10 Cellular Energetics: photosynthesis

31 10 Cellular Energetics: photosynthesis

Photosynthesis We now shift our attention to photosynthesis, the second main process for synthesizing ATP. Photosynthesis in plants occurs in chloroplasts, large organelles found mainly in leaf cells. The principal end products generated from carbon dioxide and water are two carbohydrates that are polymers of hexose (six-carbon) sugars: sucrose, a glucose-fructose disaccharide, and leaf starch, a large insoluble glucose polymer that is the primary storage carbohydrate in higher plants. Leaf starch is synthesized and stored in the chloroplast. Sucrose is synthesized in the leaf cytosol from three-carbon precursors generated in the chloroplast; it is transported to nonphotosynthetic (nongreen) plant tissues (e.g., roots and seeds), which metabolize sucrose for energy by the pathways described in the previous sections. The overall reaction of oxygen-generating photosynthesis, is the reverse of the overall reaction by which carbohydrates are oxidized to CO2 and H2O. 6 CO2+ 6 H2O 6 O2 +C6H12O2 In effect, photosynthesis in chloroplasts produces energy-rich sugars that are broken down and harvested for energy by mitochondria during the process of cellular respiration. 32

Leaf Anatomy & Chloroplasts Chloroplasts are lens shaped with an approximate diameter of 5 μm and a width of ~2.5 μm, bounded by two membranes, which do not contain chlorophyll and do not participate directly in photosynthesis Like mitochondria, plant chloroplasts are bounded by a double membrane separated by an intermembrane space. Photosynthesis occurs on a third membrane, the thylakoid membrane, which is surrounded by the inner membrane and forms a series of flattened vesicles (thylakoids) that enclose a single interconnected luminal space. The green color of plants is due to the green color of chlorophyll, all of which is localized to the thylakoid membrane. A granum is a stack of adjacent thylakoids. The stroma is the space enclosed by the inner membrane and surrounding the thylakoids. The thylakoid membrane contains a number of integral membrane proteins to which are bound several important prosthetic groups and lightabsorbing pigments, most notably chlorophyll. Carbohydrate synthesis occurs in the stroma, the soluble phase between the thylakoid membrane and the inner membrane. 33

Photosynthesis NADP=NAD+Pi Antenna Reaction center 34

Photosynthesis The photosynthetic process in plants can be divided into four stages, each localized to a defined area of the chloroplast: o In stage 1, light is absorbed by light-harvesting complexes (LHC) and reaction center of photosystem II (PSII). The LHCs transfer the absorbed energy to the reaction centers, which use it, or the energy absorbed by directly from a photon, to oxidize water to molecular oxygen and generate high-energy electrons. o In stage 2, these electrons move down an electron transport chain, which uses either lipidsoluble (Q/QH2) or water-soluble (plastocyanin, PC) electron carriers to shuttle electrons between multiple protein complexes. As electrons move down the chain, they release energy that the complexes use to generate a proton-motive force and, after additional energy is introduced by absorption of light in photosystem I (PSI), to synthesize the highenergy electron carrier NADPH. o In stage 3, movement of proteins powered by the proton-motive force drives the synthesis of ATP by an F 0 F 1 ATP synthase. Stages 1 3 in plants take place in the thylakoid membrane of the chloroplast. o In stage 4, in the chloroplast stroma, the energy stored in NADPH and ATP is used to convert CO 2 initially into three-carbon molecules (glyceraldehyde 3-phosphate), a process known as carbon fixation. These molecules are then transported to the cytosol of the cell for conversion to hexose sugars in the form of sucrose. Glyceraldehyde 3-phosphate is also used to make starch within the chloroplast.all the reactions in stages 1 3 are catalyzed by multiprotein complexes in the thylakoid membrane. The enzymes that incorporate CO 2 into chemical intermediates and then convert them to starch are soluble constituents of the chloroplast stroma; the enzymes that form sucrose from three-carbon intermediates are in the cytosol. 35

Light absorbing pigments Chlorophyll a Heme If CHO Chlorophyll b (left) Structure of chlorophyll a, the principal pigment that traps light energy. o Electrons are delocalized among three of chlorophylls a's four central rings (yellow) and the atoms that interconnect them. o In chlorophyll, a Mg 2+ ion, rather than the Fe 3+ ion found in heme, sits at the center of the porphyrin ring and an additional five-membered ring (blue) is present; otherwise, the structure of chlorophyll is similar to that of heme, found in molecules such as hemoglobin and cytochromes. o The hydrocarbon phytol tail facilitates binding of chlorophyll to hydrophobic regions of chlorophyll-binding proteins. The CH 3 group (green) is replaced by a formaldehyde (CHO) group in chlorophyll b. When chlorophyll a (or any other molecule) absorbs visible light, the absorbed light energy raises electrons in the chlorophyll a to a higher-energy (excited) state. This state differs from the ground (unexcited) state largely in the distribution of the electrons around the C and N atoms of the porphyrin ring. Excited states are unstable, and the electrons return to the ground state by one of several competing processes. For chlorophyll a molecules dissolved in organic solvents such as ethanol, the principal reactions that dissipate the excited-state energy are the emission of light (fluorescence and phosphorescence) and thermal emission (heat). When the same chlorophyll a is bound in the unique protein environment of the reaction center, dissipation of excited-state energy occurs by a quite different process that is the key to photosynthesis. 36

Energy in lights Quantum mechanics established that light, a form of electromagnetic radiation, has properties of both waves and particles. When light interacts with matter, it behaves as discrete packets of energy (quanta) called photons. The energy of a photon, ε, is proportional to the frequency of the light wave: ε = hγ, where h is Planck's constant (1.58 10 34 cal s, or 6.63 10 34 J s) and γ is the frequency of the light wave. It is customary in biology to refer to the wavelength of the light wave, λ, rather than to its frequency γ. The two are related by the simple equation γ = c λ, where c is the velocity of light (3 10 10 cm/s in a vacuum). the energy in 1 mol of photons can be denoted by E = Nε, where N is Avogadro's number (6.02 10 23 molecules or photons/mol). Thus The energy of light is considerable, as we can calculate for light with a wavelength of 550 nm (550 10 7 cm), typical of sunlight: The rate of photosynthesis is greatest at wavelengths of light absorbed by three pigments. The action spectrum of photosynthesis in plants (the ability of light of different wavelengths to support photosynthesis) is shown in black. The energy from light can be converted into ATP only if it can be absorbed by pigments in the chloroplast. Comparison of the action spectrum with the individual absorption spectra suggests that photo-synthesis at 680 nm is primarily due to light absorbed by chlorophyll a; at 650 nm, to light absorbed by chlorophyll b; and at shorter wavelengths, to light absorbed by chlorophylls a and b and by carotenoid pigments, including β-carotene. 37

Photoelectron transport, the primary event in photosynthesis The absorption of a photon of light of wavelength 680 nm by one of the two special-pair chlorophyll a molecules in the reaction center increases its energy by 42 kcal/mol (the first excited state). Such an energized chlorophyll a molecule in a plant reaction center rapidly donates an electron to an intermediate acceptor, and the electron is rapidly passed on to the primary electron acceptor, quinone Q, near the stromal surface of the thylakoid membrane. This light-driven electron transfer, called photoelectron transport, depends on the unique environment of both the chlorophylls and the acceptor within the reaction center. Photoelectron transport, which occurs nearly every time a photon is absorbed, leaves a positive charge on the chlorophyll a close to the luminal surface of the thylakoid membrane (opposite side from the stroma) and generates a reduced, negatively charged acceptor (Q ) near the stromal surface. The Q produced by photoelectron transport is a powerful reducing agent with a strong tendency to transfer an electron to another molecule, ultimately to NADP +. The positively charged chlorophyll a+, a strong oxidizing agent, attracts an electron from an electron donor on the luminal surface to regenerate the original chlorophyll a. In plants, the oxidizing power of four chlorophyll a + molecules is used, by way of intermediates, to remove four electrons from 2 H 2 O molecules bound to a site on the luminal surface to form O 2 : 38

Light-harvesting complexes and photosystems Although chlorophyll a molecules within a reaction /center that are involved directly with charge separation and electron transfer are capable of directly absorbing light and initiating photosynthesis, they most commonly are energized indirectly by energy transferred to them from other lightabsorbing and energy-transferring pigments. These other pigments, which include many other chlorophyll molecules, are involved with absorption of photons and passing the energy to the chlorophyll a molecules in the reaction center. o o Some are bound to protein subunits that are considered to be intrinsic components of the photosystem and thus are called internal antennas; others are bound to proteins complexes that bind to but are distinct from the photosystem core proteins and are called light-harvesting complexes (LHCs). Even at the maximum light intensity encountered by photosynthetic organisms (tropical noontime sunlight), each reaction-center chlorophyll a molecule absorbs only about one photon per second, which is not enough to support photosynthesis sufficient for the needs of the plant. The involvement of internal antenna and LHCs greatly increases the efficiency of photosynthesis, especially at more typical light intensities, by increasing absorption of 680-nm light and by extending the range of wavelengths of light that can be absorbed by other antenna pigments. Diagram of the membrane of a cyanobacterium, in which the multiprotein light-harvesting complex (LHC) contains 90 chlorophyll molecules (green) and 31 other small molecules, all held in a specific geometric arrangement for optimal light absorption and energy transfer. Of the six chlorophyll molecules in the reaction center, two constitute the special-pair chlorophylls (ovals, dark green) that can initiate photoelectron transport when excited (blue arrow). Resonance transfer of energy (red arrows) rapidly funnels energy from absorbed light to one of two bridging chlorophylls (squares, dark green) and thence to chlorophylls in the reaction center. 39

Light-harvesting complexes and photosystems Photosystem core proteins and LHC proteins maintain the pigment molecules in the precise orientation and position optimal for light absorption and energy transfer, thereby maximizing the very rapid and efficient resonance transfer of energy from antenna pigments to reaction-center chlorophylls. 40

The Single Photosystem photosynthesis in the green and purple bacteria does not generate oxygen, whereas photosynthesis in cyanobacteria, algae, and higher plants does. This difference is attributable to the presence of two types of photosystem (PS) in the latter organisms: PSI reduces NADP + to NADPH, and PSII forms O 2 from H2O. the green and purple bacteria have only one type of photosystem, which cannot form O 2. The three-dimensional structures of the purple bacteria photosynthetic reaction centers have been determined and similar proteins and pigments compose photosystems I and II of plants and photosynthetic bacteria. The reaction center of purple bacteria contains three protein subunits (L, M, and H) located in the plasma membrane. Bound to these proteins are the prosthetic groups that absorb light and transport electrons during photosynthesis. The prosthetic groups include a special pair of bacteriochlorophyll a molecules equivalent to the reaction-center chlorophyll a molecules in plants, as well as several other pigments and two quinones, termed Q A and Q B, that are structurally similar to mitochondrial ubiquinone. 41

The Single Photosystem cytochrome bc1: electron transport complex similar in structure to complex III in mitochondria Cyclic electron flow generates a proton-motive force but no O 2. Blue arrows indicate flow of electrons; red arrows indicate proton movement. (Left) Energy absorbed directly from light or funneled from an associated LHC (not illustrated here) energizes one of the special-pair chlorophylls in the reaction center. Photoelectron transport from the energized chlorophyll, via accessory chlorophyll, pheophytin (Ph), and quinone A (Q A ), to quinone B (Q B ) forms the semiquinone Q and leaves a positive charge on the chlorophyll. Following absorption of a second photon and transfer of a second electron to the semiquinone, the quinone rapidly picks up two protons from the cytosol to form QH 2. (Center) After diffusing through the membrane and binding to the Q o site on the exoplasmic face of the cytochrome bc 1 complex, QH 2 donates two electrons and simultaneously gives up two protons to the external medium in the periplasmic space, generating a proton electrochemical gradient (proton-motive force). Electrons are transported back to the reaction-center chlorophyll via a soluble cytochrome, which diffuses in the periplasmic space. Note the cyclic path (blue) of electrons. Operation of a Q cycle in the cytochrome bc1 complex pumps additional protons across the membrane to the external medium. 42

Chloroplasts Photosystems Linear electron flow in plants, which requires both chloroplast photosystems PSI and PSII. PSI is driven by light of wavelength 700 nm or less; PSII, only by shorter-wavelength light (<680 nm) o In chloroplasts, PSI and PSII differ in their light-absorption maxima because of differences in their protein environments. Blue arrows indicate flow of electrons; red arrows indicate proton movement. LHCs are not shown. (Left) In the PSII reaction center, two sequential light-induced excitations of the same P 680 chlorophylls result in reduction of the primary electron acceptor Q B to QH 2. On the luminal side of PSII, electrons removed from H 2 O in the thylakoid lumen are transferred to P 680+, restoring the reaction-center chlorophylls to the ground state and generating O 2. (Center) The cytochrome bf complex then accepts electrons from QH 2, coupled to the release of two protons into the lumen. Operation of a Q cycle in the cytochrome bf complex translocates additional protons across the membrane to the thylakoid lumen, increasing the proton-motive force. (Right) In the PSI reaction center, each electron released from light-excited P 700 chlorophylls moves via a series of carriers in the reaction center to the stromal surface, where soluble ferredoxin (an Fe-S protein) transfers the electron to ferredoxin-nadp + reductase (FNR). This enzyme uses the prosthetic group flavin adenine dinucleotide (FAD) and a proton to reduce NADP +, forming NADPH. P 700+ is restored to its ground state by addition of an electron carried from PSII via the cytochrome bf complex and plastocyanin, a soluble electron carrier. 43

PSII reaction system Like the bacterial reaction center, the PSII reaction center contains two molecules of chlorophyll a (P 680 ), as well as two other accessory chlorophylls, two pheophytins, two quinones (Q A and Q B ), and one nonheme iron atom. These small molecules are bound to two proteins in PSII, called D 1 and D 2 When PSII absorbs a photon with a wavelength of <680 nm, it triggers the loss of an electron from a P 680 molecule, generating P 680+. As in photosynthetic purple bacteria, the electron is transported rapidly, possibly via an accessory chlorophyll, to a pheophytin, then to a quinone (Q A ), and then to the primary electron acceptor, Q B, on the outer (stromal) surface of the thylakoid membrane. The photochemically oxidized reaction-center chlorophyll of PSII, P 680+, is the strongest biological oxidant known. The reduction potential of P 680+ is more positive than that of water, and thus it can oxidize water to generate O 2 and H + ions. The splitting of H 2 O, which provides the electrons for reduction of P 680+ in PSII, is catalyzed by a three-protein complex, the oxygen-evolving complex, located on the luminal surface of the thylakoid membrane. The oxygen-evolving complex contains four manganese (Mn) ions as well as bound Cl and Ca 2+ ions; this is one of the very few cases in which Mn plays a role in a biological system. 44

Cells use multiple methods to prevent harms from Reactive Oxygen Species (ROS) α-tocopherol Even though the PSI and PSII photosystems with their associated light-harvesting complexes are remarkably efficient at converting radiant energy to useful chemical energy in the form of ATP and NADPH, they are not perfect. Depending on the intensity of the light and the physiologic conditions of the cells, a relatively small but significant amount of energy absorbed by chlorophylls in the light-harvesting antennas and reactions centers results in the chlorophyll being converted to an activated state called triplet chlorophyll. In this state, the chlorophyll can transfer some of its energy to molecular oxygen (O 2 ), converting it from its normal, relatively unreactive ground state, called triplet oxygen ( 3 O 2 ) to a very highly reactive (ROS) singlet state form, 1 O 2 (singlet oxygen). If the 1 O 2 is not quickly quenched by reacting with specialized 1 O 2 scavenger molecules, it will react with and usually damage nearby molecules. This damage can suppress the efficiency of thylakoid activity and is called photoinhibition. Carotenoids (polymers of unsaturated isoprene groups, including beta-carotene, which gives carrots their orange color) and α-tocopherol (a form of vitamin E) are hydrophobic small molecules that play important roles as 1 O 2 quenchers to protect plants. Singlet oxygen reacts with an alkene -C=C-CH- to form the allyl hydroperoxide HO-O-R (R = alkyl), which can then be reduced to the allyl alcohol. 45

Singlet and triplet oxygen Singlet oxygen (or 1 O 2 ) is the common name used for the diamagnetic form of molecular oxygen (O 2 ), which is less stable than the normal triplet oxygen. Because of its unusual properties, singlet oxygen can persist for over an hour at room temperature, depending on the environment. Because of differences in their electron shells, singlet and triplet oxygen differ in their chemical properties. Singlet oxygen is in the same quantum state as most molecules and thus reacts readily with them, thus 46 making singlet oxygen highly reactive.

The chaperone HSP70B helps PSII recover from photoinhibition Under intense illumination, photosystem PSII is especially prone to generating 1 O 2, whereas PSI will produce other ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals. The D1 subunit in the PSII reaction center is, even under low light conditions, subjected to almost constant 1 O 2 -mediated damage. The damaged reaction center moves from the grana to the unstacked regions of the thylakoid, where the D1 subunit is degraded by a protease and replaced by newly synthesized D1 proteins in what is called the D1 protein damage-repair cycle. The rapid replacement of damaged D1, which requires a high rate of D1 synthesis, helps the PSII recover from photoinactivation and maintain sufficient activity. (Right)The chaperone HSP70B helps PSII recover from photoinhibition after exposure to intense light. The unicellular green alga Chlamydomonas reinhardtii was genetically manipulated so that it had abnormally high or low levels of the chaperone protein HSP70B. The ability of the cells to recover PSII activity depends on the levels of HSP70B the more HSP70B available, the more rapid the recovery 47

Cyclic Electron Flow in plants In some circumstances cells must generate relative amounts of ATP and NADPH that differ from those produced by linear electron flow (e.g., greater ATP-to-NADPH ratio). To do this, they photosynthetically produce ATP from PSI without concomitant NADPH production. This is accomplished using a PSII-independent process called cyclic photophosphorylation. In the NAD(P)H-dehydrogenase (Ndh)-dependent pathway for cyclic electron flow, light energy is used by PSI to transport electrons in a cycle to generate a proton-motive force and ATP without oxidizing water. The NADPH formed via the PSI/ferredoxin/FNR instead of being used to fix carbon is oxidized by Ndh. The released electrons are transferred to plastoquinone (Q) within the membrane to generate QH 2, which then transfers the electrons to the cytochrome bf complex, then to plastocyanin, and finally back to PSI, as is the case for the linear electron flow pathway. Cyclic electron flow in plants, which generates a proton-motive force and ATP but no oxygen or net NADPH. 48

Regulation of linear versus cyclic electron flow 49

Regulation of linear versus cyclic electron flow In order for PSII, which is preferentially located in the stacked grana, and PSI, which is preferentially located in the unstacked thylakoid membranes, to act in sequence during linear electron flow, the amount of light energy delivered to the two reaction centers must be controlled so that each center activates the same number of electrons. This balanced condition is called state 1. If the two photosystems are not equally excited, then cyclic electron flow occurs in PSI and PSII becomes less active (state 2). Variations in the wavelengths and intensities of ambient light (as a consequent of the time of day, clouds, etc.) can change the relative activation of the two photosystems, potentially upsetting the appropriate relative amounts of linear and cyclic electron flow necessary for production of optimal ratios of ATP and NADPH. One mechanism for regulating the relative contributions of PSI and PSII entails redistributing the light-harvesting complex LHCII between the two photosystems. The more LHCII associated with a particular photosystem, the more efficiently that system will be activated by light and the greater its contribution to electron flow. (top figure) In normal sunlight, PSI and PSII are equally activated, and the photosystems are organized in state 1. In this arrangement, light-harvesting complex II (LHCII) is not phosphorylated and is tightly associated with the PSII reaction center in the grana. As a result, PSII and PSI can function in parallel in linear electron flow. (bottom figure) When light excitation of the two photosystems is unbalanced (e.g., too much via PSII), LHCII becomes phosphorylated, dissociates from PSII, and diffuses into the unstacked membranes, where it associates with PSI and its permanently associated LHCI. In this alternative supramolecular organization (state 2), most of the absorbed light energy is transferred to PSI, supporting cyclic electron flow and ATP production but no formation of NADPH and thus no CO 2 fixation. 50

CO 2 Metabolism During Photosynthesis Chloroplasts perform many metabolic reactions in green leaves. In addition to CO 2 fixation incorporation of gaseous CO 2 into small organic molecules and then sugars the synthesis of almost all amino acids, all fatty acids and carotenes, all pyrimidines, and probably all purines occurs in chloroplasts. However, the synthesis of sugars from CO 2 is the most extensively studied biosynthetic pathway in plant cells. We first consider the unique pathway, known as the Calvin cycle (after discoverer Melvin Calvin), that fixes CO 2 into three-carbon compounds, powered by energy released during ATP hydrolysis and oxidation of NADPH. The enzyme ribulose 1,5-bisphosphate carboxylase, or rubisco, fixes CO 2 into precursor molecules that are subsequently converted into carbohydrates. This enzyme adds CO2 to the five-carbon sugar ribulose 1,5-bisphosphate to form two molecules of the three-carboncontaining 3-phosphoglycerate (as shown in the figure). Because the catalytic rate of rubisco is quite low, many copies of the enzyme are needed to fix sufficient CO 2. Indeed, this enzyme makes up almost 50 percent of the chloroplast protein and is believed to be the most abundant protein on earth. 51

After its formation in the chloroplast stroma, glyceraldehyde 3-phosphate is transported to the cytosol in exchange for phosphate. The final steps of sucrose synthesis occur in the cytosol of leaf cells. The fixation of six CO 2 molecules and the net formation of two glyceraldehyde 3-phosphate molecules require the consumption of 18 ATPs and 12 NADPHs, generated by the lightrequiring processes of photosynthesis.

In the cytosol, an exergonic series of reactions converts glyceraldehyde 3-phosphate to fructose 1,6-bisphosphate. Two molecules of fructose 1,6-bisphosphate are used to synthesize one of the disaccharide sucrose. Some glyceraldehyde 3-phosphate (not shown here) is also converted to amino acids and fats, compounds essential for plant growth. 53

Light and thioredoxin Stimulate CO 2 Fixation The Calvin cycle enzymes that catalyze CO 2 fixation are rapidly inactivated in the dark, thereby conserving ATP that is generated in the dark for other synthetic reactions, such as lipid and amino acid biosynthesis. One mechanism that contributes to this control is the ph dependence of several Calvin cycle enzymes. o Because protons are transported from the stroma into the thylakoid lumen during photoelectron transport (see p48), the ph of the stroma increases from 7 in the dark to 8 in the light. The increased activity of several Calvin cycle enzymes at the higher ph promotes CO 2 fixation in the light. A stromal protein called thioredoxin (Tx) also plays a role in controlling some Calvin cycle enzymes. o In the dark, thioredoxin contains a disulfide bond; in the light, electrons are transferred from PSI, via ferredoxin, to thioredoxin, reducing its disulfide bond: o Reduced thioredoxin then activates several Calvin cycle enzymes by reducing disulfide bonds in them. o In the dark, when thioredoxin becomes reoxidized, these enzymes are reoxidized and so inactivated. Thus these enzymes are sensitive to the redox state of the stroma, which in turn is light sensitive an elegant mechanism for regulating enzymatic activity by light. 54

CO 2 fixation and photorespiration As noted above, rubisco catalyzes the incorporation of CO 2 into ribulose 1,5-bisphosphate. It can also catalyze a second, distinct, and competing reaction with the same substrate ribulose 1,5- bisphosphate but with O 2 in place of CO 2 as a second substrate. The products of this reaction are one molecule of 3-phosphoglycerate and one molecule of the two-carbon compound phosphoglycolate. The pathway initiated by the second reaction with O 2 is called photorespiration a process that takes place in light, consumes O 2, and converts ribulose 1,5-bisphosphate in part to CO 2. The first (carbon-fixing) reaction is favored when the ambient CO 2 concentration is relatively high, whereas the second is favored when CO 2 is low and O 2 relatively high. 55

CO 2 fixation and photorespiration (Above figure) These competing pathways are both initiated by ribulose 1,5-bisphosphate carboxylase (rubisco), and both utilize ribulose 1,5-bisphosphate. CO2 fixation, pathway 1, is favored by high CO 2 and low O 2 pressures; photorespiration, pathway 2, occurs at low CO 2 and high O 2 pressures (that is, under normal atmospheric conditions). Phosphoglycolate is recycled via a complex set of reactions that take place in peroxisomes and mitochondria, as well as chloroplasts. The net result: for every two molecules of phosphoglycolate formed by photorespiration (four C atoms), one molecule of 3-phosphoglycerate is ultimately formed and recycled and one molecule of CO2 is lost. As the above figure shows, photorespiration is wasteful to the energy economy of the plant: it consumes ATP and O 2, and it generates CO 2 without fixing carbon. Indeed, when CO 2 is low and O 2 is high, much of the CO 2 fixed by the Calvin cycle is lost as the result of photorespiration. Excessive photorespiration could become a problem for plants in a hot, dry environment, because they must keep the gas-exchange pores (stomata) in their leaves closed much of the time to prevent excessive loss of moisture. As a consequence, the CO 2 level inside the leaf can fall below the Km of rubisco for CO 2. Under these conditions, the rate of photosynthesis is slowed, photorespiration is greatly favored, and the plant might be in danger of fixing inadequate amounts of CO 2. Corn, sugarcane, crabgrass, and other plants that can grow in hot, dry environments have evolved a way to avoid this problem by utilizing a two-step pathway of CO 2 fixation in which a CO 2 -hoarding step precedes the Calvin cycle. The pathway has been named the C4 pathway cause the first compound in this pathway are four-carbon compounds, such as oxaloacetate and malate, rather than the three-carbon molecules that initiate the Calvin cycle (C3 pathway). 56

Leaf anatomy of C4 plants The C4 pathway involves two types of cells: mesophyll cells, which are adjacent to the air spaces in the leaf interior, and bundle sheath cells, which surround the vascular tissue and are sequestered away from the high oxygen levels to which mesophyll cells are exposed. Bundle sheath cells line the vascular bundles containing the xylem and phloem. Mesophyll cells, which are adjacent to the substomal air spaces, can assimilate CO 2 into four-carbon molecules at low ambient CO 2 and deliver it to the interior bundle sheath cells. Bundle sheath cells contain abundant chloroplasts and are the sites of photosynthesis and sucrose synthesis. Sucrose is carried to the rest of the plant via the phloem. In C3 plants, which lack bundle sheath cells, the Calvin cycle operates in the mesophyll cells to fix CO 2. 57

C4 pathway In the mesophyll cells of C4 plants, phosphoenolpyruvate, a three-carbon molecule derived from pyruvate, reacts with CO2 to generate oxaloacetate, a four-carbon compound. The enzyme that catalyzes this reaction, phosphoenolpyruvate carboxylase, is found almost exclusively in C4 plants and unlike rubisco is insensitive to O 2. The overall reaction from pyruvate to oxaloacetate involves the hydrolysis of one ATP and has a negative ΔG. Therefore, CO 2 fixation will proceed even when the CO 2 concentration is low. The oxaloacetate formed in mesophyll cells is reduced to malate, which is transferred by a special transporter to the bundle sheath cells, where the CO 2 released by decarboxylation enters the Calvin cycle. Because of the transport of CO 2 from mesophyll cells, the CO 2 concentration in the bundle sheath cells of C4 plants is much higher than it is in the normal atmosphere. The high CO 2 and reduced O 2 concentrations in the bundle sheath cells favor the fixation of CO 2 by rubisco to form 3-phosphoglycerate and inhibit the utilization of ribulose 1,5-bisphosphate in photorespiration. 58

Summary of aerobic oxidation of glucose and fatty acids 59

Review of e - transfer chain in aerobic oxidation As shown in the figure, CoQ accepts electrons released from NADH-CoQ reductase (complex I) or succinate-coq reductase (complex II) and donates them to CoQH2 cytochrome c reductase (complex III). Importantly, reduction and oxidation of CoQ are coupled to pumping of protons. Whenever CoQ accepts electrons, it does so at a binding site on the matrix (also called the cytosolic) face of the protein complex, always picking up protons from the medium there. Whenever CoQH 2 releases its electrons, it does so at a site on the intermembrane space (also called the exoplasmic) side of the protein complex, releasing protons into the intermembrane (or exoplasmic) fluid. Thus transport of each pair of electrons by CoQ is obligatorily coupled to movement of two protons from the matrix to the intermembrane space fluid. Figure: Electrons (blue arrows) flow through four major multiprotein complexes (I IV). Electron movement between complexes is mediated either by the lipid-soluble molecule coenzyme Q (CoQ, oxidized form; CoQH 2, reduced form) or the water-soluble protein cytochrome c (cyt c). The multiple protein complexes use the energy released from passing electrons to pump protons from the matrix to the intramembrane space (red arrows). 60

Summary of Photosynthesis NADP=NAD+Pi Antenna Reaction center 61

Review of linear e- transfer chain in photosynthesis Linear electron flow in plants, which requires both chloroplast photosystems PSI and PSII. PSI is driven by light of wavelength 700 nm or less; PSII, only by shorter-wavelength light (<680 nm) o In chloroplasts, PSI and PSII differ in their light-absorption maxima because of differences in their protein environments. Blue arrows indicate flow of electrons; red arrows indicate proton movement. LHCs are not shown. (Left) In the PSII reaction center, two sequential light-induced excitations of the same P 680 chlorophylls result in reduction of the primary electron acceptor Q B to QH 2. On the luminal side of PSII, electrons removed from H 2 O in the thylakoid lumen are transferred to P 680+, restoring the reaction-center chlorophylls to the ground state and generating O 2. (Center) The cytochrome bf complex then accepts electrons from QH 2, coupled to the release of two protons into the lumen. Operation of a Q cycle in the cytochrome bf complex translocates additional protons across the membrane to the thylakoid lumen, increasing the proton-motive force. (Right) In the PSI reaction center, each electron released from light-excited P 700 chlorophylls moves via a series of carriers in the reaction center to the stromal surface, where soluble ferredoxin (an Fe-S protein) transfers the electron to ferredoxin-nadp + reductase (FNR). This enzyme uses the prosthetic group flavin adenine dinucleotide (FAD) and a proton to reduce NADP +, forming NADPH. P 700+ is restored to its ground state by addition of an electron carried from PSII via the cytochrome bf complex and plastocyanin, a soluble electron carrier. 62

Respiration & Photosynthesis When light is not available, plants can also use the same pathways as in animals or other non-photosynthetic organism respiration (aerobic or anaerobic oxidation) Respiration and photosynthesis are two of the most ancient and important metabolic pathways. In some ways, photosynthesis and respiration are almost complete opposites of each other. Photosynthetic organisms, such as plants, use the energy of sunlight to reduce carbon dioxide into carbohydrates. During respiration, glucose is oxidized back to carbon dioxide, in the process releasing energy that it is captured in the bonds of ATP. This reciprocal relationship between aerobic oxidation in mitochondria and photosynthesis in chloroplasts underlies a profound symbiotic relationship between photosynthetic and nonphotosynthetic organisms and is responsible for much of the life on earth. 63