle (Fig. 20-45), which brings about the net conversion of acetate to succinate or other four-carbon intermediate of the citric acid cycle: 2 Acetyl-CoA + NAD + + 2H2O → succinate+ 2CoA + NAD H + H FIGURE 20-45 Conversion of stored fatty acids to sucrose in germinating seeds through the glyoxylate cycle. This pathway begins in specialized peroxisomes called glyoxysomes. The citrate synthase, aconitase, and malate dehydrogenase of the glyoxylate cycle are isozymes of the citric acid cycle enzymes; isocitrate lyase and malate synthase are unique to the glyoxylate cycle. Notice that two acetyl groups enter the cycle and four carbons leave as succinate. Succinate is exported to mitochondria, where it is converted to oxaloacetate by enzymes of the citric acid cycle. Oxaloacetate enters the cytosol and serves as the starting material for gluconeogenesis and for synthesis of sucrose, the transport form of carbon in plants. In the glyoxylate cycle, acetyl-CoA condenses with oxaloacetate to form citrate, and citrate is converted to isocitrate, exactly as in the citric acid cycle. The next step, however, is not the breakdown of isocitrate by isocitrate dehydrogenase but the cleavage of isocitrate by isocitrate lyase, forming succinate and glyoxylate. The glyoxylate then condenses with a second molecule of acetyl- CoA to yield malate, in a reaction catalyzed by malate synthase. The malate is subsequently oxidized to oxaloacetate, which can condense with another molecule of acetyl-CoA to start another turn of the cycle. The succinate passes into the mitochondrial matrix, where it is converted by citric acid cycle enzymes to oxaloacetate. The oxaloacetate moves into the cytosol and can be converted to phosphoenolpyruvate by PEP carboxykinase, then to fructose 6-phosphate, the precursor of sucrose, by gluconeogenesis. Thus, reaction sequences carried out in three subcellular compartments (glyoxysomes, mitochondria, and cytosol) are integrated for the production of fructose 6-phosphate or sucrose from stored lipids. Enzymes common to the citric acid and glyoxylate cycles have two isozymes, one specific to mitochondria, the other to glyoxysomes. Physical separation of the glyoxylate cycle and β -oxidation enzymes from the mitochondrial citric acid cycle enzymes prevents further oxidation of acetyl-CoA to CO2. Each turn of the glyoxylate cycle consumes two molecules of acetyl-CoA and produces one molecule of succinate, which is then available for biosynthetic purposes. Hydrolysis of stored triacylglycerols also produces glycerol 3-phosphate, which can enter the gluconeogenic pathway, aer its oxidation to dihydroxyacetone phosphate (see Fig. 14-16). We noted in Chapter 14 that animal cells can carry out gluconeogenesis from three- and four-carbon precursors, but not from the two acetyl carbons of acetyl-CoA. Because the pyruvate dehydrogenase reaction is effectively irreversible (see Section 16.1) and animals do not have the enzymes specific to the glyoxylate cycle (isocitrate lyase and malate synthase), they have no way to convert acetyl-CoA to pyruvate or oxaloacetate. So, unlike vascular plants, animals cannot bring about the net synthesis of glucose from fatty acids. Cellulose Is Synthesized by Supramolecular Structures in the Plasma Membrane Cellulose is a major constituent of plant cell walls, providing strength and rigidity and preventing the swelling of the cell and rupture of the plasma membrane that might result when osmotic conditions favor water entry into the cell. Each year, worldwide, plants synthesize more than 1011 metric tons of cellulose, making this simple polymer one of the most abundant compounds in the biosphere. The structure of cellulose in the plant cell wall is simple: linear polymers of thousands of (β1→ 4)-linked D-glucose units, assembled into bundles of at least 18 chains, which co- crystalize to form microfibrils, which may in turn be assembled into larger macrofibrils. (Fig. 20-46). FIGURE 20-46 Cellulose structure. The plant cell wall is made up in part of cellulose molecules arranged side by side to form crystalline arrays — cellulose microfibrils. Several microfibrils may combine to form larger cellulose macrofibrils. The scanning electron microscope shows macrofibrils, 5 to 12 nm in diameter, laid down on the cell surface in several layers distinguishable by the different orientations of the fibrils. As a major component of the plant cell wall, cellulose must be synthesized from intracellular precursors but deposited and assembled outside the plasma membrane. The enzymatic machinery for initiation, elongation, and export of cellulose chains is therefore more complicated than that used to synthesize starch or glycogen (which are not exported). The complex enzymatic machinery that assembles cellulose chains spans the plasma membrane, with one part on the cytoplasmic side positioned to bind the substrate, UDP-glucose, and elongate the chains, and another part extending to the outside, responsible for exporting the cellulose molecules to the extracellular space. Freeze-fracture electron microscopy shows a cellulose synthesis complex, or rosette, composed of six large particles arranged in a regular hexagon with a diameter of about 30 nm (Fig. 20-47a). Several proteins, including the catalytic subunit of cellulose synthase, make up this structure. The structure of the plant cellulose synthase is similar to that of the bacterium Rhodobacter sphaeroides, which has been determined by x-ray crystallography (Fig. 20-47b). FIGURE 20-47 A model for the synthesis of cellulose. (a) Schematic derived from a combination of genetic, electron microscopic, and biochemical studies of Arabidopsis thaliana and other vascular plants. (b) The structure of cellulose synthase from the bacterium Rhodobacter sphaeroides. The transmembrane part of the protein provides a channel through which the lengthening cellulose polymer (red) is pushed into the periplasm as the chain grows by addition of glucose units on the inside surface of the plasma membrane. Two structures of the enzyme move during the catalytic cycle. The gating loop moves into the substrate-binding site when UDP-glucose binds, then moves out to allow UDP to leave. The finger helix touches the glucose residue at the growing polymer end, then, a er a new residue is added, moves so as to touch this new terminal glucose. The glycosyl transferase domain extends into the cytoplasm, where it binds its substrate UDP-glucose. [(b) Data from PDB ID 5EJZ, J. L. W. Morgan et al., Nature 531:329, 2016. An extension of the cellulose chain was modeled in.] In one working model of cellulose synthesis, cellulose chains are initiated by the transfer of a glucose residue from UDP-glucose to a “primer” glucose already bound to cellulose synthase on the cytoplasmic side of the plasma membrane, to form a disaccharide. As addition of further glucose residues lengthens the chain, it is extruded through a channel formed by the transmembrane helices of cellulose synthase and, on the outer surface of the plasma membrane, joins growing chains from neighboring cellulose synthase molecules to form a cellulose microfibril. Polymers of more than 6 to 8 glucose units are insoluble in water, promoting microfibril crystallization. There is no definite length for a cellulose polymer; synthesis is highly processive, and some polymers are as long as 15,000 glucose units. The UDP-glucose used for cellulose synthesis (step in Fig. 20- 47) is generated from sucrose produced during photosynthesis, in a reaction catalyzed by sucrose synthase (named for the reverse reaction): Sucrose+ U D P → U D P-glucose+ fructose A membrane-bound form of sucrose synthase may produce a high local concentration of UDP-glucose for cellulose synthesis. Each of the six particles of the rosette most likely contains three cellulose synthase molecules, each synthesizing a single cellulose chain (step ). The large enzyme complex that catalyzes this process moves along the plasma membrane with directionality oen related to the course of microtubules in the cell cortex, the cytoplasmic layer just below the membrane (step ). When these microtubules lie perpendicular to the axis of the plant’s growth, the cellulose microfibrils are laid down similarly to promote elongation. The motion of the cellulose synthase complexes is believed to be driven by energy released in the polymerization reaction, not by a molecular motor such as kinesin. The fundamental cellulose microfibril made by one rosette-type cellulose synthesis complex is thought to be composed of 18 chains lying side by side with the same (parallel) orientation of nonreducing and reducing ends. The 18 separate polymers coalesce on the outer surface of the cell and crystallize soon aer they are polymerized (step ), just prior to integrating into the cell wall. In UDP-glucose, the glucose is α -linked to the nucleotide, but in cellulose, the glucose residues are (β1→ 4)-linked, so there is an inversion of configuration at the anomeric carbon (C-1) as the glycosidic bond forms. Glycosyltransferases that invert configuration are generally assumed to use a single-displacement mechanism, with nucleophilic attack by the acceptor species at the anomeric carbon of the donor sugar (in this case, UDP- glucose). Pools of Common Intermediates Link Pathways in Different Organelles Although we have described metabolic transformations in plant cells in terms of individual pathways, these pathways interconnect so completely that we should instead consider pools of metabolic intermediates shared among these pathways and connected by readily reversible reactions (Fig. 20-48). One such metabolite pool includes the hexose phosphates glucose 1- phosphate, glucose 6-phosphate, and fructose 6-phosphate; a second includes the 5-phosphates of the pentoses ribose, ribulose, and xylulose; a third includes the triose phosphates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Metabolite fluxes through these pools change in magnitude and direction in response to changes in the circumstances of the plant, and they vary with tissue type. Transporters in the membranes of each organelle move specific compounds in and out, and the regulation of these transporters presumably influences the degree to which the pools mix. FIGURE 20-48 Pools of hexose phosphates, pentose phosphates, and triose phosphates. The compounds in each pool are readily interconvertible by reactions that have small standard free-energy changes. When one component of the pool is temporarily depleted, a new equilibrium is quickly established to replenish it. Movement of the sugar phosphates between intracellular compartments is limited; specific transporters must be present in an organelle membrane. During daylight hours, triose phosphates produced in photosynthetic leaf tissue (“source” tissues, in which there is a net fixation of CO2) move out of the chloroplast and into the cytosolic hexose phosphate pool, where they are converted to sucrose for transport via the plant phloem (sap) to nonphotosynthetic “sink” tissues (Fig. 20-49). In sink tissues such as roots, tubers, and bulbs, sucrose is converted to starch for storage or is used as an energy source via glycolysis. In growing plants, hexose phosphates are also withdrawn from the pool for the synthesis of cell walls. At night, starch is metabolized by glycolysis and oxidative phosphorylation to provide energy for both source and sink tissues.

FIGURE 20-49 Movement of sucrose between source and sink tissues. (a) In daylight, photosynthetic leaves (source tissue) fix CO2 into triose phosphates via the Calvin cycle in chloroplasts. Some of the triose phosphate is used in the chloroplasts to synthesize starch; the rest is exported to the cytosol, where it can be converted via gluconeogenesis to fructose 6-phosphate and glucose 1-phosphate. Sucrose, synthesized from UDP- glucose and fructose, is exported from leaf mesophyll cells to the plant phloem; the resulting high sucrose content draws water into the phloem by osmosis. The resulting increased turgor pressure (p. 52) pushes the solution in the phloem toward sink tissues. (b) Sucrose moves from the phloem into the sink tissues, where it is converted to starch or cell wall cellulose, or is used as fuel for glycolysis, the citric acid cycle, and oxidative phosphorylation to provide ATP for these nonphotosynthetic tissues. Sugar transport across the plasma membrane and between intracellular compartments is catalyzed by several symporters and antiporters coupled to a proton gradient. [Information from Dr. Gerald Edwards, School of Biological Sciences, Washington State University.] SUMMARY 20.6 Biosynthesis of Starch, Sucrose, and Cellulose Starch synthase in chloroplasts and amyloplasts catalyzes the addition of single glucose residues, donated by ADP-glucose, to the growing polymer chain. Sucrose is synthesized in the cytosol from UDP-glucose and fructose 1-phosphate, in two steps. The partitioning of triose phosphates between sucrose synthesis and starch synthesis is regulated by fructose 2,6- bisphosphate (F26BP). [F26BP] varies inversely with the rate of photosynthesis, and F26BP inhibits the synthesis of fructose 6- phosphate, the precursor of sucrose. The glyoxylate cycle, taking place in the glyoxysomes of germinating seeds of some plants, uses several citric acid cycle enzymes and two additional enzymes: isocitrate lyase and malate synthase. The two decarboxylation steps of the citric acid cycle are bypassed, making possible the net formation of succinate, oxaloacetate, and other cycle intermediates from acetyl-CoA. Cellulose synthase has a glycosyl transferase activity in its cytoplasmic domain and forms a transmembrane channel through which the growing cellulose chain is extruded. Glucose units are transferred from UDP-glucose to the nonreducing end of the growing chain. The plant cell shares pools of common intermediates, including hexose-, pentose-, and triose-phosphates. Transporters in the membranes of chloroplasts, mitochondria, and amyloplasts mediate the movement of sugar phosphates between organelles. The direction of metabolite flow through the pools within a leaf changes from day to night. Sucrose produced in a photosynthetic (source) tissue is exported to nonphotosynthetic (sink) tissue such as roots and tubers via the plant phloem. Chapter Review KEY TERMS Terms in bold are defined in the glossary. photosynthesis light-dependent reactions photophosphorylation chloroplast stroma thylakoid photon excited state ground state exciton exciton transfer chlorophylls accessory pigments carotenoids β -carotene action spectrum photosystem photochemical reaction center light-harvesting complexes (LHCs) cyclic electron transfer linear electron transfer ferredoxin Z scheme photosystem II (PSII) photosystem I (PSI) cytochrome b6f plastoquinone (PQA) plastocyanin phylloquinone (PQK) cyclic photophosphorylation state transition oxygen-evolving center CO2 assimilation CO2 fixation Calvin cycle reductive pentose phosphate pathway ribulose 1,5-bisphosphate 3-phosphoglycerate C3 plants ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) rubisco activase thioredoxin ferredoxin:thioredoxin reductase photorespiration 2-phosphoglycolate glycolate pathway C4 plants C4 pathway phosphoenolpyruvate carboxylase malic enzyme pyruvate phosphate dikinase CAM plants sugar nucleotide starch synthase glyoxysome glyoxylate cycle isocitrate lyase glyoxylate malate synthase cellulose synthase PROBLEMS 1. Photochemical Efficiency of Light at Different Wavelengths The rate of photosynthesis in a green plant, measured by O2 production, is higher when illuminated with light of wavelength 680 nm than with light of wavelength 700 nm. However, illumination by a combination of light of 680 nm and 700 nm gives a higher rate of photosynthesis than light of either wavelength alone. Explain. 2. Balance Sheet for Photosynthesis In 1804, Nicolas- Théodore de Saussure observed that the total weight of oxygen and dry organic matter produced by plants is greater than the weight of carbon dioxide consumed during photosynthesis. Where does the extra weight come from? 3. Role of H2S in Some Photosynthetic Bacteria Illuminated purple sulfur bacteria carry out photosynthesis in the presence of H2O and 14CO2, but only if H2S is added and O2 is absent. During photosynthesis, measured by formation of [14C] carbohydrate, the bacteria convert H2S to elemental sulfur but do not produce O2. What is the role of the conversion of H2S to sulfur? Why doesn’t photosynthesis produce O2 in these bacteria? 4. Electron Transfer through Photosystems I and II Predict how an inhibitor of electron passage through pheophytin would affect electron transfer through (a) photosystem II and (b) photosystem I. Explain your reasoning. 5. Limited ATP Synthesis in the Dark In a laboratory experiment, a researcher illuminates spinach chloroplasts in the absence of ADP and Pi. Then, the researcher turns the light off and adds ADP and Pi. ATP synthesis occurs for a short time in the dark. Explain this finding. 6. Mode of Action of the Herbicide DCMU Treating chloroplasts with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, or diuron), a potent herbicide, causes oxygen evolution and photophosphorylation to cease. Adding a Hill reagent (an external electron acceptor) restores oxygen evolution, but not photophosphorylation. How does DCMU act as a weed killer? Suggest a location for the inhibitory action of this herbicide in the scheme shown in Figure 20-12. Explain. 7. Effect of Venturicidin on Oxygen Evolution Venturicidin is a powerful inhibitor of the chloroplast ATP synthase, interacting with CFo and blocking proton passage through the CFoCF1 complex. How would venturicidin affect oxygen evolution in a suspension of well-illuminated chloroplasts? Would your answer change if the experiment were done in the presence of an uncoupling reagent such as 2,4- dinitrophenol (DNP)? Explain. 8. Light Energy for a Redox Reaction Suppose you have isolated a new photosynthetic microorganism that oxidizes H2S and passes the electrons to NAD +. What wavelength of light would provide enough energy for H2S to reduce NAD + under standard conditions? Assume 100% efficiency in the photochemical event, and use an E′° of −243 mV for H2S and −320 mV for NAD +. See Figure 20-4 for the energy equivalents of wavelengths of light. 9. Equilibrium Constant for Water-Splitting Reactions The coenzyme NAD P+ is the terminal electron acceptor in chloroplasts, according to the reaction 2H2O + 2NAD P+ → 2NAD PH + 2H+ + O2 Use information in Chapter 19 (Table 19-2) to calculate the equilibrium constant for this reaction at 25 °C. (The relationship between K′eq and ΔG′° is discussed on p. 468.) How can the chloroplast overcome this unfavorable equilibrium? 10. Energetics of Phototransduction During photosynthesis, pigment molecules in chloroplasts must absorb eight photons (four by each photosystem) for every O2 molecule they produce, according to the equation 2H2O + 2NAD P+ + 8 photons→ 2NAD PH + 2H+ + O2 The ΔG′° for the light-independent production of O2 is 400 kJ/mol. Assuming that these photons have a wavelength of 700 nm (red) and that the light absorption and use of light energy are 100% efficient, calculate the free-energy change for the process. 11. Electron Transfer to a Hill Reagent Isolated spinach chloroplasts evolve O2 when illuminated in the presence of potassium ferricyanide (a Hill reagent), according to the equation 2H2O + 4Fe3+ → O2+ 4H+ + 4Fe2+ where Fe3+ represents ferricyanide and Fe2+ represents ferrocyanide. Does this process produce NADPH? Explain. 12. How Oen Does a Chlorophyll Molecule Absorb a Photon? The amount of chlorophyll a (Mr 892) in a spinach leaf is about 20 μg/cm2 of leaf surface. In noonday sunlight (average energy reaching the leaf is 5.4 J /cm2∙min), the leaf absorbs about 50% of the radiation. How oen does a single chlorophyll molecule absorb a photon? Given that the average lifetime of an excited chlorophyll molecule in vivo is 1 ns, what fraction of the chlorophyll molecules are excited at any one time? 13. Effect of Monochromatic Light on Electron Flow Using a spectrophotometer, researchers can sometimes directly observe the extent of oxidation or reduction of an electron carrier during photosynthetic electron transfer. Illuminating chloroplasts with 700 nm light oxidizes cytochrome f, plastocyanin, and plastoquinone. Illuminating chloroplasts with 680 nm light, however, reduces these electron carriers. Explain. 14. Function of Cyclic Photophosphorylation When the [NAD PH]/[NAD P+] ratio in chloroplasts is high, photophosphorylation is predominantly cyclic (see Fig. 20- 12). Does cyclic electron transfer evolve O2? Does cyclic electron transfer produce NADPH? Explain. What is the main function of cyclic electron transfer? 15. Phases of Photosynthesis A researcher illuminates a suspension of green algae in the absence of CO2. He then incubates the algae with 14CO2 in the dark and observes the conversion of 14CO2 to [14C]glucose for a brief time. What is the significance of this observation with regard to the CO2- assimilation process, and how is it related to the light- dependent reactions of photosynthesis? Why does the conversion of 14CO2 to [14C]glucose stop aer a brief time? 16. Identification of Key Intermediates in CO2 Assimilation Calvin and his colleagues used the unicellular green alga Chlorella to study the CO2-assimilation reactions of photosynthesis. They incubated 14CO2 with illuminated suspensions of algae and followed the time course of appearance of 14C in two compounds, X and Y, under two sets of conditions. Suggest the identities of X and Y, based on your understanding of the Calvin cycle. a. They grew illuminated Chlorella with unlabeled CO2, then turned off the light and added 14CO2 (vertical dashed line in the graph below). Under these conditions, X was the first compound to become labeled with 14C; Y was unlabeled.

b. They grew illuminated Chlorella cells with 14CO2. Illumination was continued until all the 14CO2 had been taken up (vertical dashed line in the graph below). Under these conditions, X became labeled quickly but lost its radioactivity with time, whereas Y became more radioactive with time. 17. Regulation of the Calvin Cycle Iodoacetate reacts irreversibly with the free — SH groups of Cys residues in proteins. Predict which Calvin cycle enzyme(s) would be inhibited by iodoacetate, and explain why. 18. Comparison of the Reductive and Oxidative Pentose Phosphate Pathways The reductive pentose phosphate pathway generates several intermediates identical to those of the oxidative pentose phosphate pathway (Chapter 14). What role does each pathway play in cells where it is active? 19. Photorespiration and Mitochondrial Respiration Compare the oxidative photosynthetic carbon cycle, also called photorespiration, with the mitochondrial respiration that drives ATP synthesis. Why are both processes referred to as respiration? Where in the cell do they occur, and under what circumstances? What is the path of electron flow in each? 20. Pathway of CO2 Assimilation in Maize Researchers illuminate a maize (corn) plant in the presence of 14CO2. Aer about 1 second of illumination, they find more than 90% of all the radioactivity incorporated in the leaves at C-4 of malate, aspartate, and oxaloacetate. Only aer 60 seconds does 14C appear at C-1 of 3-phosphoglycerate. Explain. 21. Identifying CAM Plants Given some 14CO2 and all the tools typically present in a biochemistry research lab, how would you design a simple experiment to determine whether a plant is a typical C4 plant or a CAM plant? 22. Chemistry of Malic Enzyme: Variation on a Theme Malic enzyme, found in the bundle-sheath cells of C4 plants, carries out a reaction that has a counterpart in the citric acid cycle. What is the analogous reaction? Explain your choice. 23. Differences between C3 and C4 Plants The plant genus Atriplex includes some C3 and some C4 species. In the plots, the black curve represents species 1; the red curve represents species 2. From the data in the plots, identify which is a C3 plant and which is a C4 plant. Justify your answer in molecular terms that account for the data in all three plots.

24. Inorganic Pyrophosphatase The enzyme inorganic pyrophosphatase contributes to making many biosynthetic reactions that generate inorganic pyrophosphate essentially irreversible in cells. By keeping the concentration of PPi very low, the enzyme “pulls” these reactions in the direction of PPi formation. The synthesis of ADP-glucose in chloroplasts is one such reaction. However, the synthesis of UDP-glucose in the plant cytosol, which also produces PPi, is readily reversible in vivo. How do you reconcile these two facts? 25. Regulation of Starch and Sucrose Synthesis Sucrose synthesis occurs in the cytosol and starch synthesis occurs in the chloroplast stroma, yet the two processes are intricately balanced. What factors shi the reactions in favor of (a) starch synthesis and (b) sucrose synthesis? 26. Regulation of Sucrose Synthesis In the regulation of sucrose synthesis from the triose phosphates produced during photosynthesis, 3-phosphoglycerate and Pi play critical roles (see Fig. 20-42). Explain why the concentrations of these two regulators reflect the rate of photosynthesis. 27. Sucrose and Dental Caries The most prevalent infection in humans worldwide is dental caries, which stems from the colonization and destruction of tooth enamel by a variety of acidifying microorganisms. These organisms synthesize and live within a water-insoluble network of dextrans, called dental plaque, composed of (α1→ 6)-linked polymers of glucose with many (α1→ 3) branch points. Polymerization of dextran requires dietary sucrose, and the bacterial enzyme dextran-sucrose glucosyltransferase catalyzes the reaction. a. Write the overall reaction for dextran polymerization. b. In addition to providing a substrate for the formation of dental plaque, how does dietary sucrose also provide oral bacteria with an abundant source of metabolic energy? 28. Partitioning between the Citric Acid and Glyoxylate Cycles In an organism (such as Escherichia coli) that has both the citric acid cycle and the glyoxylate cycle, what determines which of these pathways isocitrate will enter? DATA ANALYSIS PROBLEM 29. Photophosphorylation: Discovery, Rejection, and Rediscovery In the 1930s and 1940s, researchers were beginning to make progress toward understanding the mechanism of photosynthesis. At the time, the role of “energy-rich phosphate bonds” (today, “ATP”) in glycolysis and cellular respiration was just becoming known. There were many theories about the mechanism of photosynthesis, especially about the role of light. This problem focuses on what was then called the “primary photochemical process” — that is, on what, exactly, the energy from captured light produces in the photosynthetic cell. Interestingly, one important part of the modern model of photosynthesis was proposed early on, only to be rejected, ignored for several years, then finally revived and accepted. In 1944, Emerson, Stauffer, and Umbreit proposed that “the function of light energy in photosynthesis is the formation of ‘energy-rich’ phosphate bonds” (p. 107). In their model (hereaer, the “Emerson model”), the free energy necessary to drive both CO2 fixation and reduction came from these “energy-rich phosphate bonds” (i.e., ATP), produced as a result of light absorption by a chlorophyll-containing protein. This model was explicitly rejected by Rabinowitch (1945). Aer summarizing Emerson and coauthors’ findings, Rabinowitch stated: “Until more positive evidence is provided, we are inclined to consider as more convincing a general argument against this hypothesis, which can be derived from energy considerations. Photosynthesis is eminently a problem of energy accumulation. What good can be served, then, by converting light quanta (even those of red light, which amount to about 43 kcal per Einstein) into ‘phosphate quanta’ of only 10 kcal per mole? This appears to be a start in the wrong direction — toward dissipation rather than toward accumulation of energy” (p. 228). This argument, along with other evidence, led to abandonment of the Emerson model until the 1950s, when it was found to be correct — albeit in a modified form. For each piece of information from Emerson and coauthors’ article presented in (a) through (d), answer the following three questions: 1. How does this information support the Emerson model, in which light energy is used directly by chlorophyll to make ATP, and the ATP then provides the energy to drive CO2 fixation and reduction? 2. How would Rabinowitch explain this information, based on his model (and most other models of the day), in which light energy is used directly by chlorophyll to make reducing compounds? Rabinowitch wrote: “Theoretically, there is no reason why all electronic energy contained in molecules excited by the absorption of light should not be available for oxidation-reduction” (p. 152). In this model, the reducing compounds are then used to fix and reduce CO2, and the energy for these reactions comes from the large amounts of free energy released by the reduction reactions. 3. How is this information explained by our modern understanding of photosynthesis? a. Chlorophyll contains a M g2+ ion, which is known to be an essential cofactor for many enzymes that catalyze phosphorylation and dephosphorylation reactions. b. A crude “chlorophyll protein” isolated from photo- synthetic cells showed phosphorylating activity. c. The phosphorylating activity of the “chlorophyll protein” was inhibited by light. d. The levels of several different phosphorylated compounds in photosynthetic cells changed dramatically in response to light exposure. (Emerson and coworkers were not able to identify the specific compounds involved.) As it turned out, the Emerson and Rabinowitch models were both partly correct and partly incorrect. e. Explain how the two models relate to our current model of photosynthesis. In his rejection of the Emerson model, Rabinowitch went on to say: “The difficulty of the phosphate storage theory appears most clearly when one considers the fact that, in weak light, eight or ten quanta of light are sufficient to reduce one molecule of carbon dioxide. If each quantum should produce one molecule of high-energy phosphate, the accumulated energy would be only 80–100 kcal per Einstein — while photosynthesis requires at least 112 kcal per mole, and probably more, because of losses in irreversible partial reactions” (p. 228). f. How does Rabinowitch’s value of 8 to 10 photons per molecule of CO2 reduced compare with the value accepted today? g. How would you rebut Rabinowitch’s argument, based on our current knowledge about photosynthesis? References Emerson, R.L., J.F. Stauffer, and W.W. Umbreit. 1944. Relationships between phosphorylation and photosynthesis in Chlorella. Am. J. Botany 31:107–120. Rabinowitch, E.I. 1945. Photosynthesis and Related Processes, Vol. I. New York: Interscience Publishers.