Fundamentals of Plant Physiology [1 ed.] 9781605357904, 1605357901

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Fundamentals of

Plant Physiology Lincoln Taiz • Eduardo Zeiger Ian Max M0ller • Angus Murphy

Fundamentals of

Plant Physiology

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Fundamentals of

Plant Physiology Lincoln Taiz Professor Emeritus, University of California, Santa Cruz

Eduardo Zeiger Professor Emeritus, University of California, Los Angeles

Ian Max M0l ler Professor Emeritus, Aarhus University, Denmark

Angus Murphy Professor, University of Maryland

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SINAUER ASSOCIATES

NEW YORK OXFORD OXFORD UNIVERSITY PRESS

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Xylem conduits and their interconnections. (A) Structural comparison of tracheids and vessel elements. Tracheids are elongated, hollow, dead cells with highly lignified walls. The walls contain numerous pits-regions where secondary wall is absent but primary wall remains. The shapes of pits and the patterns of wall pitting vary with species and organ type. Tracheids are present in all vascular plants. Vessels consist of a stack of two or more vessel elements. Like tracheids, vessel elements are dead cells and are connected to one another by perforation plates-regions of the wall where pores or holes have developed. Vessels are connected to other vessels and to tracheids through pits. Vessels are found in most angiosperms and are lacking in most gymnosperms. (8) Tracheids (left) and vessels (right) form a series of parallel, interconnected pathways for water movement. (C) Scanning electron micrograph showing two vessels (running diagonally from lower left to upper right). Pits are visible on the side walls, as are the scalariform end walls between vessel elements. (C © Steve Gschmeissner/Science Source.) Figure 3.6

vessel elements Nonliving water-conducting cells with perforated end walls, found only in angiosperms and a small group of gymnosperms. perforation plate The perforated end wall of a vessel element in the xylem. vessel A stack of two or more vessel elements 1n the xylem.

movement of gas bubbles. Thus, pit membranes of both types play an important role in preventing the spread of gas bubbles, ca lled emboli, within the xylem. Vessel elements tend to be shorter and wider than tracheids and have perforated end walls that form a perforation plate at each end of the cell (See Figure 3.6A). Li ke tracheids, vessel e lements have pits on their lateral walls (see Figure 3.6C). Unlike in tracheids, the perforated end walls allow vessel elements to be stacked end to end to form a much longer conduit called a vessel (see Figure

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Water Balance of Plants (A) Conifers

(8) Other vascular plants Seco nda ry cell walls

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Figure 3 .7 Pit pairs. (A) Diagram of a coniferous bordered pit with the torus centered m the pit cavity (left) or lodged to one side of the cavity (right). When the pressure difference between two tracheids is small, the pit membrane lies close to the center of the bordered pit, allowing water to flow through the porous margo region of the pit membrane; when the pressure difference between two tracheids is large, such as when one has cav1tated and the other remains filled w ith water under tension, the pit membrane is displaced such that the torus becomes lodged against the overarching walls, thereby preventing the embolism from propagating between tracheids. (B) In contrast, the pit membranes of angiosperms and other nonconiferous vascular plants are relatively homogeneous in their structure. These pit membranes have very small pores compared with those of conifers, which prevents the spread of embolism but also imparts a significant hydraulic resistance. (A after Zimmermann 1983.)

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3.68). Vessels are multicellular conduits that vary in length both within and among species. Vessels range from a few centimeters in length to many meters. The vessel elements found at the extreme ends of a vessel lack perforations in their end walls and are connected to neighboring vessels via pits. Water moves through the xylem by pressure-driven bulk flow Pressure-driven bulk flow of water is responsible for long-distance transport of water in the xylem. It also accounts for much of the water flow through the soil and through the cell walls of plant tissues. In contrast to the diffusion of water across senupermeable membranes, pressure-driven bulk flow is independent of solute concentration gradients, as long as viscosity changes are negligible. If we consider bulk flow th rough a tube, the rate of flow depends on the radius (,) of the tube, the viscosity (,1) of the liquid, and the pressure gradient (~'¥/LU) that drives the flow. Jean Leonard Marie Poiseuille {1797-1869) was a French physician and physiologist, and the relation just described is given by one form of Poiseuille's equation:

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expressed in cubic meters per second (m 3 s-1). This equation tells us that pressure-driven bu lk flow is extremely sensitive to the radius of the tube. If the radius is doubled, the volume flow rate increases by a factor of16 (2°1). Vessel elements up to 500 µm in diameter, nearly an order of m.agnitude greater than the largest tracheid s, occur in the stemsofdimbingspecies. These large-diameter vessels permit vines to h·ansport large an, ounts of water despite the slenderness of their stems. Equation 3.2 describes water flow through a cylindrical tube and thus does not take into account the fact that xylem conduits are of fin ite length, such that water must cross many pit mem branes as it flows from the soil to the leaves. All else being equal, pit membranes should impede water Oow through single-celled (and t hus shorter) tracheids to a greater extent t han through multicellu lar (and thus longer) vessels. However, the pit mem branes of conifers are much more permeable to water than are those found in other plants, allowing conifers to grow into large trees despite producing onlytracheids. Water movement through the xylem requires a smaller pressure gradient than movement through living cells The xylem provides a pathway of low resistivity for water movement. Some numerical values will help you appreciate the extraordinary efficiency of the xylem.

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Chapter 3 We will calcu late the driving force required to move water through the xylen, at a typical velocity and compare it with the driving force t hat would be needed to move water through a pathway made up of living cells at the same rate. For the purposes of this comparison, we ,,vi II use a value of 4 m m s-1 for the xylem transport velocity and 40 µmas the vessel radius. This is a high velocity for such a narrow vessel, so it will tend to exaggerate the pressure gradient required to support water flow in the xylem. Using a version of Poiseuille's equation (see Equation 3.2), we can calculate the pressure gradient needed to move water at a velocity of 4 mn1 s-1 through an ideal tube with a uniform inner radius of 40 µm. The calculation g ives a va lue of 0.02 MPa m-1 . Of course, real xylem conduits have irregular inner wall surfaces, and water flow through perforation plates and pits adds resistance to water transport. Such deviations from the idea l increase the frictiona l drag: Measu rements show that t he actual resistance is greater by approximately a factor of 2. Let's now compare this value with the driving force that would be necessary to move water at the same velocity from cell to cell, crossing the plasma membrane each time. The driving force needed to move water through a layer of cells at 4 mn1 s- 1 is 2 x 10 8 MPa m- 1. This is ten orders of magnitude greater than the driving force needed to move water through our 40-µm-radius xylem vessel. Our calculation clearly shows t hat water flow t hrough the xylem is vastly more efficient than water flow across living cells. Nevertheless, the xylem can make a sign ificant contribution to the tota I resistance to water flow through the plant.

What pressure differe nce is needed to lift w ater 100 meters t o a treetop? \!\Tith the foregoing example in mind, let's see what pressure gradient is needed to move water up to the top of a very taU tree. The tallest trees in the world are the coas t redwoods (Sequoia se111pervire11s) of North America and the mountain ash (Eucalyptus rcgnans) of Australia. Individuals o f both species can exceed 100 m. If we think of the stem of a tree as a long pipe, we can estimate the pressure diffe rence that is needed to overcome the frictiona l drag of moving water from t he soil to the top of the tree by mu ltiply ing the pressure gradient needed to move the water by the height of the tree. The pressure gradients needed to move water through the xylem of very tall trees are on the order of 0.01 MPa m- 1, sma ller than in our previous exan,ple. If we multiply this pressure gradient by the height of the tree (0.01 MPa m -1 x 100 m), we fi nd that the total pressure difference needed to overcome the frictional resistance to water movement through the stem is equal to 1 MPa. In addition to frictional resistance, we must consider gravity. As described by Equation 2.4, for a height difference of 100 m, the difference in 'Pg is approximately 1 MPa. That is, '/1' is 1 MPa higher at the top of the tree than at the g round level. 8 So the other components of water potential must be 1 MPa more negative at the top of the tree to counter the effects of gravity. To allow t ranspiration to occur, the pressure gradien t due to gravity must be added to that required to cause water movement through the xylen1. Thus, we calculate that a pressure difference of roughly 2 MPa, from the base to the top branches, is needed to carry water up the talles t trees. Th e cohesion- tension theory explains w ate r tra nsport in the xyl em In theory, the pressure gradients needed to move water through the xylem cou ld res ult from the generation of positive pressures at the base of the plant or negative pressures at the top of the plant. We mentioned previously that some roots can develop positive hydrostatic pressure in their xylem. However, root pressure is typically less than 0.1 MPa and disappears with transpiration or when soils are

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Water Balance of Plants

75

dry, so it is clearly inadequate to move water up a tall tree. Furthermore, because root pressure is generated by the accumulation of ions in the xylem, relying on this for transporting water would require a mechanism for deal ing with these solu tes once the water evaporates from the leaves. Instead, the water at the top of a tree develops a large tension {a negative hydrostatic pressure), and this tension pulls water through the xylem. This mechanism, fi rst proposed toward the end of t he n ineteenth century, is called the cohesiontension theory of sap ascent because it requires the cohesive properties of water to sustain large tensions in the xylen1 water colunms. One can readily den1onstrate xylem tension by puncturing intact xylem th rough a drop of ink on the surface of a stem from a transpiring plant. \!\Then the tension in the xylem is relieved, the ink is drawn instantly into the xylem, resulting in visible streaks along the stem . The xylem tensions needed to pull water from the soil develop in leaves as a consequence of transpiration. How does t he loss of water vapor through open stomata result in the flow of water from t he soil? When leaves open their stomata to obtain CO 2 for photosynthesis, water vapor diffuses out of the leaves. This causes water to evaporate from the surfa ce of cell wa lls inside the leaves. In tum, the loss of water from the cell walls causes the water poten tial in the walls to decrease (Figure 3.8). This creates a gradient in water poten tial that causes water to flow towa rd the sites of evaporation . O ne hypothesis for how a loss of water from cell walls results in a decrease in water potential is that as water evaporates, the surface of the remain ing water is

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Figure 3.8 The driving force for wate r movement through plants originates in leaves. One hypothesis fo r how t his occurs is that as water evaporates fro m t he surfaces of mesophyll cells, water withdraws farther into the interstices of the cell wall. Because cellulose is hydrophilic (contact angle = 0°), the force resulting from surface tension causes a negative pressure in the liquid phase. As the radius of curvature of the air- water interfaces decreases, the hydrostatic pressure becomes more negative, as calculated from Equation 3.1. (Micrograph from Gunning and Steer 1996.)

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- In contrast, low-affinity transporters. Figure 6.12 shows the rate of sucrose uptake simple diffusion through open channels is ideally by soybean cotyledon protoplasts as a function of the externa l sucrose directly proportional to the concentration of the con centration . Uptake increases sharply with concentration a nd transported solute or, fo r an ion, to the diffe rence in begins to saturate at about 10 mM. At concentra tions above 10 mM, electrochemical potential across the membrane. uptake becomes lin ear and non saturable w ithin the concentration range tested. Inhibition of ATP synthesis with metabolic poisons blocks the saturable component, but not the linear one. The interpretation of the pattern shown in Figure 6.12 is that sucrose uptake at low concen trations is an energy-dependent, ca rrier-mediated process (H+-sucrose symport). At higher concentrations, sucrose enters the cells by di ffu sion down its concentration gradient and is therefore insensitive to metabolic poisons. Consistent with these data, both H+- sucrose symporters and sucrose facilitators (i.e., transport proteins that mediate transmembrane sucrose flux down its free-energy gradien t) have been iden ti fied at the molecula r level. ~nax

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- - - - Photosynthetically - - ---+• active radiation Figure 7.12 Red drop effect. The quantum yield of oxygen evolution (upper, black curve) falls off drastically for far-red light of wavelengths greater than 680 nm, indicating that far-red light alone is inefficient in driving photosynthesis. The slight dip near 500 nm reflects the somewhat lower efficiency of photosynthesis using light absorbed by accessory pigments, carotenords.

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Photosynthesis: Th e Li ght Reactions effective as any other photon in driving photosynthesis. However, the yield drops dramatically in the far-red region of chlorophyll absorption (greater than 680 nm). This drop cannot be caused by a decrease in chlorophyll absorption, because the guantum yield measures only light that has actually been absorbed . Thus, light with a wavelength greater than 680 nm is much less efficient than light of shorter wavelengths. Another puzzling experimental result was the enhancement effect, also discovered by En, erson. He n,easured the rate of photosynthesis separately with light of two different wavelengths and then used the two beams simultaneously. When red and far-red light were given together, the rate of photosynthesis was greater than the sum of the individual rates, a startling and surprising observation. These and others observations were eventually explained by experiments performed in the 1960s that led to the discovery that two photochemical complexes, now known as photosystems I and II (PSI and PSII), operate in series to carry out the early energy-storage reactions of photosynthesis. PSI preferentially absor bs far-red light of wavelengths g reater than 680 nm; PSII preferentially absorbs red light of 680 nm and is driven very poorly by far-red light. This wavelength dependence explains the enhancement effect and the red drop effect. Another difference bet ween the photosystems is that:

enhancement effect The synergistic (higher) effect of red and far-red light on the rate of photosynthesis, as compared with the sum of the rates when the two different wavelengths are delivered separately. photosystem I (PSI) A system of photoreactions that absorbs maximally far-red light (700 nm), oxidizes plastocyanin, and reduces ferredoxm. photosystem II (PSII) A system of photoreactions that absorbs maximally red light (680 nm). oxidizes water, and reduces plastoquinone. Operates very poorly under far-red light.

• PSI produces a strong reductant, capable of reducing NADP-, and a weak oxidant. • PSII produces a very strong oxidant, capable of oxidizing water, and a weaker reductant than t he one produced by PSI.

The reductant produced by PSII re-reduces the oxidant produced by PSI. These properties of the two photosystems are shown schematically in Figure 7.13. The scheme of photosynthes is depicted in Figure 7.13, cal led the Z (for zigzag) scheme, has become the basis for understanding 0 2-evolving (oxygenic) pho tosynthetic organisms. It accounts for the operation of two physically and chemically distinct photosystems (I and II), each wi th its own antenna Strong-reductant

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Low Figure 7.17 Funneling of excitation energy from the antenna system toward the reaction center. (A) The excited-state energy of pigments increases with distance from the reaction center; that is, pigments closer to the reaction center are lower in energy than those farther from the reaction center. This energy gradient ensures that exatation transfer toward the reaction center is energetically favorable and that excitation tra nsfer back out to the peripheral portions of the antenna 1s energetically unfavorable. (8) Some energy is lost as heat to the environment by this process, but under optimal conditions almost all the excitation energy absorbed in the antenna complexes can be delivered to the reaction center. The asterisks denote excited states.

As a result of this arrangement, when excitation is transferred, for exan1ple, from a chlorophyll b molecule absorbing maximally at 650 nm to a chlorophylJ a molecu le absorbing maxin1a lly at 670 nm, the d ifference in energy between these two excited chlorophylls is Jost to the environment as heat. For t he excitation to be transferred back to the chlorophyll b, the energy lost as heat would have to be resupplied. The probability of reverse transfer is therefore sn1a1ler sin1ply because thermal energy is not sufficient to n1ake u p the deficit between the lower-energy and higher-energy pigments. This effect gives the energy-trapping process a degree of directionality orirreversibil ity and makes the delivery of excitation to the reaction center more efficient. In essence, the system sacrifices some energy from each quantum so that nearly all of the quanta can be trapped by the reaction center.

light-harvesting complex II (LHCII} The most abundant antenna protein complex, associated primarily with photosystem II. chlorophyll alb antenna proteins Chlorophyll-containing proteins associated with one or the other of the two photosystems in eukaryotic organisms. Also known as light-harvesting complex proteins (LHC proteins).

Many antenna pigment- protein complexes have a common structural motif In all eukaryotic photosynthetic organisms that contain both ch lorophyll a and chlorophyll b, the most abundant antenna proteins are members of a large family of structurally related proteins. Some of these proteins are associated primarily with PSII and are called light-harvesting complex II (LHCII) proteins; others are associated with PSI and are called LHCI proteins. These an tenna complexes are also known as chlorophyll alb antenna proteins. The structure of one of the LHCII proteins has been determined (Figure 7 .18). The protein contains three a -helical regions and binds 14 chlorophyll a and b molecules, as welI as four carotenoids. The structure of the LHCT proteins is genera Uy similar to that of the LHCII proteins. All of these proteins have significant sequence similarity and are almost certainly descendants of a common ancestral protein.

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Photosynthesis: Th e Li ght Reactions

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(C) Lumenal side

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Figure 7.18 Structure of the trimeric LHCII antenna complex from higher plants. The antenna complex is a transmembrane pigment protein; each monomer contains three helical regions that cross the nonpolar part of the membrane. The trimeric complex 1s shown (A) from the stromal side, (8) from within the membrane, and (C) from the lumenal side. Gray, polypeptide; dark blue, Chi a; green, Chi b; dark orange, lutein; light orange, neoxanthin; yellow, violaxanthin; pink, lipids. (After Barros and Kuhlbrandt 2009.)

Light absorbed by carotenoids or ch lorophyll bin the LHC proteins is rapidly transferred to chlorophyll a and then to other antenna pigments that are in timately associated with the reaction center. The LHCil complex is also involved in regulatory processes, which we discuss later in the chapter.

Mechanisms of Electron Transport Some of the evidence that led to the idea of two photochem ical reactions operating in series was discussed earlier in this chapter. In this section we consider in detail the chemical reactions involved in electron transfer during photosynthesis. We discuss the excitation of chlorophyll by light and the reduction of the first electron acceptor, the flow of electrons through photosystems II and I, the oxidation of water as the primary source of electrons, and the reduction of the final electron acceptor (NADp+)_ The chemiosmotic mechanism that n1edia tes ATP synthesis is discussed in detail later in the chapter {see the section Proton Transport a11d ATP Synthesis in the Chloroplast). Electrons from chlorophyll travel through the carriers organized in the Z scheme Figure 7. 19 shows a current version of the Z schen1e, in which all the electron carriers known to function in electron flow from H 2O to NADp+ are arranged vertically at their midpoint redox potentials. Components known to react with each other are connected by arrows, so the Z scheme is really a synthesis of both kinetic and thermodynamic inforn, ation. The large vertical arrows represent the input of light energy into the system.

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1.5 ~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Photosystem II Phot osyst em I Figure 7.19 Detailed Z scheme for 0 2 -evolving photosynthetic organisms. The redox carriers are placed at their midpoint redox potentials (at pH 7). (1) The vertical arrows represent photon absorption by the reaction center chlorophylls: P680 for photosystem II (PSII) and P700 f or photosystem I (PSI). The excited PSII reaction center chlorophyll, P680*. transfers an electron to pheophytin (Pheo). (2) On the oxidizing side of PSII (to the left of the arrow Joining P680 with P680*), P680 oxidized by pheophytin after light excitation is re-reduced by Y z• which has received electrons from oxidation of water. (3) On the reducing side of PSII (to the right of the arrow joining P680 with P680*), pheophytin t ransfers electrons t o the acceptors PQA and PQ 8,

whteh are plastoquinones. (4) The cytochrome b5' complex transfers electrons t o plastocyanin (PC), a soluble protein, which in turn reduces P7QQ+ (oxidized P700). (5) The acceptor of electrons from P700* (Ac,) is thought to be a chlorophyll, and the next acceptor (A1) is a quinone. A series of membrane-bound iron-sulfur proteins (FeSx, FeSA• and FeS8) transfers electrons to soluble ferredoxin (Fd). (6) The soluble flavoprot ein ferredoxinNAOP+ reductase (FNR) reduces NAOP+ to NAOPH, which is used in the Calvin-Benson cycle to reduce CO 2 (see Chapter 8). The dashed line indicates cyclic electron flow around PSI. (After Blankenship and Prince 1985.)

Photons excite the specialized chlorophyll of t he reaction centers {P680 for PSII; P700 for PSI), and an electron is ejected. The electron then passes through a series of electron carriers and eventually reduces P700 (for electrons from PSII) or NADp+ (for electrons from PSI). Much of the following discussion describes the journeys of these electrons and the natu re of their carriers. Almost all the chemical processes that make up the light reactions of photosynthesis are carried out by fou r major protein complexes: PSII, the cytochrome b6f complex, PSI, an d ATP synthase. These four integral membrane complexes are vectorially oriented in the thylakoid membrane to function as follows (Figure 7.20; also see Figure 7.16): • PSII oxidizes water to 0 2 in the thylak oid lu men and in the process releases protons into the lu1nen. The reduced product of photosystem ll is plastohydroquinone (PQHi). • Cytochrome b6f oxidizes PQH2 molecules tha t were reduced by PSII and delivers elect rons to PSI via the soluble copper protein plastocyanin. The oxidation of PQH2 is coupled to proton transfer in to the lumen from t he stroma, generating a proton motive force. • PSI reduces NADr+ to NADPH in the stroma by the action of ferredoxin (Fd) and the flavoprotein ferredoxin- NADp+ reductase (FNR).

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Transfer of electrons and protons in the thylakoid membrane is carried out vectorially by four protein complexes (see Figure 7 .16B for structures). Water 1s oxidized and protons are released in the lumen by PSII. PSI reduces NADp+ to NADPH in the stroma, via the action of f erredoxin (Fd) and the flavoprotein ferredoxin-NADP+ reductase (FNR). Protons are also transported into the lumen by the action of the cytochrome

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complex and contribute to the electrochemical proton gradient. These protons must then diffuse to the ATP synt hase enzyme, where their d1ffus1on down the electrochemical potential gradient is used to synthesize ATP in the stroma. Reduced plastoquinone (PQH 2) and plastocyanin transfer electrons to cytochrome and to PSI, respectively. The dashed lines represent electron transfer; solid lines represent proton movement.

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• ATP synthase produces ATP as protons diffuse back through it from the lumen into the stron1.a.

Energy is captured when an excited chlorophyll reduces an electron acceptor molecule As discussed earlier, the function of light is to excite a specialized chlorophyll in the reaction center, either by direct absorption or, more frequently, via energy transfer from an antenna pigment. This excitation process can be envisioned as the promotion of an electron from the highest-energy fill ed orbital of the chlorophyll to the lowest-energy unfi lled orbital (Figure 7.21). The electron in the upper orbital is only loosely bound to the chlorophyll and is easily lost if a molecule that can accept the electron is nearby. The first reaction that converts electron energy into chemical energy-that is, the primary photochemical event- is the transfer of an electron from t he excited state of a chlorophyll in the reaction center to an acceptor molecule. An equivalent way to view this process is that the absorbed photon causes an elect ron rearrangen1.ent in the reaction center chlorophyll, followed by an electron transfer process in which part of the energy in the ph oton is captured in the form of redox energy. In,mediately a fter the photoche1nical event, the reaction cen ter chlorophyll is in an oxidized s tate (electron deficient, or positively charged), and t he nearby electron acceptor molecule is reduced (electron rich, or negatively charged). The system is now at a critica l juncture. The lower-energy orbital of the positively charged oxidized reaction center chlorophyll shown in Figure 7.21 has a vacancy

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Chapter 7

and can accept an elect ron. If the acceptor molecule dona tes its elect ron back to the reaction center chlorophyll, the system will be returned to the state that exis ted before the light Poor Good excita tion, and all the absorbed energy will be converted Acceptor oxidizing Donor reducing orbital orbital agent agent into heat. This wasteful recombination process, however, does not appear to occur to any substantia l degree in functioning reacLight tion centers. Instead, the acceptor transfers its extra electron Donor to a secondary acceptor and so on down t he electron transport Acceptor Good Poor orbital orbital oxidizing reducing chain. The oxidized reaction center of the chlorophyll that ~ ~ agent agent had donated an electron is re-reduced by a secondary donor, Ground-state Excited-state wh ich in turn is reduced by a tertiary donor. In plants, the chlorophyll chlorophyll ultima te electron donor is H 20, and the ultimate electron acceptor is NADp+ (see Figure 7.19). Figure 7.21 Orbital occupation diagram for the ground and The essence of photosynthetic energy storage is t hus the excited states of reaction center chlorophyll. In the ground initial t ransfer of an electron from an excited chlorophyll to an state the molecule is a poor reducing agent (loses electrons from a low-energy orbital) and a poor oxidizing agent (accepts acceptor molecule, followed by a very rapid series of secondary electrons only into a high-energy orbital). In the excited state chen1ical reactions that separate the positive and negative the situation is markedly different, and an electron can be charges. These secondary reactions separate the charges to lost from the high-energy orbital, making the molecule an opposite sides of t he thylakoid membran e in approximately extremely powerful reducing agent. This is the reason for the 200 picoseconds (1 picosecond = 10- 12 s). extremely negative excited-state redox potential shown by W ith the charges thus separated, the reversal reaction P680* and P700* in Figure 7 .19. The excited state can also is ma ny orders of magnitude slower, and t he energy has act as a strong oxidant by accepting an electron into the lower-energy orbital, although this pathway is not significant m been captured. Each of the secondary electron transfers reaction centers. (After Blankenship and Prince 1985.) is accompanied by a loss of some energy, thus making the process effectively irreversible. The quantum y ield for the production of stable products in p urified reaction centers from photosynthetic bacteria has been measured as 1.0; that is, every photon produces stable products, and no reversal reactions occur. Measured quantum requiremen ts for 0 2 production in higher plants under optimal conditions Oow-intensity light) indicate that the va lues for the primary photochemical events are also very close to 1.0. The structure of th e reaction center appears to be extremely fine- tuned for maximum rates of productive reactions and minimum rates of energy-wasting reactions. Redox properties of ground and excited states of reaction center chlorophyll

i

*

bleaching The loss of chlorophyll's characteristic absorbance due to its conversion mto another structural state, often by oxidation.

P700 The chlorophyll of the photosystem I reaction center that absorbs maximally at 700 nm in its neutral state. The P stands for pigment. P680 The chlorophyll of the photosystem II reaction center that absorbs maximally at 680 nm in its ne utral state.

t-

Th e reaction center chlorophylls of the two photosystems absorb at different w avelengths As discussed earlier in the chapter, PSI and PSII have distinct absorp tion characteristics. P recise measurements of absorption maxima a re made possible by optical changes in the reaction center chlorophylls in the reduced and oxidized states. The reaction center chlorophyll is transiently in an oxidized state after losing an electron and before being re-reduced by its electron donor. In the oxidized state, chlorophylls lose their characteristic strong light absorbance in the red region of the spectru m; t hey become bleached. It is therefore possible to monitor the redox state of these chlorophylls by time-resolved optical absorbance measurements in which this bleaching is monitored directly. Using such techniques, it was found that t he reaction center chlorophyll of PSI absorbs maximally at 700 nm in its reduced state. Accordingly, t his chlorophyll is named P700 (the P stands for pigment). The analogous optical transient of PSII is at 680 nm, so its reaction center chlorophyll is known as P680. T he prima ry donor of PSI, P700, is also a dimer of chlorophyll a molecules. PSII a lso contains a dimer of chlorophylls, although the primary electron transfer event may not originate from these pigments. In the oxidized state, reaction center ch lorophylls contain an unpaired electron.

.

"Cl

Photosyn th esis: Th e Li ght Reactions Molecules with unpaired electrons can often be detected by a magnetic-resonance technique known as electron spin resonance (ESR) spectroscopy. ESR studies, along with the spectroscopic measurements already described, have led to the discovery of many intermediate electron carriers in the photosynthetic electron transport system.

The PSII reaction center is a multi-subunit pigment-protein complex PSII is contained in a multi-subunit protein supercomplex. In higher plants, the supercomplex has two con1plete reaction centers and some antenna complexes. The core of the reaction center consists of two membra ne proteins known as D1 and 02, as well as other proteins, as shown in Figure 7.22. The primary donor ch lorophyll, additiona l chlorophylls, carotenoids, pheophytins, and plasto quinones (two electron acceptors described below) are

201

electron spin resonance (ESR) spectroscopy A magnetic-resonance technique that detects unpaired electrons in molecules. Instrumental measurements that identify intermediate electron carriers in t he photosynthetic or respiratory electron transport system.

(A)

Nonheme Fe

STROMA

Figure 7.22 Structure of t he PSII reaction center from the cyanobacterium Thermosynechococcus elongatus, resolved at 0.35 nm. The structure includes the D1 (yellow) and D2 (orange) core reaction center proteins, the CP43 (green) and CP47 (red) antenna proteins, cytochromes b 559 and c550, the extrinsic 33-kDa protein PsbO (dark blue), and the pigme nts and other cofactors. (A) Side view parallel to t he membrane plane. (B) View from t he lumenal surface, perpendicular to the plane of the membrane. (C) Detail of the Mn-containing oxygen-evolving complex (OEC). (A and B from Ferreira et al. 2004; C from Umena et al. 2011.)

Two-fold axis

(B)

rwo-iold syrnrn_etr\l

---

3)(IS

Psbl

,,

,

His 332

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.

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202

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Chapter 7

pheophytin A chlorophyll in which the central magnesium atom has been replaced by two hydrogen atoms. plastohydroquinone (PQH 2) The fully reduced form of plastoquinone. cytochrome b6 f complex A large multi-subunit protein complex containing two b-type hemes, one c-type heme (cytochrome f), and a Rieske iron-sulfur protein. A relatively immobile complex distributed equally between the grana and the stroma regions of the thylakoid membranes. cyt ochrome f A subunit in the cytochrome b 6 f complex that plays a role in electron transport between photosystems I and II. Rieske iron- sulfur protein A protein subunit in the cytochrome b 6 f complex, in which two iron atoms are bridged by two sulfur atoms, with two histidine and two cysteine ligands.

N

bound to the membrane proteins D1 and D2. Other protei ns serve as antenna complexes or are involved in oxygen evolution. Some, such as cytochrome b55 9' have no known function but may be involved in a protective cycle around PSil.

Water is oxidized to oxygen by PSII Water is oxidized according to the following chemical reaction: 2 H 20

-+

0 2 + 4 H• + 4 e-

(7.8)

This equation indicates that fo ur electrons are removed from two water molecules, generati ng an oxygen molecule and four hydrogen ions. Water is a very stable molecule. Oxidation of water to form molecular oxygen is very difficult: The photosynthetic oxygen-evolving complex (0 EC) is the o nly known biochemical system that carries out this reaction, and is the source of almost all the oxygen in Earth's atmosphere. The protons produced by water oxidation are released into the lumen of the thylakoid, not directly into the stromal compartment (see Figure 7.20). They are released into the lumen because of the vectorial nature of t he membrane and the fact that the oxygen-evolving complex is localized near the interior surface of the thylakoid membrane (see Figure 7.22A). These protons are eventua lly transferred from the lun1en to the stroma by t ranslocation through the ATP synthase. In this way, the pro tons released during water oxidation contribute to the electrochemical potential driving ATP formation (see Figure 7.20). lt has been known for many years that manganese (Mn) is an essential cofactor in the water-oxidizingprocess (see 01apter 4), and a classic hypothesis in photosynthesis research postulates that Mn ions undergo a series of oxidations-known as S states and labeled S0, S1, S2, S3, and S4- that are perhaps linked to H 2Ooxidation and the generation of 0 2 . Thjs hypothesis has received strong support from a variety of experiments, most notably X-ray absorption and ESR studies, both of which detect the manganese ions directly. Analytical experiments indicate that four Mn ions are associated with each oxygen-evolving complex. Other experiments have shown that c1- and Ca 2• ions are essential for 0 2 evolution. The detailed chemical n1echanism of the oxidation of water to 0 2 is not yet well understood, but with structural information now available, rapid progress is being made in this area. One e lectron carrier, generally identified as Yz, functions between theorygen-evolving complex and P680 (see Figure 7.19). To fu nction in this region, Yz nee ds to have a very strong tendency to retain its electrons. This species has been identified as a radical formed from a tyrosine residue in the D1 protein of the PSII reaction center.

Pheophytin and two quinones accept electrons from PSII Spectral and ESR studies have revea led the structura l arrangement of the carriers in the electron acceptor complex. Pheophytin, a chlorophyll in w hich the central magnesium ion has been replaced by two hydrogen ions, acts as an early acceptor in PSII. The structural change gives pheophytin chemica l and spectra l properties that are slightly different from those of Mg-based chlorophylls. Pheophytin passes electrons to a complex of two plastoquinones in close proximity to an iron ion. The two plastoquinones, PQA and PQa, are bound to the reaction center a nd rece ive electrons fron1 pheophytin in a sequential fashion. Transfer of the two electrons to PQ5 reduces it to PQ/-, and the reduced PQl- takes two protons fron1 the strom a side of the medium, yielding a fu lly reduced plastohydroqu inone (PQH 2) (Figure 7 .23). The PQH2 then dissociates from the reaction center complex and enters the hydrocarbon portion of the membrane, where it in turn transfers its electrons to the cytochrome b6f complex. Unlike the large protein complexes of the thylakoid membrane, PQH 2 is a s mall, non polar molecule that d iffuses readily i n the nonpolar core of the membrane bilayer.

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Photosynthesis: Th e Li ght Reactions (A)

203

Figure 7.23 Structure and reactions of plastoquinones that operate in PSII. (A) The plastoquinone consists of a qurno1d head and a long nonpolar tail that anchors it in the membrane. (B) Redox reactions of plastoquinone. The fully oxidized plastoquinone (PQ), anionic plastosemiquinone (PQ• ), and reduced plastohydroquinone (PQH 2) forms are shown; R represents the side chain. 0

Plastoquinone (B)

o•

0

H3 C

R

+

e-

H3 C

H3 C

OH R

+

1 e-

2 H+

"":::

R

+

H3C 0

H3C

/7

H3C

OH

0-

Plastoquinone

Plastosemiquinone

(PQ)

(PQ• )

Plastohydroquinone (PQH 2)

Electron flow through the cytochrome b6 f complex also transports protons

The cytochrome b 6f complex is a large mu lti-subunit protein with severa l prosthetic groups (Figure 7. 24) . It contains two b-type hemes and one c-type heme (cytochrome f ). In c-type cytochromes the heme is covalently attached to the peptide; in b-type cytochromes the chemically sim ilar protohe1ne group is not covalently attached. In addition, the complex contains a Rieske iron-sulfur protein (named for the scientist who discovered it), in which two iron ions are bridged by two s ulfide ions. The functional roles of all these cofactors are reasonably well understood, as described below. However, the cytochrome b6f complex also

PQH2

e-

e-

--------------LUMEN

/

L

------------· ,.. -------------2H+

'-......[2Fe- 2S]

e-

, cluster

Hemet

Figure 7.24 Structure of the cytochrome b6f complex from cyanobactena. The diagram on the right shows the arrangement o f the proteins and cofactors in the complex. Cytochrome b 6 protein is shown in blue, cytochrome fprotein in red, Rieske iron-sulfur protein in yellow, and other

smaller subunits in green and purple. On the left, the proteins have been omitted to more clearly show the posit ions of the cofactors. [2 Fe-2S] cluster, part of the Rieske iron-sulfur protein; PC, plastocyanin; PQ, plastoquinone; PQH 2 , plastohydroqu1none. (After Kurisu et al. 2003 .)

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Chapter 7

Q cycle A mechanism for oxidation of plastohydroquinone (reduced plastoquinone, also called plastoquinoO in chloroplasts and of ubihydroquinone (reduced ubiquinone, also called ubiquinol) in mitochondria.

FeSR An iron- and sulfur-containing subunit of the cytochrome b5' complex, involved in electron and proton transfer.

contains additional cofactors-including an additional heme group (called heme c11), a chlorophyll, and a carotenoid- whose functions are yet to be resolved. The s tructures of the cytochrome b6 f complex and the related cytochrome bc1 complex in the mitochondrial electron transport chain (see Chapter 11) suggest a mechan ism for electron and proton flow. The precise way by which electrons and protons flow through the cytochrome b6 f c01nplex is not yet fully understood, but a mechan ism known as the Q cycle accounts for most of the observations. In t his mechanism, plastohydroguinone (also ca lled plastoguinol) (PQH2) is oxidized, and one of the two electrons is passed along a linear electron transport chain toward PSI, while the other electron goes through a cyclic process that increases t he number of protons pumped across the membrane (Figure 7 .25). In the linear electron transport chain, the oxidized Rieske protein (FeSR) accepts an electron from PQH2 and transfers it to cytochrome f (see Figure 7.25A). Cytochrome f then transfers an electron to the blue-colored copper protein plastocyani:n (PC), which in turn reduces oxidized P700 of PSI. In the cyclic part of the process (see Figure 7.25B), the plastosemiquinone (see Figure 7.23) tra nsfers its other electron to one of the b-type hemes, releasing both of its protons to the lumenal side of the membrane. The first b-type heme transfers its electron through the second b-type heme to an oxidized plastoguinone molecule, reducing it to the semiquinone form near the stron1al surface of the con1plex. Another similar sequence of electron flow (see Figure 7.25B) fully reduces the plastoquinone, which picks up protons from the stromal side of the membrane and is released from the b6f complex as plastohydroquinone. (A) First QH 2 oxidize d Cytochrome b6' complex

STROMA

PQ PQ•~...., Heme Cn •, Cyt b 1 - - - - - - - - - ~

Thylakoid membrane

Figure 7.25 Mechanism of electron and proton transfer in the cytochrome b/ complex. This complex contains two b-type cytochromes (Cyt b), a c-type cytochrome (Cyt c, historically called cytochrome f), a Rieske Fe-S protein (FeSR), and two qumone ox1dat1on- reductmn srtes. (A) The noncyclic or linear processes: A plastohydroquinone (PQH 2) molecule produced by the action of PSII (see Figures 7 .20, 7 .23) IS oxidized near the lumenal side of the complex, transferring its two electrons to t he Rieske Fe-S protein and one of the b-type cytochromes and simultaneously expelling two protons to the lumen. The electron transferred to FeSR is passed to cytochrome f (Cyt f) and then to plastocyanin (PC). which reduces P700 of PSI. The reduced b-type cytochrome transfers an electron to the other b-type cytochrome, which reduces a plastoquinone (PQ) to the plastosemiquinone (PQ•) state (see Figure 7.23). (8) The cyclic processes: A second PQH 2 is oxidized, with one electron going from FeSR to PC and finally to P700. The second electron goes through the two b-type cytochromes and reduces the plastosemiquinone to the plastohydroquinone, at the same time picking up two protons from the stroma. Overall, four protons are transported across the membrane for every two electrons delivered to P700.

~ PQH2

~

0

,,•··► Cyt b ...••••

PQ .,_-7 •..

, - - - - - - ---,r-1 •►

®

,:(

(t

,t. •:

FeSR ······~yt f \

...___ __ _ __.,_,,. 2 H+ LUMEN

(B) Second QH2 oxidized STRO MA Thylakoid membrane

2 H+

2 H+

LUMEN

PC

· ••••,,'

o ···;lastocyanin

.

"Cl

Photosynthesis: Th e Li ght Reactions The overa II result of two turnovers of the complex is that two electrons are transferred to P700, two plastohydroguinones are oxidized to the plastoguinone form, and one oxidized plastoguinone is reduced to the plastohydroquinone form. In the process of oxidizing the plastohydroqu inones, four protons a re transferred from the stromal to the lumenal side of the membrane. By this n1echanisn1, electron flow connecting the acceptor side of the PSII reaction center to the donor side of the PSI reaction center also gives rise to an electrochemical potential across the n1embrane, due in part to H+ concentration differences on the two sides of the membrane. This electrochemical potential is used to power the synthesis of ATP. The cyclic electron flow through the cytochrome band plastoguinone increases t he number of protons pumped per electron beyond what could be achieved in a strictly linear seguence. Plastoquinone and plastocyanin carry electrons between photosystem II and photosystem I The location of the two photosystems at different sites on the thylakoid membranes (see Figure 7.16) reguires that at least one component is capable of moving along or within the n1embrane in order to deliver electrons produced by PSII to PSI. The cytochrome b6f complex is distributed egua Uy between the grana and the stron1a regions of the membranes, but its large size makes it unlikely that it is the mobile carrier. Instead, plastoquinone or plastocyanin or possibly both are thought to serve as mobile carriers to connect the two photosystems. Plastocyanin (PQ is a small (10.5 kDa), water-soluble, copper-containing protein that transfers electrons between the cytochron1e b6f con1plex and P700. This protein is found in the lumenal space (see Figure 7.25). Th e PSI reaction center reduces NADP• The PSI reaction center complex is a large multi-subunit complex (Figure 7 .26). Unlike in PSil, in which the antenna chlorophylls are associated with the reaction center but are present on separate pign1ent-proteins, a core antenna consisting of about 100 chlorophylls is an integral part of the PSI reaction center. The core antenna and P700 are bound to two proteins, PsaA and PsaB, with molecular masses in the range of 66 to 70 kDa. The PSI reaction center complex from pea contains four LHCI complexes in addition to the core structure similar to that found in cyanobacteria {see Figure 7.26). The tota I number of ch lorophyl I molecules in this con1plex is nearly 200. The core antenna pigments form a bowl surrounding the electron transfer cofac tors, which are in the center of the complex. In their reduced form, the electron carriers that function in the acceptor region of PSI are all extremely strong reducing agents. These reduced species are very unstable and thus difficult to identify. Evidence indicates that one of these early acceptors is a chlorophyll molecule, and another is a guinone species, phylloquinone, also known as vitamin K1. Additiona l electron acceptors indude a series of three membrane-associated iron- sulfur proteins, also known as Fe-S centers: FeSx, FeSA, and Fe S8 (see Figure 7.26). FeSx is part of the P700-binding protein; FeSA and FeS 8 reside on an 8- kDa protein that is part of the PSI reaction center con1plex. Electrons are transferred through FeS A and FeS 8 to ferred oxin (Fd), a small, water-soluble iron-sulfur protein (see Figures 7.19 and 7.26). The membrane-associated flavoprotein f erredoxin- NADP• reductase (FNR) reduces NADp+ to NADPH, thus completing the seguence of noncyclic elect ron transport that begins with the oxidation of water. Tn add ition to the reduction ofNAD-ri-, reduced ferredoxin produced by PSI has several other functions in the chloroplast, such as supplying reductant for nitrate reduction and regulating some of the carbon fixation enzymes (see Chapter 8).

205

plastocyan in (PC) A small (10.5 kDa), water-soluble, copper-containing protein that transfers electrons between the cytochrome b6 f complex and P700. This protein is found in the lumenal space. Fe-S centers Prosthetic groups consisting of inorganic iron and sulfur that are abundant in proteins in respiratory and photosynthetic electron transport. FeSx, FeSA' FeS 8 Membrane-bound iron-sulfur proteins that transfer electrons between photosystem I and ferredoxin. ferredoxin (Fd) A small, water-soluble iron-sulfur protein mvolved in electron transport in photosystem I. ferredoxin- NADP• re ductase (FNR) A membrane-assoaated flavoprotein that receives electrons from photosystem I and reduces NADp+ to NADPH.

N 0

u,

.

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206

N 0

Chapter 7

(A)

0)

(8)

Ferredoxin

STROMA

D

FeS cluster

Major protein PsaA

Major protein PsaB

L I

FeSx

,A,

Phylloquinone

A(_-+l'--l- Chlorophyl l J

HG

molecule

Minor protein PsaN N Light

Chlorophyll molecule

Protein ribbon for LHCI complex

Figure 7.26

Structure of PSI. (A) Structural m odel of the PSI react ion center from higher plants. Components of the PSI reaction center are organized around two major core proteins, PsaA and PsaB. Minor proteins PsaC to PsaN are labeled C to N. Electrons are transferred from plastocyanin (PC) to P700 (see Figures 7 .19 and 7 .20) and then to a chlorophyll molecule (Ao), to phylloquinone (A 1), to t he Fe-S centers FeSx, FeSA, and FeS8 , and finally to the soluble iron- sulfur protein ferredoxin (Fd). (B) Structure of the PSI reaction center complex from pea at 0.44 nm resolution, including the LHCI antenna complexes. This is viewed from the stromal side of t he membrane. (A after Buchanan et al. 2000; B after Nelson and Ben-Shem 2004.)

Cyclic electron flow generates ATP but no NADPH Some of the cytochrome b6f complexes are fou nd in the s troma region of the n1embrane, where PSI is located. Under certain cond itions, cyclic electron flow is known to occur from the reducing side of PSI via plastohydroquinone and the b6f complex and back to P700. This cyclic electron flow is coupled to pro ton pumping into the lumen, which can be used for ATP synthesis but does not oxidize water or reduce NADP" (see Figure 7.16B). Cyclic electron flow is especially important as an ATP source in the bundle sheath chloroplasts of some plants that carry out C4 carbon fixation (see Chapter 8). The molecular mechanism of cyclic electron flow is not well understood.

cyclic electron flow In photosystem I, the flow of electrons from the electron acceptors through the cytochrome b 6 f complex and back to P700, coupled to proton pumping into the lumen. This electron flow energizes ATP synt hesis but does not oxidize water or reduce NADp+_

Some herbicides block photosynthetic electron flow The use of herbicides to kill unwanted plants is widespread in modern agriculture. Many different classes of herbicides have been developed. Some act by blocking amino acid, carotenoid, or lipid biosynthesis or by disrupting cell division. Other herbicides, such as dichlorophenyldimethylurea (DCMU, also known as diuron) and paraquat, block photosynthetic electron flow (Figure 7.27). DCMU blocks electron flow at the qui none acceptors of PSII, by con1peting for the binding site of plastoquinone that is normally occupied by PQ8 . Paraquat accepts electrons from the early acceptors of PSI and then reacts with oxygen to form superoxide, 0 2•, a reactive oxygen s pecies that is very damaging to chloroplast components.

.

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Photosynthesis: Th e Li ght Reactions

207

-N+O-QN+-

CH 3

CH 3

2

er

Paraquat (methyl viologen) DCMU (diuron) (3.4-di ch loroph enyl di methylurea) (B)

Paraquat

" ::

f"NAD":J

!"(

NADPH

P680

Chemical structure and mechanism of action of two important herbicides. (A) Chemical structure of dichlorophenyldimethylurea (DCMU) and methyl viologen (paraquat, a cloride salt), two herbicides that block photosynthetic electron flow. DCMU is also known as diuron. (B) Sites of action of the two herbicides. DCMU blocks electron flow at the plastoquinone acceptors of PSII by competing for the binding site of plastoquinone. Paraquat acts by accepting electrons from the early acceptors of PSI.

Figure 7.27

Proton Transport and ATP Synthesis in the Chloroplast In the preceding sections we learned how captured light energy is used to reduce NAO? to NADPH. Another fraction of the captured light energy is used for light-dependent ATP synthesis, which is known as photophosphorylation. This process was discovered by Daniel Arnon and his coworkers in the 1950s. Under norma l cellular conditions, photophosphorylation requires electron flow, a lthough under some conditions e lectron flow and photophosphorylation can take place independently of each other. Electron flow without accompanying phosphorylation is said to be uncoupled . Tt is now widely accepted that photophosphorylation \Vorks via the chemiosmotic mechanism. This mechanism was first proposed in the 1960s by Peter Mitchell. The same general mechanism drives phosphorylation during aerobic respiration in bacteria and mitochondria (see Chapter 11), as well as the transfer of many ions and metabolites across membranes {see Chapter 6). Chemiosmosis appears to be a unifying aspect of membrane processes in all forms of life. In Chapter 6 we discussed the role of ATPases in chemiosmosis and ion transport at the cell's plasma membrane. The ATP used by the plasma rnembrane ATPase is synthesized by photophosphorylation in the chloroplast and oxidative phosphorylation in the mitochondrion. Here we are concerned w ith chemiosmosis and transmembrane proton concentration differences used to make ATP in the chloroplast. The basic principle of chemiosmosis is that ion concentration differences and electrical potential differences across membranes are sources of free energy

The formation of ATP from ADP and inorganic phosphate (Pi), catalyzed by the CFoF,ATP synthase and using light energy stored in the proton gradient across the thylakoid membrane. photophosphorylation

A process by which coupled reactions are separated in such a way that the free energy released by one reaction is not available to drive the other reaction. uncoupling

N 0 -.J

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208

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Chapter 7

co that can be used by the cell. As described by the second law of thermodynamics, any nonuniform distribution of matter or energy represents a source of energy. Differences in chemical potential of any molecular species whose concentrations are not the same on opposite sides of a membrane provide such a source of energy. The asyn1me tric nat ure of the photosynthetic n1embrane and the fact that proton flow from one side of the membrane to the other accomp anies electron flow were discussed earlier. The direction of proton translocation is such that the stroma becomes more alkaline (fewer H+ ions) and the lumen becomes more acidic (more H+ ions) as a result of electron transport (see Figures 7.20 and 7.25). Some of the early evidence supporting a chen1iosmotic n1echanism of photosynthetic ATP formation was provided by an elegant experiment carried out by Andre Jagendorf and coworkers (Figure 7.28). They suspended chloroplast thyla koids in a pH 4 buffer, and the buffer diffused across the membrane, causing the interior, as well as the exterior, of the thylakoid to equilibrate at this acidic pH. They then rapidly transferred t he t hylakoids to a pH 8 buffer, thereby creating a pH difference of four units across the thylakoid me1nbrane, with the inside acidic relative to the outside. They found that large an,ounts of ATP were formed from ADP and P; by this process, with no light input or electron transport. This result supports the predictions of the chemiosmotic hypothesis, described in the paragraphs that follow. Mitchell proposed that the total energy available for ATP synthesis, which he ca lled the proton motive force (6.p), is the sum of a proton chemical potential and a transmembrane electrica l potential. These two components of the proton motive force from the outside of the membrane to the inside are given by the following equation:

chemical potential The free energy associated with a substance that is available to perform work. proton motive force (PMF) The e nergetic effect of the electrochemical H+ gradient across a membrane, expressed in units of electrical potential.

(7.9) where ti£ is the transmembrane electrical potential, and pH1 - pH0 (or 6 pH) is the pH difference across the membrane. The constant of proportiona lity (at

(~

I

~