Is it so small a thing
To have enjoyed the sun,
To have lived light in the spring,
To have loved, to have thought, to have

Matthew Arnold, Empedocles on Etna (1852)

“Sunflowers,” by Claude Monet (1840-1926), French,
Metropolitan Museum of Art, New York City/Superstock, Inc.

Chapter 22

Photosynthesis

The vast majority of energy consumed by living organisms stems from solar energy captured by the process of photosynthesis. Only chemolithotropic bacteria (Chapter 18) are independent of this energy source. Of the 1.5x10²² kJ of energy reaching the earth each day from the sun, 1% is absorbed by photosynthetic organisms and transduced into chemical energy.¹ This energy, in the form of biomolecules, becomes available to other members of the biosphere through food chains. The transduction of solar, or light, energy into chemical energy is often expressed in terms of carbon dioxide fixation, in which hexose is formed from carbon dioxide and oxygen is evolved:

                                                                   Light

                               6 CO₂+6 H₂O ® C₆H₁₂O₆+6 O₂                                              (22.1)

Estimates indicate that 10¹¹ tons of carbon dioxide are fixed globally per year, of which one-third is fixed in the oceans, primarily by photosynthetic marine microorganisms.
      Although photosynthesis is traditionally equated with CO₂ fixation, light energy (or rather the chemical energy derived from it) can be used to drive virtually any cellular process. The assimilation of inorganic forms of nitrogen and sulfur into organic molecules (Chapter 27) represents two other metabolic conversions driven by light energy in green plants. Our previous considerations of aerobic metabolism (Chapters 19 through 21) treated cellular respiration (precisely the reverse of Equation [22.1]) as the central energy-releasing process in life. It necessarily follows that the formation of hexose from carbon dioxide and water, the products of cellular respiration, must be endergonic. The necessary energy comes from light. Note that in the carbon dioxide fixation reaction described, light is used to drive a chemical reaction against its thermodynamic potential.

22.1 × General Aspects of Photosynthesis Photosynthesis Occurs in Membranes

Figure 22.1 · Electron micrograph of a representative chloroplast. (James Dennis/CNRI/Phototake NYC)

Organisms capable of photosynthesis are very diverse, ranging from simple prokaryotic forms to the largest organisms of all, Sequoia gigantea, the giant redwood trees of California. Despite this diversity, we find certain generalities regarding photosynthesis. An important one is that photosynthesis occurs in membranes. In photosynthetic prokaryotes, the photosynthetic membranes fill up the cell interior; in photosynthetic eukaryotes, the photosynthetic membranes are localized in large organelles known as chloroplasts (Figures 22.1 and 22.2).

Figure 22.2 · Schematic diagram of an idealized chloroplast.

Chloroplasts are one member in a family of related plant-specific organelles known as plastids. Chloroplasts themselves show a range of diversity, from the single, spiral chloroplast that gives Spirogyra its name to the multitude of ellipsoidal plastids typical of higher plant cells (Figure 22.3).

Figure 22.3 · (a) Spirogyra—a freshwater green alga. (b) A higher plant cell. (a, Michael Siegel/Phototake NYC; b, Biophoto Associates/Science Source.)

      Characteristic of all chloroplasts, however, is the organization of the inner membrane system, the so-called thylakoid membrane. The thylakoid membrane is organized into paired folds that extend throughout the organelle, as in Figure 22.1. These paired folds, or lamellae, give rise to flattened sacs or disks, thylakoid vesicles (from the Greek thylakos, meaning “sack”), which occur in stacks called grana. A single stack, or granum, may contain dozens of thylakoid vesicles, and different grana are joined by lamellae that run through the soluble portion, or stroma, of the organelle. Chloroplasts thus possess three membrane-bound aqueous compartments: the intermembrane space, the stroma, and the interior of the thylakoid vesicles, the so-called thylakoid space (also known as the thylakoid lumen). As we shall see, this third compartment serves an important function in the transduction of light energy into ATP formation. The thylakoid membrane has a highly characteristic lipid composition and, like the inner membrane of the mitochondrion, is impermeable to most ions and molecules. Chloroplasts, like their mitochondrial counterparts, possess DNA, RNA, and ribosomes and consequently display a considerable amount of autonomy. However, many critical chloroplast components are encoded by nuclear genes, so autonomy is far from absolute.

Photosynthesis Consists of Both Light Reactions and Dark Reactions

If a chloroplast suspension is illuminated in the absence of carbon dioxide, oxygen is evolved. Furthermore, if the illuminated chloroplasts are now placed in the dark and supplied with CO₂, net hexose synthesis can be observed (Figure 22.4). Thus, the evolution of oxygen can be temporally separated from CO₂ fixation and also has a light dependency that CO₂ fixation lacks. The light reactions of photosynthesis, of which O₂ evolution is only one part, are associated with the thylakoid membranes. In contrast, the light-independent reactions, or so-called dark reactions, notably CO₂ fixation, are located in the stroma. A concise summary of the photosynthetic process is that radiant electromagnetic energy (light) is transformed by a specific photochemical system located in the thylakoids to yield chemical energy in the form of reducing potential (NADPH) and high-energy phosphate (ATP). NADPH and ATP can then be used to drive the endergonic process of hexose formation from CO₂ by a series of enzymatic reactions found in the stroma (see Equation 22.3, which follows).

Figure 22.4 · The light-dependent and light-independent reactions of photosynthesis. Light reactions are associated with the thylakoid membranes, and light-independent reactions are associated with the stroma.

Water Is the Ultimate e^- Donor for Photosynthetic NADP⁺ Reduction

In green plants, water serves as the ultimate electron donor for the photosynthetic generation of reducing equivalents. The reaction sequence

                                                                  nhv

describes the process, where nhv symbolizes light energy (n is some number of photons of energy hv, where h is Planck’s constant and v is the frequency of the light). Light energy is necessary to make the unfavorable reduction of NADP⁺ by H₂O (E_o' = -1.136 V; DG_o' = +219 kJ/mol NADP⁺) thermodynamically favorable. Thus, the light energy input, nhv, must exceed 219 kJ/mol NADP⁺. The stoichiometry of ATP formation depends on the pattern of photophosphorylation operating in the cell at the time and on the ATP yield in terms of the chemiosmotic ratio, ATP/H⁺, as we will see later. Nevertheless, the stoichiometry of the metabolic pathway of CO₂ fixation is certain:

12 NADPH + 12 H⁺ + 18 ATP + 6 CO₂ + 12 H₂O ®
                                          C₆H₁₂O₆ + 12 NADP⁺ + 18 ADP + 18 P_i   (22.3)

A More Generalized Equation for Photosynthesis

In 1931, comparative study of photosynthesis in bacteria led van Niel to a more general formulation of the overall reaction:

                                                            Light                              CO₂   +   2 H₂A  ^®  (CH₂O) +  2A  + H₂O                     (22.4)

                     Hydrogen     Hydrogen              Reduced    Oxidized                        acceptor          donor                  acceptor       donor

      In photosynthetic bacteria, H₂A is variously H₂S (photosynthetic green and purple sulfur bacteria), isopropanol, or some similar oxidizable substrate. [(CH₂O) symbolizes a carbohydrate unit.]

                              CO₂ + 2 H₂S ^® (CH₂O) + H₂O + 2 S

In cyanobacteria and the eukaryotic photosynthetic cells of algae and higher plants, H₂A is H₂O, as implied earlier, and 2 A is O₂. The accumulation of O₂ to constitute 20% of the earth’s atmosphere is the direct result of eons of global oxygenic photosynthesis.

22.2 × Photosynthesis Depends on the Photoreactivity of Chlorophyll

Chlorophylls are magnesium-containing substituted tetrapyrroles whose basic structure is reminiscent of heme, the iron-containing porphyrin (Chapters 5 and 21). Chlorophylls differ from heme in a number of properties: magnesium instead of iron is coordinated in the center of the planar conjugated ring structure; a long-chain alcohol, phytol, is esterified to a pyrrole ring substituent; and the methine bridge linking pyrroles III and IV is substituted and cross-linked to ring III, leading to the formation of a fifth five-membered ring. The structures of chlorophyll a and b are shown in Figure 22.5.

Figure 22.5 · Structures of chlorophyll a and b. Chlorophylls are structurally related to hemes, except Mg²⁺ replaces Fe²⁺ and ring II is more reduced than the corresponding ring of the porphyrins. The chlorophyll tetrapyrrole ring system is known as a chlorin. R=CH₃ in chlorophyll a; R=CHO in chlorophyll b. Note that the aldehyde C=O bond of chlorophyll b introduces an additional double bond into conjugation with the double bonds of the tetrapyrrole ring system. Ring V is the additional ring created by interaction of the substituent of the methine bridge between pyrroles III and IV with the side chain of ring III. The phytyl side chain of ring IV provides a hydrophobic tail to anchor the chlorophyll in membrane protein complexes.

      Chlorophylls are excellent light absorbers because of their aromaticity. That is, they possess delocalized p electrons above and below the planar ring structure. The energy differences between electronic states in these p orbitals correspond to the energies of visible light photons. When light energy is absorbed, an electron is promoted to a higher orbital, enhancing the potential for transfer of this electron to a suitable acceptor. Loss of such a photo-excited electron to an acceptor is an oxidation-reduction reaction. The net result is the transduction of light energy into the chemical energy of a redox reaction.

Figure 22.6 · Absorption spectra of chlorophylls a and b.

Chlorophylls and Accessory Light-Harvesting Pigments

The absorption spectra of chlorophylls a and b (Figure 22.6) differ somewhat. Plants that possess both chlorophylls can harvest a wider spectrum of incident energy. Other pigments in photosynthetic organisms, so-called accessory light-harvesting pigments (Figure 22.7),

Figure 22.7 · Structures of representative accessory light-harvesting pigments in photosynthetic cells. (a) b-Carotene, an accessory light-harvesting pigment in leaves. Note the many conjugated double bonds. (b) Phycocyanobilin, a blue pigment found in cyanobacteria. It is a linear or open pyrrole.

increase the possibility for absorption of incident light of wavelengths not absorbed by the chlorophylls. These accessory pigments, such as carotenoids and phycobilins, are also responsible for the magnificent colors of autumn. They persist longer after leaf death than the green chlorophylls, finally imparting their particular hues to the plant. These pigments, like chlorophyll, possess many conjugated double bonds and thus absorb visible light.

The Fate of Light Energy Absorbed by Photosynthetic Pigments

Each photon represents a quantum of light energy. A quantum of light energy absorbed by a photosynthetic pigment has four possible fates (Figure 22.8):

Figure 22.8 · Possible fates of the quantum of light energy absorbed by photosynthetic pigments.

A. Loss as heat. The energy can be dissipated as heat through redistribution into atomic vibrations within the pigment molecule.

B. Loss of light. Energy of excitation reappears as fluorescence (light emission); a photon of fluorescence is emitted as the e^- returns to a lower orbital. This fate is common only in saturating light intensities. For thermodynamic reasons, the photon of fluorescence is of longer wavelength and hence lower energy than the quantum of excitation.

C. Resonance energy transfer. The excitation energy can be transferred by esonance energy transfer, a radiationless process, to a neighboring molecule if their energy level difference corresponds to the quantum of excitation energy. In this process, the quantum, or so-called exciton, is transferred, raising an electron in the receptor molecule to a higher energy state as the photo-excited e^- in the original absorbing molecule returns to ground state. This so-called Förster resonance energy transfer is the mechanism whereby quanta of light falling anywhere within an array of pigment molecules can be transferred ultimately to specific photochemically reactive sites.

D. Energy transduction. The energy of excitation, in raising an electron to a higher energy orbital, dramatically changes the standard reduction potential, E_o', of the pigment such that it becomes a much more effective electron donor. That is, the excited-state species, by virtue of having an electron at a higher energy level through light absorption, has become a potent electron donor. Reaction of this excited-state electron donor with an electron acceptor situated in its vicinity leads to the transformation, or transduction, of light energy (photons) to chemical energy (reducing power, the potential for electron-transfer reactions). Transduction of light energy into chemical energy, the photochemical event, is the essence of photosynthesis.

Photosynthetic Units Consist of Many Chlorophyll Molecules but Only a Single Reaction Center

In the early 1930s, Emerson and Arnold investigated the relationship between the amount of incident light energy, the amount of chlorophyll present, and the amount of oxygen evolved by illuminated algal cells (this relationship is called the quantum yield of photosynthesis). Their studies gave an unexpected result: When algae were illuminated with very brief light flashes that could excite every chlorophyll molecule at least once, only one molecule of O₂ was evolved per 2400 chlorophyll molecules. This result implied that not all chlorophyll molecules are photochemically reactive, and it led to the concept that photosynthesis occurs in functionally discrete units. Chlorophyll serves two roles in photosynthesis. It is involved in light harvesting and the transfer of light energy to photoreactive sites by exciton transfer, and it participates directly in the photochemical events whereby light energy becomes chemical energy. A photosynthetic unit can be envisioned as an antenna of several hundred light-harvesting chlorophyll molecules plus a special pair of photochemically reactive chlorophyll a molecules called the reaction center. The purpose of the vast majority of chlorophyll in a photosynthetic unit is to harvest light incident within the unit and funnel it, via resonance energy transfer, to special reaction center chlorophyll molecules that are photochemically active. Most chlorophyll thus acts as a large light-collecting antenna, and it is at the reaction centers that the photochemical event occurs (Figure 22.9). Oxidation of chlorophyll leaves a cationic free radical, Chl×⁺, whose properties as an electron acceptor have important consequences for photosynthesis. Note that the Mg²⁺ ion does not change in valence during these redox reactions.

Figure 22.9 · Schematic diagram of a photosynthetic unit. The light-harvesting pigments, or antenna molecules (green), absorb and transfer light energy to the specialized chlorophyll dimer that constitutes the reaction center (orange).

22.3 × Eukaryotic Phototrophs Possess Two Distinct Photosystems

The existence of two separate but interacting photosystems in photosynthetic eukaryotes was demonstrated through analysis of the photochemical action spectrum of photosynthesis, in which the oxygen-evolving capacity as a function of light wavelength was determined (Figure 22.10).

Figure 22.10 · The photochemical action spectrum of photosynthesis. The quantum yield of photosynthesis as a function of wavelength of incident light shows an abrupt decrease above 680 nm, the so-called red drop.

      Although chlorophyll a has some capacity to absorb 700-nm light, light of this wavelength is relatively inefficient in driving photosynthesis. However, if light of shorter wavelength (less than 680 nm) is used to supplement 700-nm light, an enhancement of photosynthetic quantum yield, the so-called Emerson enhancement effect, is observed. In other words, these two wavelengths are synergistic: When given together, these wavelengths elicit more O₂ evolution than expected from the sum of the amounts when each wavelength of light is given alone. One interpretation is that two light reactions participate in oxygen-evolving photosynthetic cells, one using light of 700 nm and the other using light of wavelength 680 nm or less. The existence of two light reactions established the presence of two photosystems, I and II. Photosystem I (PSI) is defined as containing reaction center chlorophylls with maximal red light absorption at 700 nm; PSI is not involved in O₂ evolution. Photosystem II (PSII) functions in O₂ evolution, using reaction centers that exhibit maximal red light absorption at 680 nm.
      All photosynthetic cells contain some form of photosystem. Photosynthetic bacteria, unlike cyanobacteria and eukaryotic phototrophs, have only one photosystem. Interestingly, bacterial photosystems resemble eukaryotic PSII more than PSI, even though photosynthetic bacteria lack O₂-evolving capacity.

P700 and P680 Are the Reaction Centers of PSI and PSII, Respectively

Precise spectrophotometric measurements showed that a small amount of pigment absorbing 700-nm light (P700) is bleached when light of this wavelength is used to illuminate suspensions of eukaryotic photosynthetic cells. Because bleaching, or disappearance, of the 700-nm absorbance can be mimicked by adding an electron acceptor such as ferricyanide, bleaching is correlated with electron loss from P700. The concentration of P700 is small, only 0.25% of the total amount of chlorophyll in plants. However, this low concentration is consistent with the notion of reaction centers (specific photoreactive sites). P700 is the reaction center of photosystem I. Similar studies using shorter-wavelength light identified an analogous pigment, P680, which constitutes the reaction center of photosystem II. Both P700 and P680 are chlorophyll a dimers situated within specialized protein complexes.

Chlorophyll Exists in Plant Membranes in Association with Proteins

Detergent treatment of a suspension of thylakoids dissolves the membranes, releasing complexes containing both chlorophyll and protein. These chlorophyll-protein complexes represent integral components of the thylakoid membrane, and their organization reflects their roles as either light-harvesting complexes (LHC), PSI complexes, or PSII complexes. All chlorophyll is apparently localized within these three macromolecular assemblies.

Figure 22.11 · Roles of the two photosystems, PSI and PSII.

The Roles of PSI and PSII

What are the roles of the two photosystems, and what is their relationship to each other? Photosystem I provides reducing power in the form of NADPH. Photosystem II splits water, producing O₂, and feeds the electrons released into an electron transport chain that couples PSII to PSI. Electron transfer between PSII and PSI pumps protons for chemiosmotic ATP synthesis. As summarized by Equation (22.2), photosynthesis involves the reduction of NADP⁺, using electrons derived from water and activated by light, hv. ATP is generated in the process. The standard reduction potential for the NADP⁺/NADPH couple is -0.32 V. Thus, a strong reductant with E_o' more negative than -0.32 V is required to reduce NADP⁺ under standard conditions. By similar reasoning, a very strong oxidant will be required to oxidize water to oxygen because O₂/H₂O) is +0.82 V. Separation of the oxidizing and reducing aspects of Equation (22.2) is accomplished in nature by devoting PSI to NADP⁺ reduction and PSII to water oxidation. PSI and PSII are linked via an electron transport chain so that the weak reductant generated by PSII can provide an electron to reduce the weak oxidant side of P700 (Figure 22.11). Thus, electrons flow from H₂O to NADP⁺, driven by light energy absorbed at the reaction centers. Oxygen is a by-product of the photolysis, literally “light-splitting,” of water. Accompanying electron flow is production of a proton gradient and ATP synthesis (see Section 22.7). This light-driven phosphorylation is termed photophosphorylation.

22.4 × The Z Scheme of Photosynthetic Electron Transfer

Photosystems I and II contain unique complements of electron carriers, and these carriers mediate the stepwise transfer of electrons from water to NADP⁺. When the individual redox components of PSI and PSII are arranged as an e^- transport chain according to their standard reduction potentials, the zigzag result resembles the letter Z laid sideways (Figure 22.12). The various electron carriers are indicated as follows: “Mn complex” symbolizes the manganese-containing oxygen-evolving complex; D is its e^- acceptor and the immediate e^- donor to P680⁺; Q_A and Q_B represent special plastoquinone molecules (see Figure 22.15) and PQ the plastoquinone pool; Fe-S stands for the Rieske iron-sulfur center, and cyt f, cytochrome f. PC is the abbreviation for plastocyanin, the immediate e^- donor to P700^-; and F_A, F_B, and F_X represent the membrane-associated ferredoxins downstream from A₀ (a specialized Chl a) and A₁ (a specialized PSI quinone). Fd is the soluble ferredoxin pool that serves as the e^- donor to the flavoprotein (Fp), called ferredoxin-NADP⁺ reductase, which catalyzes reduction of NADP⁺ to NADPH. Cyt(b₆)_n,(b₆)_p symbolizes the cytochrome b₆ moieties functioning to transfer e^- from F_A/F_B back to P700⁺ during cyclic photophosphorylation (the pathway symbolized by the dashed arrow).
      Overall photosynthetic electron transfer is accomplished by three membrane-spanning supramolecular complexes, composed of intrinsic and extrinsic polypeptides (shown as shaded boxes bounded by solid black lines in Figure 22.12). These complexes are the PSII complex, the cytochrome b₆/cytochrome f complex, and the PSI complex. The PSII complex is aptly described as a light-driven water:plastoquinone oxidoreductase; it is the enzyme system responsible for photolysis of water, and as such, it is also referred to as the oxygen-evolving complex, or OEC. Within this complex, a manganese-containing protein is intimately involved in the evolution of oxygen, perhaps through formation of a tetrametallic center consisting of 4 Mn²⁺ coordinating two equivalents of water. Both protons and electrons are abstracted from these water molecules, and O₂ is released as P680 undergoes four cycles of oxidation (Figure 22.13).

Oxygen Evolution Requires the Accumulation of Four Oxidizing Equivalents in PSII

When isolated chloroplasts that have been held in the dark are illuminated with very brief flashes of light, O₂ evolution reaches a peak on the third flash and every fourth flash thereafter (Figure 22.14a). The oscillation in O₂ evolved dampens over repeated flashes and converges to an average value. These data are interpreted to mean that the P680 reaction center complex cycles through five different oxidation states, numbered S₀ to S₄. One electron and one prot on are removed photochemically in each step. When S₄ is attained, an O₂ mole-cule is released (Figure 22.14b) as PSII returns to oxidation state S₀ and two new water molecules bind. The reason the first pulse of O₂ release occurred on the third flash (Figure 22.14a) is that the PSII reaction centers in the isolated chloroplasts were already poised at S₁ reduction level.

Figure 22.12 · The Z scheme of photosynthesis. (a) The Z scheme is a diagrammatic representation of photosynthetic electron flow from H₂O to NADP⁺. The energy relationships can be derived from the E_o' scale beside the Z diagram, with lower standard potentials and hence greater energy as you go from bottom to top. Energy input as light is indicated by two broad arrows, one photon appearing in P680 and the other in P700. P680* and P700* represent photoexcited states. Electron loss from P680* and P700* creates P680⁺ and P700⁺. The representative components of the three supramolecular complexes (PSI, PSII, and the cytochrome b₆/cytochrome f complex) are in shaded boxes enclosed by solid black lines. Proton translocations that establish the proton-motive force driving ATP synthesis are illustrated as well. (b) Figure showing the functional relationships among PSII, the cytochrome b/cytochrome f complex, PSI, and the photosynthetic CF₁CF₀ ATP synthase within the thylakoid membrane. Note that e^- acceptors Q_A (for PSII) and A₁ (for PSI) are at the stromal side of the thylakoid membrane, whereas the e^- donors to P680⁺ and P700⁺ are situated at the lumenal side of the membrane. The consequence is charge separation (2_stroma, 1_lumen) across the membrane. Also note that protons are translocated into the thylakoid lumen, giving rise to a chemiosmotic gradient that is the driving force for ATP synthesis by CF₁CF₀ ATP synthase.

Light-Driven Electron Flow from H₂O Through PSII

The events intervening between H₂O and P680 involve D, the name assigned to a specific protein tyrosine residue that mediates e^- transfer from H₂O via the Mn complex to P680⁺ (Figure 22.12). The oxidized form of D is a tyrosyl free radical species, D?¹. To begin the cycle, an exciton of energy excites P680 to P680*, whereupon P680* donates an electron to a special molecule of pheophytin, symbolized by “Pheo” in Figure 22.12. Pheophytin is like chlorophyll a, except 2 H⁺ replace the centrally coordinated Mg²⁺ ion. This special pheophytin is the direct electron acceptor from P680^*. Loss of an electron from P680* creates P680⁺, the electron acceptor for D. Electrons flow from Pheo via specialized molecules of plastoquinone, represented by “Q” in Figure 22.12, to a pool of plastoquinone within the membrane. Because of its lipid nature, plastoquinone is mobile within the membrane and hence serves to shuttle electrons from the PSII supramolecular complex to the cytochrome b₆/cytochrome f complex. Alternate oxidation-reduction of plastoquinone to its hydroquinone form involves the uptake of protons (Figure 22.15). The asymmetry of the thylakoid membrane is designed to exploit this proton uptake and release so that protons (H⁺) accumulate within the thylakoid vesicle, establishing an electrochemical gradient. Note that plastoquinone is an analog of coenzyme Q, the mitochondrial electron carrier (Chapter 21).

Figure 22.13 · Suggested interaction of four manganese atoms in forming a tetra-metallic center that could coordinate two water molecules and oxidize them to yield a molecule of O₂. This photo-oxidation, or photolysis, of water would proceed as P680 undergoes four cycles of light-induced oxidation. The four oxidizing equivalents accumulate in the manganese-containing active site of the O₂-evolving complex. (Adapted from Hoganson, C. W., and  Babcock, G. T., 1997. A metalloradical mechanism for the generation of oxygen in photosynthesis. Science 277:1953-1956.)

Figure 22.14 · Oxygen evolution requires the accumulation of four oxidizing equivalents in PSII. (a) Dark-adapted chloroplasts show little O₂ evolution after two brief light flashes. Oxygen evolution then shows a peak on the third flash and every fourth flash thereafter. The oscillation in O₂ evolution is dampened by repeated flashes and converges to an average value after 20 or so flashes. (b) The oscillation in O₂ evolution per light flash is due to the cycling of the PSII reaction center through five different oxidation states, S₀ to S₄. When S₄ is reached, O₂ is released. One e^- is removed photochemically at each light flash, moving the reaction center successively through S₁, S₂, S₃, and S₄. S₄ decays spontaneously to S₀ by oxidizing 2 H₂O to O₂. The peak of O₂ evolution at flash 3 in part (a) is due to the fact that the isolated chloroplast suspension is already at the S₁ stage.

Figure 22.15 · The structures of plastoquinone and its reduced form, plastohydroquinone (or plastoquinol). The oxidation of the hydroquinone releases 2 H⁺ as well as 2 e^-. The form shown (plastoquinone A) has nine isoprene units and is the most abundant plastoquinone in plants and algae. Other plastoquinones have different numbers of isoprene units and may vary in the substitutions on the quinone ring.

Electron Transfer Within the Cytochrome b₆/Cytochrome f Complex

The cytochrome b₆/cytochrome f or plastoquinol:plastocyanin oxidoreductase is a large (210 kD) multimeric protein possessing 22 to 24 transmembrane a-helices. It includes the two heme-containing electron transfer proteins for which it is named as well as iron-sulfur clusters (Chapter 21), which also participate in electron transport. The purpose of this complex is to mediate the transfer of electrons from PSII to PSI and to pump protons across the thylakoid membrane via a plastoquinone-mediated Q cycle, analogous to that found in mitochondrial e^- transport (Chapter 21). Cytochrome f (f from the Latin folium, meaning “foliage”) is a c-type cytochrome, with an a-absorbance band at 553 nm and a reduction potential of +0.365 V. Cytochrome b₆ apparently does not lie directly on the pathway of electron transfer from PSII to PSI. This cytochrome, whose a-absorbance band lies at 559 nm and whose standard reduction potential is -0.06 V, is thought to participate in an alternative cyclic e^- transfer pathway. Under certain conditions, electrons derived from P700* are not passed on to NADP⁺ but instead cycle down an alternative path via ferredoxins in the PSI complex to cytochrome b₆, plastoquinone, and ultimately back to P700⁺. This cyclic flow yields no O₂ evolution or NADP⁺reduction but can lead to ATP synthesis via so-called cyclic photophosphorylation, discussed later.

Electron Transfer from the Cytochrome b₆/Cytochrome f Complex to PSI

Plastocyanin (“PC” in Figure 22.12) is an electron carrier capable of diffusion along the inside of the thylakoid and migration in and out of the membrane, aptly suited to its role in shuttling electrons between the cytochrome b₆/cyto-chrome f complex and PSI. Plastocyanin is a low-molecular-weight (10.4 kD) protein containing a single copper atom. PC functions as a single-electron carrier (E_o' =10.32 V) as its copper atom undergoes alternate oxidation-reduction between the cuprous (Cu¹) and cupric (Cu²⁺ ) states. PSI is a light-driven plastocyanin:ferredoxin oxidoreductase. When P700, the specialized chlorophyll a dimer of PSI, is excited by light and oxidized by transferring its e^- to an adjacent chlorophyll a molecule that serves as its immediate e^- acceptor, P700⁺ is formed. (The standard reduction potential for the P700⁺/P700 couple lies near +0.45 V.) P700⁺ readily gains an electron from plastocyanin.
      The immediate electron acceptor for P700* is a special molecule of chlorophyll. This unique Chl a (A₀) rapidly passes the electron to a specialized quinone (A₁), which in turn passes the e^- to the first in a series of membrane-bound ferredoxins (Fd, Chapter 21). This Fd series ends with a soluble form of ferredoxin, Fd_s, which serves as the immediate electron donor to the flavoprotein (Fp) that catalyzes NADP⁺ reduction, namely, ferredoxin: NADP⁺ reductase.

The Initial Events in Photosynthesis Are Very Rapid Electron-Transfer Reactions

Electron transfer from P680 to Q and from P700 to Fd occurs on a pico-second-to-microsecond time scale. The necessity for such rapid reaction becomes obvious when one realizes that light-induced Chl excitation followed by electron transfer leads to separation of opposite charges in close proximity, as in P700⁺:A₀^-. Accordingly, subsequent electron transfer reactions occur rapidly in order to shuttle the electron away quickly, before the wasteful back reaction of charge recombination (and dissipation of excitation energy), as in return to P700:A₀, can happen.

22.5 × The Molecular Architecture of Photosynthetic Reaction Centers

What molecular architecture couples the absorption of light energy to rapid electron-transfer events, in turn coupling these e^- transfers to proton translocations so that ATP synthesis is possible? Part of the answer to this question lies in the membrane-associated nature of the photosystems. Membrane proteins have been difficult to study due to their insolubility in the usual aqueous solvents employed in protein biochemistry. A major breakthrough occurred in 1984 when Johann Deisenhofer, Hartmut Michel, and Robert Huber reported the first X-ray crystallographic analysis of a membrane protein. To the great benefit of photosynthesis research, this protein was the reaction center from the photosynthetic purple bacterium Rhodopseudomonas viridis. This research earned these three scientists the 1984 Nobel Prize in chemistry.

Structure of the R. viridis Photosynthetic Reaction Center

Figure 22.16 · Model of the structure and activity of the R. viridis reaction center. Four polypeptides (designated cytochrome, M, L, and H) make up the reaction center, an integral membrane complex. The cytochrome maintains its association with the membrane via a diacylglyceryl group linked to its N-terminal Cys residue by a thioether bond. M and L both consist of five membrane-spanning a-helices; H has a single membrane-spanning a-helix. The prosthetic groups are spatially situated so that rapid e^- transfer from P870* to Q_B is facilitated. Photoexcitation of P870 leads in less than 1 picosecond (psec) to reduction of the L-branch BChl only. P870⁺ is re-reduced via an e^- provided through the heme groups of the cytochrome.

Rhodopseudomonas viridis is a photosynthetic prokaryote with a single type of photosystem. The reaction center (145 kD) of the R. viridis photosystem is localized in the plasma membrane of these photosynthetic bacteria and is composed of four different polypeptides, designated L (273 amino acid residues), M (323 residues), H (258 residues), and cytochrome (333 amino acid residues). L and M each consist of five membrane-spanning a-helical segments; H has one such helix, the majority of the protein forming a globular domain in the cytoplasm (Figure 22.16). The cytochrome subunit contains four heme groups; the N-terminal amino acid of this protein is cysteine. This cytochrome is anchored to the periplasmic face of the membrane via the hydrophobic chains of two fatty acid groups that are esterified to a glyceryl moiety joined via a thioether bond to the Cys (Figure 22.16). L and M each bear two bacteriochlorophyll molecules (the bacterial version of Chl) and one bacteriopheophytin. L also has a bound quinone molecule, Q_A. Together, L and M coordinate an Fe atom. The photochemically active species of the R. viridis reaction center, P870, is composed of two bacteriochlorophylls, one contributed by L and the other by i.

Photosynthetic Electron Transfer in the R. viridis Reaction Center

The prosthetic groups of the R. viridis reaction center (P870, BChl, BPheo, and the bound quinones) are fixed in a spatial relationship to one another that favors photosynthetic e^- transfer (Figure 22.16). Photoexcitation of P870 (creation of P870*) leads to e^- loss (P870⁺) via electron transfer to the nearby bacteriochlorophyll (BChl).The e^- is then transferred via the L bacteriopheophytin (BPheo) to Q_A, which is also an L prosthetic group. The corresponding site on M is occupied by a loosely bound quinone, Q_B, and electron transfer from Q_A to Q_B takes place. An interesting aspect of the system is that no electron transfer occurs through M, even though it has components apparently symmetrical to and identical with the L e^- transfer pathway.
      The reduced quinone formed at the Q_B site is free to diffuse to a neighboring cytochrome b/cytochrome c₁ membrane complex, where its oxidation is coupled to H⁺ translocation (and, hence, ultimately to ATP synthesis) (Figure 22.17). Cytochrome c₂, a periplasmic protein, serves to cycle electrons back to P870⁺ via the four hemes of the reaction center cytochrome subunit. A specific tyrosine residue of L (Try¹⁶²) is situated between P870 and the closest cytochrome heme. This Tyr is the immediate e^- donor to P870⁺ and completes the light-driven electron transfer cycle. The structure of the R. viridis reaction center (derived from X-ray crystallographic data) is modeled in Figure 22.18.

Figure 22.17 · The R. viridis reaction center is coupled to the cytochrome b/c₁ complex through the quinone pool (Q). Quinone molecules are photoreduced at the reaction center Q_B site (2 e^- [2 hn] per Q reduced) and then diffuse to the cytochrome b/c₁ complex, where they are reoxidized. Note that e^- flow from cytochrome b/c₁ back to the reaction center occurs via the periplasmic protein cytochrome c₂. Note also that 3 to 4 H⁺ are translocated into the periplasmic space for each Q molecule oxidized at cytochrome b/c₁. The resultant proton-motive force drives ATP synthesis by the bacterial F₁F₀ ATP synthase. (Adapted from Deisenhofer, J., and Michel, H., 1989. The photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis. Science 245:1463.)

Figure 22.18 · Model of the R. viridis reaction center. (a, b) Two views of the ribbon diagram of the reaction center. M and L subunits appear in purple and blue, respectively. Cytochrome subunit is brown; H subunit is green. These proteins provide a scaffold upon which the prosthetic groups of the reaction center are situated for effective photosynthetic electron transfer. Panel (c) shows the spatial relationship between the various prosthetic groups (4 hemes, P870, 2 BChl, 2 BPheo, 2 quinones, and the Fe atom) in the same view as in (b), but with protein chains deleted.

Eukaryotic Reaction Centers: The Molecular Architecture of PSII

PSII of higher plants and green algae contains more than 20 subunits and is considerably more complex than the R. viridis reaction center. Nevertheless, the R. viridis reaction center is a fairly good model for the core structure of PSII. P680 and its two equivalents of pheophytin (Pheo) are located on a pair of integral membrane proteins designated D1 (38 kD) and D2 (39.4 kD) (Figure 22.19). The tyrosine species D is Tyr¹⁶¹ in the D1 amino acid sequence. Complexed to D2 is a tightly bound plastoquinone molecule, Q_A. Electrons flow from P680* to Pheo on D1 and thence to Q_A on D2 and then on to a second plastoquinone situated in the Q_B site on D1. Electron transfer from Q_A and Q_B is assisted by an iron atom located between them. Each plastoquinone (PQ) that enters the Q_B site accepts two electrons derived from water and two H⁺ from the stroma before it is released into the membrane as the hydroquinone, PQH₂. The stoichiometry of the overall reaction catalyzed by PSII is 2 H₂O+2 PQ+4 hv ® O₂+2 PQH₂. A cytochrome species, cytochrome b₅₅₉, composed of two polypeptides (4.4 kD and 9.3 kD), is associated with PSII; its function is as yet unclear. Two chlorophyll-binding proteins (47 and 43 kD) harvest light and deliver exciton energy to P680. A Mn-containing extrinsic membrane protein, the OEC or oxygen-evolving complex (whose principal subunits are 33-, 23-, and 16-kD polypeptides) is located on the lumenal side of the thylakoid membrane.

Figure 22.19 · The molecular architecture of PSII. The core of the PSII complex consists of the two polypeptides (D1 and D2) that bind P680, pheophytin (Pheo), and the quinones, Q_A and Q_B. Additional components of this complex include cytochrome b₅₅₉, two additional intrinsic proteins (47 and 43 kD) that serve an accessory light-harvesting function, and an extrinsic protein complex that is essential to O₂ evolution.

Figure 22.20 · The molecular architecture of PSI. PsaA and PsaB constitute the reaction center dimer, an integral membrane complex; P700 is located at the lumenal side of this dimer. PsaC, which bears Fe-S centers F_A and F_B, and PsaD, the interaction site for ferredoxin, are on the stromal side of the thylakoid membrane. PsaF, which provides the plastocyanin interaction site, is on the lumenal side. (Adapted from Golbeck, J. H., 1992. Annual Review of Plant Physiology and Plant Molecular Biology 43:293-324.)

The Molecular Architecture of PSI

The structure of PSI from the cyanobacterium Synechococcus elongatus has been solved by X-ray crystallography, and this structure shows strong similarities to the R. viridis reaction center and our emerging view of the eukaryotic PSII, both discussed earlier. Because of direct correlations with information about eukaryotic PSI, this cyanobacterial PSI provides a general model for all P700-dependent photosystems (Figure 22.20). Although PSI consists of 11 different protein subunits, all the electron-transferring prosthetic groups essential to PSI function are localized to just three polypeptides. Two of these, PsaA and PsaB (83 kD each), compose the reaction center heterodimer, a structural pattern that now seems universal in photosynthetic reaction centers. PsaA and PsaB each have 11 transmembrane a-helices. PSI has approximately 100 chlorophyll molecules, including the two composing P700 and two positioned some 16 Å from P700, one of which functions as A₀, the immediate e^- acceptor for P700 (Figure 22.20). Quinones are found in association with PSI, including one that functions as A₁, an intermediate e^- carrier. The Fe-S center designated F_x bridges PsaA and PsaB. The third protein, PsaC (9 kD), bears two additional Fe-S clusters designated F_A and F_B ; PsaC (along with two other proteins designated PsaD and PsaE) lies on the stromal face of the reaction center complex. PsaD is the site of ferredoxin binding in eukaryotic PSI systems. PsaF, with three transmembrane a-helices, provides the interaction site for plastocyanin (on the lumenal side of the membrane).
      The overall structure of S. elongatus PSI thus consists of a core reaction center surrounded by and connected to a large Chl-based antenna system. Three equivalents of such PSI complexes occur together to form a trimeric structure. Photochemistry begins with exciton absorption at P700, almost instantaneous electron transfer and charge separation (P700⁺:A₀^- ), followed by transfer of the electron from A₀ to A₁ and on to F_x and then F_A/F_B, where it is used to reduce a ferredoxin molecule at the “stromal” side of the membrane. The positive charge at P700⁺ and the e^- at F_A/F_B represent a charge separation across the membrane, an energized condition created by light.

22.6 × The Quantum Yield of Photosynthesis

The quantum yield of photosynthesis, the amount of product formed per equiva­lent of light input, has traditionally been expressed as the ratio of CO₂ fixed or O₂ evolved per quantum absorbed. At each reaction center, one photon or quantum yields one electron. Interestingly, an overall stoichiometry of one H⁺ translocated into the thylakoid vesicle for each photon has also been observed. Two photons per center would allow a pair of electrons to flow from H₂O to NADP⁺ (Figure 22.12), resulting in the formation of 1 NADPH and  O₂. If one ATP were formed for every 3 H⁺ translocated during photosynthetic electron transport,  ATP would be synthesized. More appropriately, 4 hv per center (8 quanta total) would drive the evolution of 1 O₂, the reduction of 2 NADP⁺, and the phosphorylation of  ATP.
      The energy of a photon depends on its wavelength, according to the equation E=hv=hc/l , where E is energy, c is the speed of light, and l is its wavelength. Expressed in molar terms, an Einstein is the amount of energy in Avogadro’s number of photons: E=Nhc/l . Light of 700-nm wavelength is the longest-wavelength and the lowest-energy light acting in the eukaryotic photo- systems discussed here. An Einstein of 700-nm light is equivalent in energy to approximately 170 kJ. Eight Einsteins of this light, 1360 kJ, theoretically generate 2 moles of NADPH,  moles of ATP, and 1 mole of O₂.

Photosynthetic Energy Requirements for Hexose Synthesis

The fixation of carbon dioxide to form hexose, the dark reactions of photosynthesis, requires considerable energy. The overall stoichiometry of this process (Eq. 22.3) involves 12 NADPH and 18 ATP. To generate 12 equivalents of NADPH necessitates the consumption of 48 Einsteins of light, minimally 170 kJ each. However, if the preceding ratio of  ATP per NADPH were correct, insufficient ATP for CO₂ fixation would be produced. Six additional Einsteins would provide the necessary two additional ATP. From 54 Einsteins, or 9180 kJ, one mole of hexose would be synthesized. The standard free energy change, DG°' , for hexose formation from carbon dioxide and water (the exact reverse of cellular respiration) is +2870 kJ/mol.

22.7 × Light-Driven ATP Synthesis—Photophosphorylation

Light-driven ATP synthesis, termed photophosphorylation, is a fundamental part of the photosynthetic process. The conversion of light energy to chemical energy results in electron-transfer reactions leading to the generation of reducing power (NADPH). Coupled with these electron transfers, protons are driven across the thylakoid membranes from the stromal side to the lumenal side. These proton translocations occur in a manner analogous to the proton translocations accompanying mitochondrial electron transport that provide the driving force for oxidative phosphorylation (Chapter 21). Figure 22.12 indicates that proton translocations can occur at a number of sites. For example, protons may be translocated by reactions between H₂O and PSII as a consequence of the photolysis of water. The oxidation-reduction events as electrons pass through the plastoquinone pool and the Q cycle are another source of proton translocations. The proton transfer accompanying NADP⁺ reduction also can be envisioned as protons being taken from the stromal side of the thylakoid vesicle. The current view is that two protons are translocated for each electron that flows from H₂O to NADP⁺. Because this electron transfer requires two photons, one falling at PSII and one at PSI, the overall yield is one proton per quantum of light.

The Mechanism of Photophosphorylation Is Chemiosmotic

The thylakoid membrane is asymmetrically organized, or “sided,” like the mitochondrial membrane. It also shares the property of being a barrier to the passive diffusion of H⁺ ions. Photosynthetic electron transport thus establishes an electrochemical gradient, or proton-motive force, across the thylakoid membrane with the interior, or lumen, side accumulating H⁺ ions relative to the stroma of the chloroplast. Like oxidative phosphorylation, the mechanism of photophosphorylation is chemiosmotic.
      A proton-motive force of approximately -250 mV is needed to achieve ATP synthesis. This proton-motive force, Dp, is composed of a membrane potential, DC, and a pH gradient, DpH (Chapter 21). The proton-motive force is defined as the free energy difference, DG, divided by, Faraday’s constant:

                               Dp=DG/=DC-(2.3RT/)DpH                                                         (22.5)

In chloroplasts, the value of DC is typically -50 to -100 mV, and the pH gradient is equivalent to about 3 pH units, so that -(2.3 RT/)DpH=2200 mV. This situation contrasts with the mitochondrial proton-motive force, where the membrane potential contributes relatively more to Dp than does the pH gradient.

Critical Developments in Biochemistry
Experiments with Isolated Chloroplasts Provided the First Direct Evidence for the Chemiosmotic Hypothesis
Experimental proof that the mechanism of photophosphorylation is chemiosmotic was provided in an elegant experiment by Andre Jagendorf and Ernest Uribe in 1966 (see figure). Jagendorf and Uribe reasoned that if photophosphorylation were indeed driven by an electrochemical gradient established by photosynthetic electron transfer reactions, they might artificially generate such a gradient by first incubating chloroplasts in an acid bath in the dark and then quickly raising the pH of the external medium. The resulting inequality in hydrogen ion electrochemical activity across the membrane should mimic the conditions normally found upon illumination of chloroplasts and should provide the energized condition necessary to drive ATP formation. To test this interpretation, Jagendorf and Uribe bathed isolated chloroplasts in a weakly acidic (pH 4) medium for 60 seconds, allowing the pH inside the chloroplasts to equilibrate with the external medium. The pH of the solution was then quickly raised to slightly	alkaline pH (pH 8), artificially creating a pH gradient across the thylakoid membranes. When ADP and Pi were added, ATP synthesis was observed as the pH gradient collapsed. This classic experiment was the first real proof of Mitchell’s chemiosmotic hypothesis and directed the scientific community to a greater acceptance of Mitchell’s interpretations. Mitchell’s chemiosmotic hypothesis for ATP synthesis now occupies the position of dogma as the weight of evidence has accumulated in its favor. Photophosphorylation then can be concisely summarized by noting that thylakoid vesicles accumulate H1 upon illumination and that the electrochemical gradient thus created represents an energized state that can be tapped to drive ATP synthesis. Collapse of the gradient—that is, equilibration of the ion concentration difference across the membrane—is the energy-transducing mechanism: the chemical potential of a concentration difference is transduced into synthesis of ATP.
The mechanism of photophosphorylation is chemiosmotic. In 1966, Jagendorf and Uribe experi­mentally demonstrated for the first time that establishment of an electrochemical gradient across the membrane of an energy-transducing organelle could lead to ATP synthesis. They equilibrated isolated chloroplasts for 60 seconds in a pH 4 bath, adjusted the pH to 8 in the presence of ADP and Pi, and allowed phosphorylation to proceed for 15 seconds. The entire experiment was carried out in the dark.

CF₁CF₀ ATP Synthase Is the Chloroplast Equivalent of the Mitochondrial F₁F₀ ATP Synthase

The transduction of the electrochemical gradient into the chemical energy represented by ATP is carried out by the chloroplast ATP synthase, which is highly analogous to the mitochondrial F₁F₀ ATP synthase. The chloroplast enzyme complex is called CF₁CF₀ ATP synthase, “C” symbolizing chloroplast. Like the mitochondrial complex, CF₁CF₀ ATP synthase is a heteromultimer of a, b, g, d, e, a, b, and c subunits (Chapter 21), consisting of a knoblike structure some 9 nm in diameter (CF₁) attached to a stalked base (CF₀) embedded in the thylakoid membrane. The mechanism of action of CF₁CF₀ ATP synthase in coup­ling ATP synthesis to the collapse of the pH gradient is similar to that of the mitochondrial ATP synthase described in Chapter 21. The mechanism of photophosphorylation is summarized schematically in Figure 22.21.

Figure 22.21 · The mechanism of photophosphorylation. Photosynthetic electron transport establishes a proton gradient that is tapped by the CF₁CF₀ ATP synthase to drive ATP synthesis. Critical to this mechanism is the fact that the membrane-bound components of light-induced electron transport and ATP synthesis are asymmetrical with respect to the thylakoid membrane so that vectorial discharge and uptake of H¹ ensue, generating the proton-motive force.

Cyclic and Noncyclic Photophosphorylation

Photosynthetic electron transport, which pumps H⁺ into the thylakoid lumen, can occur in two modes, both of which lead to the establishment of a transmembrane proton-motive force. Thus, both modes are coupled to ATP synthesis and are considered alternative mechanisms of photophosphorylation even though they are distinguished by differences in their electron transfer pathways. The two modes are cyclic and noncyclic photophosphorylation. Noncyclic photophosphorylation has been the focus of our discussion and is represented by the scheme in Figure 22.21, where electrons activated by quanta at PSII and PSI flow from H₂O to NADP⁺, with concomitant establishment of the proton-motive force driving ATP synthesis. Note that in noncyclic photophosphorylation, O₂ is evolved and NADP⁺ is reduced.

Cyclic Photophosphorylation

In cyclic photophosphorylation, the “electron hole” in P700⁺ created by electron loss from P700 is filled not by an electron derived from H₂O via PSII but by a cyclic pathway in which the photoexcited electron returns ultimately to P700⁺. This pathway is schematically represented in Figure 22.12 by the dashed line connecting F_B and cytochrome b₆. Thus, the function of cytochrome b₆ (b₅₆₃) is to couple the bound ferredoxin carriers of the PSI complex with the cytochrome b₆/cytochrome f complex via the plastoquinone pool. This pathway diverts the activated e^- from NADP⁺ reduction back through plastocyanin to re-reduce P700⁺ (Figure 22.22).

Figure 22.22 · The pathway of cyclic photophosphorylation by PSI. (Adapted from Arnon, D. I., 1984. Trends in Biochemical Sciences 9:258.)

      Proton translocations accompany these cyclic electron transfer events, so ATP synthesis can be achieved. In cyclic photophosphorylation, ATP is the sole product of energy conversion. No NADPH is generated, and, because PSII is not involved, no oxygen is evolved. The maximal rate of cyclic photophosphorylation is less than 5% of the rate of noncyclic photophosphorylation. Cyclic photophosphorylation depends only on PSI.

22.8 × Carbon Dioxide Fixation

As we began this chapter, we saw that photosynthesis traditionally is equated with the process of CO₂ fixation, that is, the net synthesis of carbohydrate from CO₂. Indeed, the capacity to perform net accumulation of carbohydrate from CO₂ distinguishes the phototrophic (and autotrophic) organisms from heterotrophs. Although animals possess enzymes capable of linking CO₂ to organic acceptors, they cannot achieve a net accumulation of organic material by these reactions. For example, fatty acid biosynthesis is primed by covalent attachment of CO₂ to acetyl-CoA to form malonyl-CoA (Chapter 25). Nevertheless, this “fixed CO₂” is liberated in the very next reaction, so no net CO₂ incorporation occurs.
      Elucidation of the pathway of CO₂ fixation represents one of the earliest applications of radioisotope tracers to the study of biology. In 1945, Melvin Calvin and his colleagues at the University of California, Berkeley, were investigating photosynthetic CO₂ fixation in Chlorella. Using ¹⁴CO₂, they traced the incorporation of radioactive ¹⁴C into organic products and found that the earliest labeled product was 3-phosphoglycerate (see Figure 18.13). Although this result suggested that the CO₂ acceptor was a two-carbon compound, further investigation revealed that, in reality, two equivalents of 3-phosphoglycerate were formed following addition of CO₂ to a five-carbon (pentose) sugar:

CO₂+5-carbon acceptor ^® [6-carbon intermediate] ^®
                                                                                   Two 3-phosphoglycerates

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

The five-carbon CO₂ acceptor was identified as ribulose-1,5-bisphosphate (RuBP), and the enzyme catalyzing this key reaction of CO₂ fixation is ribulose bisphosphate carboxylase/oxygenase, or, in the jargon used by workers in this field, rubisco. The name ribulose bisphosphate carboxylase/oxygenase reflects the fact that rubisco catalyzes the reaction of either CO₂ or, alternatively, O₂ with RuBP. Rubisco is found in the chloroplast stroma. It is a very abundant enzyme, constituting more than 15% of the total chloroplast protein. Given the preponderance of plant material in the biosphere, rubisco is probably the world’s most abundant protein. Rubisco is large: in higher plants, rubisco is a 550-kD heteromultimeric (a₈b₈) complex consisting of eight identical large subunits (55 kD) and eight small subunits (15 kD) (Figure 22.23). The large subunit is the catalytic unit of the enzyme. It binds both substrates (CO₂ and RuBP) and Mg²⁺ (a divalent cation essential for enzymatic activity). The small subunit modulates the activity of the enzyme, increasing k_cat more than 100-fold.²

Figure 22.23 · Schematic diagram of the subunit organization of ribulose bisphos hate carboxylase as revealed by X-ray crystallography. The enzyme consists of eight equiva­lents each of two types of subunits, large L (55 kD) and small S (15 kD). Clusters of four small subunits are located at each end of the symmetrical octamer formed by four L₂ dimers. (From Knight, S., Andersson, I., and Branden, C. I., 1990. Journal of Molecular Biology 215:113-160.)

The Ribulose-1,5-Bisphosphate Carboxylase Reaction

The addition of CO₂ to ribulose-1,5-bisphosphate results in the formation of an enzyme-bound intermediate, 2-carboxy,3-keto-arabinitol (Figure 22.24). This intermediate arises when CO₂ adds to the enediol intermediate generated from ribulose-1,5-bisphosphate. Hydrolysis of the C₂-C₃ bond of the intermediate generates two molecules of 3-phosphoglycerate. The CO₂ ends up as the carboxyl group of one of the two molecules.

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

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity

Rubisco exists in three forms: an inactive form designated E; a carbamylated, but inactive, form designated EC; and an active form, ECM, which is carbamylated and has Mg²⁺ at its active sites as well. Carbamylation of rubisco takes place by addition of CO₂ to its Lys²⁰¹ e-NH₂ groups (to give e¾NH¾COO^- derivatives). The CO₂ molecules used to carbamylate Lys residues do not become substrates. The carbamylation reaction is promoted by slightly alkaline pH (pH 8). Carbamylation of rubisco completes the formation of a binding site for the Mg²⁺ that participates in the catalytic reaction. Once Mg²⁺ binds to EC, rubisco achieves its active ECM form. Activated rubisco displays a K_m for CO₂ of 10 to 20 mM.³
      Substrate RuBP binds much more tightly to the inactive E form of rubisco (K_D=20 nM) than to the active ECM form (K_m for RuBP=20 mM). Thus, RuBP is also a potent inhibitor of rubisco activity. Release of RuBP from the active site of rubisco is mediated by rubisco activase. Rubisco activase is a regulatory protein; it binds to E-form rubisco and, in an ATP-dependent reaction, promotes the release of RuBP. Rubisco then becomes activated by carbamylation and Mg²⁺ binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco.

22.9 × The Calvin-Benson Cycle

The immediate product of CO₂ fixation, 3-phosphoglycerate, must undergo a series of transformations before the net synthesis of carbohydrate is realized. Among carbohydrates, hexoses (particularly glucose) occupy center stage. Glucose is the building block for both cellulose and starch synthesis. These plant polymers constitute the most abundant organic material in the living world, and thus, the central focus on glucose as the ultimate end product of CO₂ fixation is amply justified. Also, sucrose (a-D-glucopyranosyl-(1 ® 2)-b-D-fructofuranoside) is the major carbon form translocated out of leaves to other plant tissues. In nonphotosynthetic tissues, sucrose is metabolized via glycolysis and the TCA cycle to produce ATP.
      The set of reactions that transforms 3-phosphoglycerate into hexose is named the Calvin-Benson cycle (often referred to simply as the Calvin cycle) for its discoverers. The reaction series is indeed cyclic because not only must carbohydrate appear as an end product, but the 5-carbon acceptor, RuBP, must be regenerated to provide for continual CO₂ fixation. Balanced equations that schematically represent this situation are
            6(1) + 6(5) ® 12(3)
          12(3)  ®^{1(6) + 6(5)}
Net:     6(1)  ®¹⁽⁶⁾

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

The Enzymes of the Calvin Cycle

The Calvin cycle enzymes serve three important ends:

1. They constitute the predominant CO₂ fixation pathway in nature.
2. They accomplish the reduction of 3-phosphoglycerate, the primary product of CO₂ fixation, to glyceraldehyde-3-phosphate so that carbohydrate synthesis becomes feasible.
3. They catalyze reactions that transform 3-carbon compounds into 4-, 5-, 6-, and 7-carbon compounds.

      Most of the enzymes mediating the reactions of the Calvin cycle also participate in either glycolysis (Chapter 19) or the pentose phosphate pathway (Chapter 23). The aim of the Calvin scheme is to account for hexose formation from 3-phosphoglycerate. In the course of this metabolic sequence, the NADPH and ATP produced in the light reactions are consumed, as indicated earlier in Equation (22.3).
      The Calvin cycle of reactions starts with ribulose bisphosphate carboxylase catalyzing formation of 3-phosphoglycerate from CO₂ and RuBP and concludes with ribulose-5-phosphate kinase (also called phosphoribulose kinase), which forms RuBP (Figure 22.25 and Table 22.1). The carbon balance is given at the right side of the table. Several features of the reactions in Table 22.1 merit discussion. Note that the 18 equivalents of ATP consumed in hexose formation are expended in reactions 2 and 15: 12 to form 12 equivalents of 1,3-bisphosphoglycerate from 3-phosphoglycerate by a reversal of the normal glycolytic reaction catalyzed by 3-phosphoglycerate kinase, and six to phosphorylate Ru-5-P to regenerate 6 RuBP. All 12 NADPH equivalents are used in reaction 3. Plants possess an NADPH-specific glyceraldehyde-3-phosphate dehydrogenase, which contrasts with its glycolytic counterpart in its specificity for NADP over NAD and in the direction in which the reaction normally proceeds.

Balancing the Calvin Cycle Reactions To Account for Net Hexose Synthesis

When carbon rearrangements are balanced to account for net hexose synthesis, five of the glyceraldehyde-3-phosphate molecules are converted to dihydroxyacetone phosphate (DHAP). Three of these DHAPs then condense with three glyceraldehyde-3-P via the aldolase reaction to yield 3 hexoses in the form of fructose bisphosphate (Figure 22.25). (Recall that the DG°' for the aldolase reaction in the glycolytic direction is +23.9 kJ/mol. Thus, the aldolase reaction running “in reverse” in the Calvin cycle would be thermodynamically favored under standard-state conditions.) Taking one FBP to glucose, the desired product of this scheme, leaves 30 carbons, distributed as two fructose-6-phosphates, four glyceraldehyde-3-phosphates, and 2 DHAP. These 30 Cs are reorganized into 6 RuBP by reactions 9 through 15. Step 9 and steps 12 through 14 involve carbohydrate rearrangements like those in the pentose phosphate pathway (see Chapter 23). Reaction 11 is mediated by sedoheptulose-1,7-bisphosphatase. This phosphatase is unique to plants; it generates sedoheptulose-7-P, the seven-carbon sugar serving as the transketolase substrate. Likewise, phosphoribulose kinase carries out the unique plant function of providing RuBP from Ru-5-P (reaction 15). The net conversion accounts for the fixation of six equivalents of carbon dioxide into one hexose at the expense of 18 ATP and 12 NADPH.

Figure 22.25 · The Calvin-Benson cycle of reactions. The number associated with the arrow at each step indicates the number of molecules reacting in a turn of the cycle that produces one molecule of glucose. Reactions are numbered as in Table 22.1.

22.10 × Regulation of Carbon Dioxide Fixation

Figure 22.26 · Light regulation of CO₂ fixation prevents a substrate cycle between cellular respiration and hexose synthesis by CO₂ fixation. Because plants possess mitochondria and are capable of deriving energy from hexose catabolism (glycolysis and the citric acid cycle), regulation of photosynthetic CO₂ fixation by light activation controls the net flux of carbon between these opposing routes.

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

Light-Induced pH Changes in Chloroplast Compartments

Figure 22.27 · Light-induced pH changes in chloroplast compartments. Illumination of chloroplasts leads to proton pumping and pH changes in the chloroplast, such that the pH within the thylakoid space falls and the pH of the stroma rises. These pH changes modulate the activity of key Calvin cycle enzymes.

As discussed in Section 22.7, illumination of chloroplasts leads to light-driven pumping of protons into the thylakoid lumen, which causes pH changes in both the stroma and the thylakoid lumen (Figure 22.27). The stromal pH rises, typically to pH 8. Because rubisco and rubisco activase are more active at pH 8, CO₂ fixation is activated as stromal pH rises. Fructose-1,6-bisphosphatase, ribulose-5-phosphate kinase, and glyceraldehyde-3-phosphate dehydrogenase all have alkaline pH optima. Thus, their activities increase as a result of the light-induced pH increase in the stroma.

Light-Induced Generation of Reducing Power

Figure 22.28 · The pathway for light regulation of Calvin cycle enzymes. Light-generated reducing power (Fd_red=reduced ferredoxin) provides e² for reduction of thioredoxin (T) by FTR (ferredoxin-thioredoxin reductase). Several Calvin cycle enzymes have pairs of Cys residues that are involved in the disulfide-sulfhydryl transition between an inactive (-S-S-) form and an active (-SH HS-) form, as shown here. These enzymes include fructose-1,6-bisphosphatase (residues Cys¹⁷⁴ and Cys¹⁷⁹), NADP⁺-malate dehydrogenase (residues Cys¹⁰ and Cys¹⁵), and ribulose-5-P kinase (residues Cys¹⁶ and Cys⁵⁵).

Illumination of chloroplasts initiates photosynthetic electron transport, which generates reducing power in the form of reduced ferredoxin and NADPH. Several enzymes of CO₂ fixation, notably fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and ribulose-5-phosphate kinase, are activated upon reduction of specific Cys-Cys disulfide bonds to cysteine sulfhydryls. The reduced form of thioredoxin mediates this reaction. Thioredoxin is a small (12 kD) protein possessing in its reduced state a pair of sulfhydryls (-SH HS-), which upon oxidation form a disulfide bridge (-S-S-). Thioredoxin serves as the hydrogen carrier between NADPH or Fd_red and enzymes regulated by light (Figure 22.28).

Light-Induced Mg²⁺ Efflux from Thylakoid Vesicles

When light-driven proton pumping across the thylakoid membrane occurs, a concomitant efflux of Mg²⁺ ions from vesicles into the stroma is observed. This efflux of Mg²⁺ somewhat counteracts the charge accumulation due to H⁺ influx and is one reason why the membrane potential change in response to proton pumping is less in chloroplasts than in mitochondria (Eq. 22.5). Both ribulose bisphosphate carboxylase and fructose-1,6-bisphosphatase are Mg²⁺ -activated enzymes, and Mg²⁺ flux into the stroma as a result of light-driven proton pumping stimulates the CO₂ fixation pathway at these key steps. Activity measurements have indicated that fructose bisphosphatase may be the rate-limiting step in the Calvin cycle. The recurring theme of fructose bisphosphatase as the target of the light-induced changes in the chloroplasts implicates this enzyme as a key point of control in the Calvin cycle.

22.11 × The Ribulose Bisphosphate Oxygenase Reaction: Photorespiration

As indicated, ribulose bisphosphate carboxylase/oxygenase catalyzes an alternative reaction in which O₂ replaces CO₂ as the substrate added to RuBP (Figure 22.29a). The ribulose-1,5-bisphosphate oxygenase reaction diminishes plant productivity because it leads to loss of RuBP, the essential CO₂ acceptor. The K_m for O₂ in this oxygenase reaction is about 200 mM. Given the relative abundance of CO₂ and O₂ in the atmosphere and their relative K_m values in these rubisco-mediated reactions, the ratio of carboxylase to oxygenase activity in vivo is about 3 or 4 to 1.

Figure 22.29 · The oxygenase reaction of rubisco. (a) The reaction of ribulose bisphosphate carboxylase with O₂ in the presence of ribulose bisphosphate leads to wasteful cleavage of RuBP to yield 3-phosphoglycerate and phosphoglycolate. (b) Conversion of phosphoglycolate to glycine. In mitochondria, two glycines from photorespiration are converted into one serine plus CO₂. This step is the source of the CO₂ evolved in photorespiration. Transamina-tion of glyoxylate to glycine by the product serine yields hydroxypyruvate; reduction of hydroxypyruvate yields glycerate, which can be phosphorylated to 3-phosphoglycerate. 3-Phosphoglycerate can fuel resynthesis of ribulose bisphosphate by the Calvin cycle (Figure 22.25).

      The products of ribulose bisphosphate oxygenase activity are 3-phosphoglycerate and phosphoglycolate. Dephosphorylation and oxidation convert phosphoglycolate to glyoxylate, the a-keto acid of glycine (Figure 22.29b). Transamination yields glycine. Other fates of phosphoglycolate are also possible, including oxidation to CO₂, with the released energy being dissipated as heat. Obviously, agricultural productivity is dramatically lowered by this phenomenon, which, because it is a light-related uptake of O₂ and release of CO₂, is termed photorespiration. As we shall see, certain plants, particularly tropical grasses, have evolved means to circumvent photorespiration. These plants are more efficient users of light for carbohydrate synthesis.

22.12 × The C-4 Pathway of CO₂ Fixation

Tropical grasses are less susceptible to the effects of photorespiration, as noted earlier. Studies employing ¹⁴CO₂ as a tracer indicated that the first organic intermediate labeled in these plants was not a three-carbon compound but a four-carbon compound. Hatch and Slack, two Australian biochemists, first discovered this C-4 product of CO₂ fixation, and the C-4 pathway of CO₂ incorporation is named the Hatch-Slack pathway after them. The C-4 pathway is not an alternative to the Calvin cycle series of reactions or even a net CO₂ fixation scheme. Instead, it functions as a CO₂ delivery system, carrying carbon dioxide from the relatively oxygen-rich surface of the leaf to interior cells where oxygen is lower in concentration and hence less effective in competing with CO₂ in the rubisco reaction. Thus, the C-4 pathway is a means of avoiding photorespiration by sheltering the rubisco reaction in a cellular compartment away from high [O₂]. The C-4 compounds serving as CO₂ transporters are malate or aspartate.
      Compartmentation of these reactions to prevent photorespiration involves the interaction of two cell types, mesophyll cells and bundle sheath cells. The mesophyll cells take up CO₂ at the leaf surface, where O₂ is abundant, and use it to carboxylate phosphoenolpyruvate to yield OAA in a reaction catalyzed by PEP carboxylase (Figure 22.30). This four-carbon dicarboxylic acid is then either reduced to malate by an NADPH-specific malate dehydrogenase or transaminated to give aspartate in the mesophyll cells.⁴ The 4-C CO₂ carrier (malate or aspartate) then is transported to the bundle sheath cells, where it is decarboxylated to yield CO₂ and a 3-C product. The CO₂ is then fixed into organic carbon by the Calvin cycle localized within the bundle sheath cells, and the 3-C product is returned to the mesophyll cells, where it is reconverted to PEP in preparation to accept another CO₂ (Figure 22.30). Plants that use the C-4 pathway are termed C4 plants, in contrast to those plants with the conventional pathway of CO₂ uptake (C3 plants).

Figure 22.30 · Essential features of the compartmentation and biochemistry of the Hatch-Slack pathway of carbon dioxide uptake in C4 plants. Carbon dioxide is fixed into organic linkage by PEP carboxylase of mesophyll cells, forming OAA. Either malate (the reduced form of OAA) or aspartate (the aminated form) serves as the carrier transporting CO₂ to the bundle sheath cells. Within the bundle sheath cells, CO₂ is liberated by decarboxylation of malate or aspartate; the C-3 product is returned to the mesophyll cell. Formation of PEP by pyruvate;P_i dikinase reinitiates the cycle. The CO₂ liberated in the bundle sheath cell is used to synthesize hexose by the conventional rubisco-Calvin cycle series of reactions.

Intercellular Transport of Each CO₂ via a C-4 Intermediate Costs 2 ATP

The transport of each CO₂ requires the expenditure of two high-energy phosphate bonds. The energy of these bonds is expended in the phosphorylation of pyruvate to PEP (phosphoenolpyruvate) by the plant enzyme pyruvate-P_i di-kinase; the products are PEP, AMP, and pyrophosphate (PP_i). This represents a unique phosphotransferase reaction in that both the b- and g-phosphates of a single ATP are used to phosphorylate the two substrates, pyruvate and P_i. The reaction mechanism involves an enzyme phosphohistidine intermediate. The g-phosphate of ATP is transferred to P_i, whereas formation of E-His-P occurs by addition of the b-phosphate from ATP:

             E¾His + AMP_a¾P_b¾P_g + P_i ^®E¾His-P_b + AMP_a + P_gP_i

                        E¾His-P_b + pyruvate ^® PEP + E¾His

             Net:  ATP + pyruvate + P_i ^® AMP + PEP + PP_i

      Pyruvate-P_i dikinase is regulated by reversible phosphorylation of a threonine residue, the nonphosphorylated form being active. Interestingly, ADP is the phosphate donor in this interconvertible regulation. Despite the added metabolic expense of two phosphodiester bonds for each equivalent of carbon dioxide taken up, CO₂ fixation is more efficient in C4 plants, provided that light intensities and temperatures are both high. (As temperature rises, photorespiration in C3 plants rises and efficiency of CO₂ fixation falls.) Tropical grasses that are C4 plants include sugarcane, maize, and crabgrass. In terms of photosynthetic efficiency, cultivated fields of sugarcane represent the pinnacle of light-harvesting efficiency. Approximately 8% of the incident light energy on a sugarcane field appears as chemical energy in the form of CO₂ fixed into carbohydrate. This efficiency compares dramatically with the estimated photosynthetic efficiency of 0.2% for uncultivated plant areas. Research on photorespiration is actively pursued in hopes of enhancing the efficiency of agriculture by controlling this wasteful process. Only 1% of the 230,000 different plant species known are C4 plants; most are in hot climates.

22.13 × Crassulacean Acid Metabolism

In contrast to C4 plants, which have separated CO₂ uptake and fixation into distinct cells in order to minimize photorespiration, succulent plants native to semiarid and tropical environments separate CO₂ uptake and fixation in time. Carbon dioxide (as well as O₂) enters the leaf through microscopic pores known as stomata, and water vapor escapes from plants via these same openings. In nonsucculent plants, the stomata are open during the day, when light can drive photosynthetic CO₂ fixation, and closed at night. Succulent plants, such as the Cactaceae (cacti) and Crassulaceae, cannot open their stomata during the heat of day because any loss of precious H₂O in their arid habitats would doom them. Instead, these plants open their stomata to take up CO₂ only at night, when temperatures are lower and water loss is less likely. This carbon dioxide is immediately incorporated into PEP to form OAA by PEP carboxylase; OAA is then reduced to malate by malate dehydrogenase and stored within vacuoles until morning. During the day, the malate is released from the vacuoles and decarboxylated to yield CO₂ and a 3-C product. The CO₂ is then fixed into organic carbon by rubisco and the reactions of the Calvin cycle. Because this process involves the accumulation of organic acids (OAA, malate) and is common to succulents of the Crassulaceae family, it is referred to as crassulacean acid metabolism, and plants capable of it are called CAM plants.