Topics 23: Photosynthesis

Topics 23: Photosynthesis • • • • • • 1. 2. 3. 4. Identify components of chloroplasts in drawings, photomicrographs and electron micrographs Explain the absorption and action spectra of photosynthetic pigments With reference to the chloroplast structure, describe and explain how light energy is harnessed and converted into chemical energy during the light-dependent reactions of photosynthesis Outline chemiosmosis in photosynthesis Outline the three phases of the Calvin cycle in C3 plants: (i) CO 2 fixation, (ii) PGA reduction and (iii) ribulose bisphosphate (RuBP) regeneration, indicating the roles of rubisco, ATP and reduced NADP in these processes that ultimately allow synthesis of sugars Discuss limiting factors in photosynthesis and carry out investigations on the effect of limiting factors such as temperature, light intensity and carbon dioxide concentration on the rate of photosynthesis The Leaf Light Photosynthesis Process Factors affecting the rate of photosynthesis 1. The Leaf • • • The major photosynthetic organ Structure and function are closely linked The adaptation of the leaf for photosynthesis are: o To obtain solar energy o To obtain and remove gases (carbon dioxide and oxygen) o To obtain and remove liquids (water and sugar solution) 1.1. Chloroplasts • • • • Plastids: Organelles that function in photosynthesis or storage in plants Three common types: Chromoplast (store pigments), chloroplast (*primary focus), leukoplast (food) All green parts of a plant, including green stems and unripened fruit, have chloroplasts, but the leaves are the major sites of photosynthesis in most plants. Chloroplasts are found mainly in the cells of the mesophyll, the tissue in the interior of the leaf. Carbon dioxide enters the leaf, and oxygen exits, by way of microscopic pores called stomata. Water absorbed by the roots is delivered to the leaves in veins. Leaves also use veins to export sugar to roots and other nonphotosynthetic parts of the plant. A chloroplast has two membranes surrounding a dense fluid called the stroma. Suspended within the stroma is a third membrane system, made up of sacs called thylakoids, which segregates the stroma from Continuous to thylakoid • the thylakoid space inside these sacs. (thylakoid space is similar to mitochondrial intermembrane space where both accumulate H+) In some places, thylakoid sacs are stacked in columns called grana (singular, granum). Chlorophyll, the green pigment that gives leaves their color, resides in the thylakoid membranes of the chloroplast. It is the light energy absorbed by chlorophyll that drives the synthesis of organic molecules in the chloroplast. Chloroplasts are chemical factories powered by the sun. Their thylakoids transform light energy into the chemical energy of ATP and NADPH, which will be used to synthesize glucose and other molecules that can be used as energy sources 1.2. Photosynthetic Pigments • • • • Chemical compounds that reflect only certain wavelengths of visible light. Act as energy-receiving antennas Two basic classes: o Chlorophyll o Carotenoid When light meets matter, it may be reflected, transmitted, or absorbed. Substances that absorb visible light are known as pigments. Different pigments absorb light of different wavelengths, and the wavelengths that are absorbed disappear. If a pigment is illuminated with white light, the color we see is the color most reflected or transmitted by the pigment. (If a pigment absorbs all wavelengths, it appears black.) We see green when we look at a leaf because chlorophyll absorbs violet-blue and red light while transmitting and reflecting green light (Figure 10.8). 1.2.1. Chlorophyll • • • A pigment that absorbs mainly red and blue-violet light, reflecting green light Two parts: o Porphyrin ring (Light absorption) o Hydrophobic phytol tail (Anchors to the thylakoid membrane) Two types o Chlorophyll a ▪ Major pigment ▪ Can participate directly in the Light Reaction o Chlorophyll b ▪ Common accessory pigment ▪ Only can transfer energy to other pigments 1.2.2. Carotenoid absorb wavelengths of light (mainly blue and blue-green light) that chlorophyll does not absorb efficiently. The absorbed energy is then transferred to chlorophyll, helping • • • • 2. Light • • the plant capture more sunlight for photosynthesis. Accessory pigment Two main types: Carotene and Xanthophylls Absorb light in the 460 to 550 nm of the visible light Two main functions o Broadening spectrum of light for photosynthesis o Photoprotection (Absorbing excessive light) receives more light energy than it can use for photosynthesis, the excess energy can damage chlorophyll and other cellular components by generating harmful reactive oxygen species. Light has a wave nature, forming part of electromagnetic spectrum Visible light: Part of the radiant energy with wavelengths that are visible to human eye • • • • Three features of light: o Spectral quality (wavelength) o Intensity (amount of light/distance from light source) o Duration (amount of time) A single quantum of light is called a photon Energy of photon is inversely proportional to the wavelength Light is a form of energy known as electromagnetic energy, also called electromagnetic radiation. Electromagnetic energy travels in rhythmic waves analogous to those created by dropping a pebble into a pond. Electromagnetic waves, however, are disturbances of electric and magnetic fields rather than disturbances of a material medium such as water. The distance between the crests of electromagnetic waves is called the wavelength. Wavelengths range from less than a nanometer (for gamma rays) to more than a kilometer (for radio waves). This entire range of radiation is known as the electromagnetic spectrum (Figure 10.7). The segment most important to life is the narrow band from about 380 nm to 750 nm in wavelength. This radiation is known as visible light because it can be detected as various colors by the human eye. The model of light as waves explains many of light’s properties, but in certain respects light behaves as though it consists of discrete particles, called photons. Photons are not tangible objects, but they act like objects in that each of them has a fixed quantity of energy. The amount of energy is inversely related to the wavelength of the light: The shorter the wavelength, the greater the energy of each photon of that light. Thus, a photon of violet light packs nearly twice as much energy as a photon of red light (see Figure 10.7). Although the sun radiates the full spectrum of electromagnetic energy, the atmosphere acts like a selective window, allowing visible light to pass through while screening out a substantial fraction of other radiation. The part of the spectrum we can see—visible light—is also the radiation that drives photosynthesis. 2.1. Absorption Spectra and Action Spectra • • • • • Absorption Spectra: Amount of light absorbed at different wavelength by a pigment Action Spectra: Effectiveness of different wavelengths if light driving photosynthesis The ability of a pigment to absorb various wavelengths of light can be measured with an instrument called a spectrophotometer. This machine directs beams of light of different wavelengths through a solution of the pigment and measures the fraction of the light transmitted at each wavelength. A graph plotting a pigment’s light absorption versus wavelength is called an absorption spectrum (Figure 10.9). The absorption spectra of chloroplast pigments provide clues to the relative effectiveness of different wavelengths for driving photosynthesis, since light can perform work in chloroplasts only if it is absorbed. Figure 10.10a shows the absorption spectra of three types of pigments in chloroplasts: chlorophyll a, the key light-capturing pigment that participates directly in the light reactions; the accessory pigment chlorophyll b; and a separate group of accessory pigments called carotenoids. The spectrum of chlorophyll a suggests that • • violet-blue and red light work best for photosynthesis, since they are absorbed, while green is the least effective color. This is confirmed by an action spectrum for photosynthesis (Figure 10.10b), which profiles the relative effectiveness of different wavelengths of radiation in driving the process. An action spectrum is prepared by illuminating chloroplasts with light of different colors and then plotting wavelength against some measure of photosynthetic rate, such as CO2 consumption or O2 release. The action spectrum for photosynthesis was first demonstrated by Theodor W. Engelmann, a German botanist, in 1883. Before equipment for measuring O2 levels ha d even been invented, Engelmann performed a clever experiment in which he used bacteria to measure rates of photosynthesis in filamentous algae (Figure 10.10c). His results are a striking match to the modern action spectrum shown in Figure 10.10b. Notice by comparing Figures 10.10a and 10.10b that the action spectrum for photosynthesis is much broader than the absorption spectrum of chlorophyll a. The absorption spectrum of chlorophyll a alone underestimates the effectiveness of certain wavelengths in driving photosynthesis. This is partly because accessory pigments with different absorption spectra also present in chloroplasts—including chlorophyll b and carotenoids— broaden the spectrum of colors that can be used for photosynthesis. Figure 10.11 shows the structure of chlorophyll a compared with that of chlorophyll b. A slight structural difference between them is enough to cause the two pigments to absorb at slightly different wavelengths in the red and blue parts of the spectrum (see Figure 10.10a). As a result, chlorophyll a appears blue green and chlorophyll b olive green under visible light. Other accessory pigments include carotenoids, hydrocarbons that are various shades of yellow and orange because they absorb violet and blue-green light (see Figure 10.10a). Carotenoids may broaden the spectrum of colors that can drive photosynthesis. However, a more important function of at least some carotenoids seems to be photoprotection: These compounds absorb and dissipate excessive light energy that would otherwise damage chlorophyll or interact with oxygen, forming reactive oxidative molecules that are dangerous to the cell. Interestingly, carotenoids similar to the photoprotective ones in chloroplasts have a photoprotective role in the human eye. (Carrots, known for aiding night vision, are rich in carotenoids.) These and related molecules are, of course, found naturally in many vegetables and fruits. They are also often advertised in health food products as “phytochemicals” (from the Greek phyton, plant), some of which have antioxidant properties. Plants can synthesize all the antioxidants they require, but humans and other animals must obtain some of them from their diets. 3. Photosynthesis • • • A process of energy transduction Light energy is converted to chemical energy Three main phases: o Light Harvesting Stage o Light-dependent Stage / Light Reaction o Light-independent Stage / Dark Reaction / Calvin Cycle Tracking atoms through Photosynthesis: • In the presence of light, the green parts of plants produce organic compounds and oxygen from carbon dioxide and water. Using molecular formulas, we can summarize the complex series of chemical reactions in photosynthesis with this chemical equation: • We use glucose (C6H12O6) here to simplify the relationship between photosynthesis and respiration, but the direct product of photosynthesis is actually a three-carbon sugar that can be used to make glucose. Water appears on both sides of the equation because 12 molecules are consumed and 6 molecules are newly formed during photosynthesis. We can simplify the equation by indicating only the net consumption of water: 6 CO2 + 6 H2O + Light energy -> C6H12O6 + 6 O2 Writing the equation in this form, we can see that the overall chemical change during photosynthesis is the reverse of the one that occurs during cellular respiration. Both of these metabolic processes occur in plant cells. However, chloroplasts do not synthesize sugars by simply reversing the steps of respiration. Now let’s divide the photosynthetic equation by 6 to put it in its simplest possible form: CO2 + H2O -> [CH2O] + O2 Here, the brackets indicate that CH2O is not an actual sugar but represents the general formula for a carbohydrate. In other words, we are imagining the synthesis of a sugar molecule one carbon at a time (with six repetitions theoretically adding up to a glucose molecule—C6H12O6). • The Splitting of Water • • • • • One of the first clues to the mechanism of photosynthesis came from the discovery that the O2 given off by plants is derived from H2O and not from CO2. The chloroplast splits water into hydrogen and oxygen. Before this discovery, the prevailing hypothesis was that photosynthesis split carbon dioxide (CO2 -> C + O2) and then added water to the carbon (C + H2O -> [CH2O]). This hypothesis predicted that the O2 released during photosynthesis came from CO2. This idea was challenged in the 1930s by C. B. van Niel, of Stanford University. Van Niel was investigating photosynthesis in bacteria that make their carbohydrate from CO2 but do not release O2. He concluded that, at least in these bacteria, CO2 is not split into carbon and oxygen. One group of bacteria used hydrogen sulfide (H2S) rather than water for photosynthesis, forming yellow globules of sulfur as a waste product. Here is the chemical equation for photosynthesis in these sulfur bacteria: CO2 + 2 H2S -> [CH2O] + H2O + 2 S Van Niel reasoned that the bacteria split H2S and used the hydrogen atoms to make sugar. He then generalized that idea, proposing that all photosynthetic organisms require a hydrogen source but that the source varies: o Sulfur bacteria: CO2 + 2 H2S -> [CH2O] + H2O + 2 S o Plants: CO2 + 2 H2O -> [CH2O] + H2O + O2 o General: CO2 + 2 H2X -> [CH2O] + H2O + 2 X Thus, van Niel hypothesized that plants split H2O as a source of electrons from hydrogen atoms, releasing O2 as a by-product. Nearly 20 years later, scientists confirmed van Niel’s hypothesis by using oxygen-18 (18O), a heavy isotope, as a tracer to follow the path of oxygen atoms during photosynthesis. The experiments showed that the O2 from plants was labeled with 18O only if water was the source of the tracer (experiment 1). If the 18O was introduced to the plant in the form of CO2, the label did not turn up in the released O2 (experiment 2). In the following summary, magenta denotes labeled atoms of oxygen (18O): o Experiment 1: CO2 + 2 H2O S [CH2O] + H2O + O2 o Experiment 2: CO2 + 2 H2O S [CH2O] + H2O + O2 A significant result of the shuffling of atoms during photosynthesis is the extraction of hydrogen from water and its incorporation into sugar. The waste product of photosynthesis, O2, is released to the atmosphere. Figure 10.5 shows the fates of all atoms in photosynthesis. Photosynthesis as a Redox Process • Let’s briefly compare photosynthesis with cellular respiration. Both processes involve redox reactions. During cellular respiration, energy is released from sugar when electrons associated with hydrogen are transported by carriers to oxygen, forming water as a by-product. The electrons lose potential energy as they “fall” down the electron transport chain toward electronegative oxygen, and the mitochondrion harnesses that energy to synthesize ATP. Photosynthesis reverses the direction of electron flow. Water is split, and its electrons are transferred along with hydrogen ions (H+) from the water to carbon dioxide, reducing it to sugar. Because the electrons increase in potential energy as they move from water to sugar, this process requires energy—in other words, it is endergonic. This energy boost that occurs during photosynthesis is provided by light. • Actually, photosynthesis is not a single process, but two processes, each with multiple steps. These two stages of photosynthesis are known as the light reactions (the photo part of photosynthesis) and the Calvin cycle (the synthesis part). The light reactions are the steps of photosynthesis that convert solar energy to chemical energy. Water is split, providing a source of electrons and protons (H+ ) and giving off O2 as a by-product. Light absorbed by chlorophyll drives a transfer of the electrons and hydrogen ions from water to an acceptor called NADP+ (nicotinamide adenine dinucleotide phosphate), where they are temporarily stored. (The electron acceptor NADP+ is first cousin to NAD+ , which functions as an electron carrier in cellular respiration; the two molecules differ only by the presence of an extra phosphate group in the NADP+ molecule.) The light reactions use solar energy to reduce NADP+ to NADPH by adding a pair of electrons along with an H+ . The light reactions also generate ATP, using chemiosmosis to power the addition of a phosphate group to ADP, a process called photophosphorylation. Thus, light energy is initially converted to chemical energy in the form of two compounds: NADPH and ATP. NADPH, a source of electrons, acts as “reducing power” that can be passed along to an electron acceptor, reducing it, while ATP is the versatile energy currency of cells. Notice that the light reactions produce no sugar; that happens in the second stage of photosynthesis, the Calvin cycle. The Calvin cycle begins by incorporating CO2 from the air into organic molecules already present in the chloroplast. This initial incorporation of carbon into organic compounds is known as carbon fixation. The Calvin cycle then reduces the fixed carbon to carbohydrate by the addition of electrons. The reducing power is provided by NADPH, which acquired its cargo of electrons in the light reactions. To convert CO2 to carbohydrate, the Calvin cycle also requires chemical energy in the form of ATP, which is also generated by the light reactions. Thus, it is the Calvin cycle that makes sugar, but it can do so only with the help of the NADPH and ATP produced by the light reactions. The metabolic steps of the Calvin cycle are sometimes referred to as the dark reactions, or light independent reactions, because none of the steps requires light directly. Nevertheless, the Calvin cycle in most plants occurs during daylight, for only then can the light reactions provide the NADPH and ATP that the Calvin cycle requires. In essence, the chloroplast uses light energy to make sugar by coordinating the two stages of photosynthesis. • • • As Figure 10.6 indicates, the thylakoids of the chloroplast are the sites of the light reactions, while the Calvin cycle occurs in the stroma. On the outside of the thylakoids, molecules of NADP+ and ADP pick up electrons and phosphate, respectively, and NADPH and ATP are then released to the stroma, where they play crucial roles in the Calvin cycle. 3.1. Phase 1: Light Harvesting Stage • • Light which strikes a leaf may be: o Reflected o Transmitted o Absorbed (mostly) But only a small part is used in photosynthesis 3.1. LHS: Excitation of Chlorophyll • • Excited molecule (unstable) tends to return to its original, unexcited state in one of the three ways: o Energy is lost in the process as heat and light of longer wavelength o Energy is transferred to a neighbouring chlorophyll: Resonance energy transfer o High energy electron is transferred to another molecule (electron acceptor) What exactly happens when chlorophyll and other pigments absorb light? The colors corresponding to the photolysis absorbed wavelengths disappear from the spectrum of the transmitted and reflected light, but energy cannot disappear. When a molecule absorbs a photon of light, one of the molecule’s electrons is elevated to an orbital where it has more potential energy. When the electron is in its normal orbital, the pigment molecule is said to be in its ground state. Absorption of a photon boosts an electron to an orbital of higher energy, and the pigment molecule is then said to be in an excited state. The only photons absorbed are those whose energy is exactly equal to the energy difference between the ground state and an excited state, and this energy difference varies from one kind of molecule to another. Thus, a particular compound absorbs only photons corresponding to specific wavelengths, which is why each pigment has a unique absorption spectrum. In the leaf, most of the chlorophyll electrons excited by photon absorption are used to power the reactions of photosynthesis. • Once absorption of a photon raises an electron to an excited state, the electron cannot stay there long. The excited state, like all high-energy states, is unstable. Generally, when isolated pigment molecules absorb light, their excited electrons drop back down to the ground-state orbital in a billionth of a second, releasing their excess energy as heat. This conversion of light energy to heat is what makes the top of an automobile so hot on a sunny day. (White cars are coolest because their paint reflects all wavelengths of visible light.) In isolation, some pigments, including chlorophyll, emit light as well as heat after absorbing photons. As excited electrons fall back to the ground state, photons are given off, an afterglow called fluorescence. An illuminated solution of chlorophyll isolated from chloroplasts will fluoresce in the red part of the spectrum and also give off heat (Figure 10.12). This is best seen by illuminating with ultraviolet light, which chlorophyll can also absorb (see Figures 10.7 and 10.10a). Viewed under visible light, the fluorescence would be difficult to see against the green of the solution. 3.1.1. LHS: Photosystems • • • Large multiprotein complexes in the thylakoid membrane Two closely linked components o Antenna complexes (200 to 300 pigment molecules) o A reaction centre (a pair of chlorophyll a molecules) Two photosystems (PSI and PSII) A Photosystem: a reaction-Center Complex associated with Light-harvesting Complexes • • • Chlorophyll molecules excited by the absorption of light energy produce very different results in an intact chloroplast than they do in isolation (see Figure 10.12). In their native environment of the thylakoid membrane, chlorophyll molecules are organized along with other small organic molecules and proteins into complexes called photosystems. A photosystem is composed of a reaction-center complex surrounded by several light-harvesting complexes (Figure 10.13). The reaction-center complex is an organized association of proteins holding a special pair of chlorophyll a molecules and a primary electron acceptor. Each light-harvesting complex consists of various pigment molecules (which may include chlorophyll a, chlorophyll b, and multiple carotenoids) bound to proteins. The number and variety of pigment molecules enable a photosystem to harvest light over a larger surface area and a larger portion of the spectrum than could any single pigment molecule alone. Together, these light-harvesting complexes act as an antenna for the reaction-center complex. When a pigment molecule absorbs a photon, the energy is transferred from pigment molecule to pigment molecule within a lightharvesting complex, until it is passed to the pair of chlorophyll a molecules in the reaction-center complex. The pair of chlorophyll a molecules in the reaction-center complex are special because their molecular environment—their location and the other molecules with which they are associated—enables them to use the energy from light not only to boost one of their electrons to a higher energy level, but also to transfer it to a different molecule—the primary electron acceptor, which is a molecule capable of accepting electrons and becoming reduced. The solar-powered transfer of an electron from the reaction-center chlorophyll a pair to the primary electron acceptor is the first step of the light reactions. As soon as the chlorophyll electron is excited to a higher energy level, the primary electron acceptor captures it; this is a redox reaction. In the flask shown in Figure 10.12b, isolated chlorophyll fluoresces because there is no electron acceptor, so electrons of photoexcited • chlorophyll drop right back to the ground state. In the structured environment of a chloroplast, however, an electron acceptor is readily available, and the potential energy represented by the excited electron is not dissipated as light and heat. Thus, each photosystem—a reactioncenter complex surrounded by light-harvesting complexes— functions in the chloroplast as a unit. It converts light energy to chemical energy, which will ultimately be used for the synthesis of sugar. The thylakoid membrane is populated by two types of photosystems that cooperate in the light reactions of photosynthesis: photosystem II (PS II) and photosystem I (PS I). (They were named in order of their discovery, but photosystem II functions first in the light reactions.) Each has a characteristic reaction-center complex—a particular kind of primary electron acceptor next to a special pair of chlorophyll a molecules associated with specific proteins. The reaction-center chlorophyll a of photosystem II is known as P680 because this pigment is best at absorbing light having a wavelength of 680 nm (in the red part of the spectrum). The chlorophyll a at the reaction-center complex of photosystem I is called P700 because it most effectively absorbs light of wavelength 700 nm (in the far-red part of the spectrum). These two pigments, P680 and P700, are nearly identical chlorophyll a molecules. However, their association with different proteins in the thylakoid membrane affects the electron distribution in the two pigments and accounts for the slight differences in their light-absorbing properties. Now let’s see how the two types of photosystems work together in using light energy to generate ATP and NADPH, the two main products of the light reactions. Linear electron Flow Light drives the synthesis of ATP and NADPH by energizing the two types of photosystems embedded in the thylakoid membranes of chloroplasts. The key to this energy transformation is a flow of electrons through the photosystems and other molecular components built into the thylakoid membrane. This is called linear electron flow, and it occurs during the light reactions of photosynthesis, as shown in Figure 10.14. The numbered steps in the text correspond to the numbered steps in the figure. 1. A photon of light strikes one of the pigment molecules in a light-harvesting complex of PS II, boosting one of its electrons to a higher energy level. As this electron falls back to its ground state, an electron in a nearby pigment molecule is simultaneously raised to an excited state. The process continues, with the energy being relayed to other pigment molecules until it reaches the P680 pair of chlorophyll a molecules in the PS II reaction-center complex. It excites an electron in this pair of chlorophylls to a higher energy state. 2. This electron is transferred from the excited P680 to the primary electron acceptor. We can refer to the resulting form of P680, missing the negative charge of an electron, as P680+ 3. An enzyme catalyzes the splitting of a water molecule into two electrons, two hydrogen ions (H+ ), and an oxygen atom. The electrons are supplied one by one to the P680+ pair, each electron replacing one transferred to the primary electron acceptor. (P680+ is the strongest biological oxidizing agent known; its electron “hole” must be filled. This greatly facilitates the transfer of electrons from the split water molecule.) The H+ are released into the thylakoid space (interior of the thylakoid). The oxygen atom immediately combines with an oxygen atom generated by the splitting of another water molecule, forming O2. 4. Each photoexcited electron passes from the primary electron acceptor of PS II to PS I via an electron transport chain, the components of which are similar to those of the electron transport chain that functions in cellular respiration. The electron transport chain between PS II and PS I is made up of the electron carrier plastoquinone (Pq), a cytochrome complex, and a protein called plastocyanin (Pc). Each component carries out redox reactions as electrons flow down the electron transport chain, releasing free energy that is used to pump protons (H+ ) into the thylakoid space, contributing to a proton gradient across the thylakoid membrane. 5. The potential energy stored in the proton gradient is used to make ATP in a process called chemiosmosis, to be discussed shortly. 6. Meanwhile, light energy has been transferred via lightharvesting complex pigments to the PS I reactioncenter complex, exciting an electron of the P700 pair of chlorophyll a molecules located there. The photoexcited electron is then transferred to PS I’s primary electron acceptor, creating an electron “hole” in the P700—which we now can call P700+ . In other words, P700+ can now act as an electron acceptor, accepting an electron that reaches the bottom of the electron transport chain from PS II. 7. Photoexcited electrons are passed in a series of redox reactions from the primary electron acceptor of PS I down a second electron transport chain through the protein ferredoxin (Fd). (This chain does not create a proton gradient and thus does not produce ATP.) 8. The enzyme NADP+ reductase catalyzes the transfer of electrons from Fd to NADP+ . Two electrons are required for its reduction to NADPH. Electrons in NADPH are at a higher energy level than they are in water (where they started), so they are more readily available for the reactions of the Calvin cycle. This process also removes an H+ from the stroma. The energy changes of electrons during their linear flow through the light reactions are shown in a mechanical analogy in Figure 10.15. Although the scheme shown in Figures 10.14 and 10.15 may seem complicated, do not lose track of the big picture: The light reactions use solar power to generate ATP and NADPH, which provide chemical energy and reducing power, respectively, to the carbohydrate-synthesizing reactions of the Calvin cycle. Figure 10.14 How linear electron flow during the light reactions generates ATP and NAdPH. The gold arrows trace the flow of light-driven electrons from water to NADPH. The black arrows trace the transfer of energy from pigment molecule to pigment molecule. Input: Water, light End products: NADPH. ATP System 1&2 work together for ETC, chemiosmosis 3.1.1.1. LHS: Photosystems I (PSI) • • • • Absorbs light energy independently of PSII Antenna pigments: 100 molecules of Chl a and Chl b (ratio 4:1) Reaction centre is called P700 (max absorption at 700 nm) Function: To oxidise reduced plastocyanin and transfer the electrons to a soluble form of Fe-S protein (ferredoxin) (Fd: receive electrons from plastocyanin) 3.1.1.2. LHS: Photosystems II (PSII) • • • • Absorbs light energy independently of PSI Antenna pigments: 250 molecules of Chl a and Chl b (ratio 1:1) and numerous xanthophylls Reaction centre is called P680 (max absorption at 680 nm) Function: To use light energy (ps1) to reduce plastoguinone (NADP to NADPH) using electrons from photolysis of water (ps2) 3.2. Phase 2: Light-Dependent Reaction • • • • • Also known as light reaction Three main stages: o Charge separation o Electron Transport o Chemiosmosis Functions: To synthesise NADPH and ATP The NADPH and ATP are used to fix carbon dioxide and trap energy in the form of glucose Two main ways: o Non-cyclic photophosphorylation (pre-dominant route) (impt!!!) o Cyclic photophosphorylation 3.2.1. Non-cyclic Photophosphorylation (Linear electron flow) • • Involved both PSI and PSII in the Z-scheme of electron flow ATP and NADPH are produced 3.2.2. Cyclic Photophosphorylation • • Only involved PSI Only ATP is produced Cyclic electron Flow • In certain cases, photoexcited electrons can take an alternative path called cyclic electron flow, which uses photosystem I but not photosystem II. You can see in Figure 10.16 that cyclic flow is a short circuit: The • • • electrons cycle back from ferredoxin (Fd) to the cytochrome complex, then via a plastocyanin molecule (Pc) to a P700 chlorophyll in the PS I reaction-center complex. There is no production of NADPH and no release of oxygen that results from this process. On the other hand, cyclic flow does generate ATP. Rather than having both PS II and PS I, several of the currently existing groups of photosynthetic bacteria are known to have a single photosystem related to either PS II or PS I. For these species, which include the purple sulfur bacteria (see Figure 10.2e) and the green sulfur bacteria, cyclic electron flow is the one and only means of generating ATP during the process of photosynthesis. Evolutionary biologists hypothesize that these bacterial groups are descendants of ancestral bacteria in which photosynthesis first evolved, in a form similar to cyclic electron flow. Cyclic electron flow can also occur in photosynthetic species that possess both photosystems; this includes some prokaryotes, such as the cyanobacteria shown in Figure 10.2d, as well as the eukaryotic photosynthetic species that have been tested thus far. Although the process is probably in part an “evolutionary leftover,” research suggests it plays at least one beneficial role for these organisms. Plants with mutations that render them unable to carry out cyclic electron flow are capable of growing well in low light, but do not grow well where light is intense. This is evidence for the idea that cyclic electron flow may be photoprotective. Whether ATP synthesis is driven by linear or cyclic electron flow, the actual mechanism is the same. 3.2.3. Chemiosmosis A Comparison of Chemiosmosis in Chloroplasts and Mitochondria • Chloroplasts and mitochondria generate ATP by the same basic mechanism: chemiosmosis (see Figure 9.15). An electron transport chain pumps protons (H+ ) across a membrane as electrons are passed through a series of carriers that are progressively more (Build up protons) Protons used for phosphorylation • • • • • • electronegative. Thus, electron transport chains transform redox energy to a proton-motive force, potential energy stored in the form of an H+ gradient across a membrane. An ATP synthase complex in the same membrane couples the diffusion of hydrogen ions down their gradient to the phosphorylation of ADP, forming ATP. Some of the electron carriers, including the iron-containing proteins called cytochromes, are very similar in chloroplasts and mitochondria. The ATP synthase complexes of the two organelles are also quite similar. But there are noteworthy differences between photophosphorylation in chloroplasts and oxidative phosphorylation in mitochondria. Both work by way of chemiosmosis, but in chloroplasts, the high-energy electrons dropped down the transport chain come from water, while in mitochondria, they are extracted from organic molecules (which are thus oxidized). Chloroplasts do not need molecules from food to make ATP; their photosystems capture light energy and use it to drive the electrons from water to the top of the transport chain. In other words, mitochondria use chemiosmosis to transfer chemical energy from food molecules to ATP, whereas chloroplasts use it to transform light energy into chemical energy in ATP. Although the spatial organization of chemiosmosis differs slightly between chloroplasts and mitochondria, it is easy to see similarities in the two. Electron transport chain proteins in the inner membrane of the mitochondrion pump protons from the mitochondrial matrix out to the intermembrane space, which then serves as a reservoir of hydrogen ions. Similarly, electron transport chain proteins in the thylakoid membrane of the chloroplast pump protons from the stroma into the thylakoid space, which functions as the H+ reservoir. If you imagine the cristae of mitochondria pinching off from the inner membrane, this may help you see how the thylakoid space and the intermembrane space are comparable spaces in the two organelles, while the mitochondrial matrix is analogous to the stroma of the chloroplast. In the mitochondrion, protons diffuse down their concentration gradient from the intermembrane space through ATP synthase to the matrix, driving ATP synthesis. In the chloroplast, ATP is synthesized as the hydrogen ions diffuse from the thylakoid space back to the stroma through ATP synthase complexes (Figure 10.18), whose catalytic knobs are on the stroma side of the membrane. Thus, ATP forms in the stroma, where it is used to help drive sugar synthesis during the Calvin cycle. The proton (H+ ) gradient, or pH gradient, across the thylakoid membrane is substantial. When chloroplasts in an experimental setting are illuminated, the pH in the thylakoid space drops to about 5 (the H+ concentration increases), and the pH in the stroma increases to about 8 (the H+ concentration decreases). This gradient of three pH units corresponds to a thousandfold difference in H+ concentration. If the lights are then turned off, the pH gradient is abolished, but it can quickly be restored by turning the lights back on. Experiments such as this provided strong evidence in support of the chemiosmotic model. The currently accepted model for the organization of the light-reaction “machinery” within the thylakoid membrane is based on several research studies. Each of the molecules and molecular complexes in the figure is present in numerous copies in each thylakoid. Notice that NADPH, like ATP, is produced on the side of the membrane facing the stroma, where the Calvin cycle reactions take place. Let’s summarize the light reactions. Electron flow pushes electrons from water, where they are at a state of low potential energy, ultimately to NADPH, where they are stored at a state of high potential energy. The light-driven electron flow also generates ATP. Thus, the equipment of the thylakoid membrane converts light Synthesis Synthesis ATP for energy to ATP for light-independent chemical cell use stage energy stored in ATP and NADPH. Oxygen is produced as a by- (a) decrease the pH outside the mitochondrion (thus increasing the product. H+ concentration) (b) increase the pH in the chloroplast stroma (thus decreasing the H+ concentration). In both cases, this would generate an H+ gradient across the membrane that would cause ATP synthase to synthesize ATP. Build up H+ -> power the synthesis of ATP By product of photolysis 3.2.3. Comparisons (Summary) • • • • • Chloroplasts and mitochondria generate ATP by chemiosmosis, but use different sources of energy Mitochondria transfer chemical energy from food to ATP; chloroplasts transform light energy into the chemical energy of ATP Spatial organization of chemiosmosis differs between chloroplasts and mitochondria but also shows similarities In mitochondria, protons are pumped to the intermembrane space and drive ATP synthesis as they diffuse back into the mitochondrial matrix In chloroplasts, protons are pumped into the thylakoid space and drive ATP synthesis as they diffuse back into the stroma 3.3. Phase 3: Light-Independent Reaction • • • • Also known as dark reaction or Calvin cycle Occurs in the stroma Function: To reduce carbon dioxide using ATP and NADPH Four main stages: o Carbon Dioxide Fixation o Reduction and Sugar Formation o Regeneration of carbon dioxide acceptor (RuBP) o Product Synthesis The Calvin cycle uses the chemical energy of ATP and NADPH to reduce Co2 to sugar • • • • • • The Calvin cycle is similar to the citric acid cycle in that a starting material is regenerated after some molecules enter and others exit the cycle. However, the citric acid cycle is catabolic, oxidizing acetyl CoA and using the energy to synthesize ATP, while the Calvin cycle is anabolic, building carbohydrates from smaller molecules and consuming energy. Carbon enters the Calvin cycle in the form of CO2 and leaves in the form of sugar. The cycle spends ATP as an energy source and consumes NADPH as reducing power for adding high-energy electrons to make the sugar. As we mentioned in Concept 10.1, the carbohydrate produced directly from the Calvin cycle is not glucose. It is actually a three-carbon sugar called glyceraldehyde 3-phosphate (G3P). For the net synthesis of one molecule of G3P, the cycle must take place three times, fixing three molecules of CO2—one per turn of the cycle. (Recall that the term carbon fixation refers to the initial incorporation of CO2 into organic material.) As we trace the steps of the Calvin cycle, keep in mind that we are following three molecules of CO2 through the reactions. Figure 10.19 divides the Calvin cycle into three phases: carbon fixation, reduction, and regeneration of the CO2 acceptor. Phase 1: Carbon fixation. The Calvin cycle incorporates each CO2 molecule, one at a time, by attaching it to a fivecarbon sugar named ribulose bisphosphate (abbreviated RuBP). The enzyme that catalyzes this first step is RuBP carboxylase-oxygenase, or rubisco. (This is the most abundant protein in chloroplasts and is also thought to be the most abundant protein on Earth.) The product of the reaction is a six-carbon intermediate that is short-lived because it is so energetically unstable that it immediately splits in half, forming two molecules of 3-phosphoglycerate (for each CO2 fixed). Phase 2: Reduction. Each molecule of 3-phosphoglycerate receives an additional phosphate group from ATP, becoming 1,3-bisphosphoglycerate. Next, a pair of electrons donated from NADPH reduces 1,3bisphosphoglycerate, which also loses a phosphate group in the process, becoming glyceraldehyde 3phosphate (G3P). Specifically, the electrons from NADPH reduce a carboyxl group on 1,3bisphosphoglycerate to the aldehyde group of G3P, which stores more potential energy. G3P is a sugar—the same three-carbon sugar formed in glycolysis by the splitting of glucose (see Figure 9.9). Notice in Figure 10.19 that for every three molecules of CO2 that enter the cycle, there are six molecules of G3P formed. But only one molecule of this three-carbon sugar can be counted as a net gain of carbohydrate because the rest are required to complete the cycle. The cycle began with 15 carbons’ worth of carbohydrate in the form of three molecules of the five-carbon sugar RuBP. Now there are 18 carbons’ worth of carbohydrate in the form of six molecules of G3P. One molecule exits the cycle to be used by the plant cell, but the other five molecules must be recycled to regenerate the three molecules of RuBP. Phase 3: Regeneration of the CO2 acceptor (RuBP). In a complex series of reactions, the carbon skeletons of five molecules of G3P are rearranged by the last steps of the Calvin cycle into three molecules of RuBP. To accomplish this, the cycle spends three more molecules of ATP. The RuBP is now prepared to receive CO2 again, and the cycle continues. For the net synthesis of one G3P molecule, the Calvin cycle consumes a total of nine molecules of ATP and six molecules of NADPH. The light reactions regenerate the ATP and NADPH. The G3P spun off from the Calvin cycle becomes the starting material for metabolic pathways that synthesize other organic compounds, including glucose (from two molecules of G3P), the disaccharide sucrose, and other carbohydrates. Neither the light reactions nor the Calvin cycle alone can make sugar from CO2. Photosynthesis is an emergent property of the intact chloroplast, which integrates the two stages of photosynthesis. 4. Factors Affecting the Rate of Photosynthesis • • • • Light intensity Amount of carbon dioxide Temperature Amount of chlorophyll Questions 1. Compare the roles of CO2 and H2O in cellular respiration and photosynthesis. CO2 and H2O are the products of cellular respiration; they are the reactants in photosynthesis. In respiration, glucose is oxidized to CO2 and electrons are passed through an electron transfer chain from glucose to O2, producing H2O. In photosynthesis, H2O is the source of electrons, which are energized by light, temporarily stored in NADPH, and used to reduce CO2 to carbohydrate. 2. The absorption spectrum of chlorophyll a differs from the action spectrum of photosynthesis. Explain this observation. The action spectrum of photosynthesis shows that some wavelengths of light that are not absorbed by chlorophyll a are still effective at promoting photosynthesis. The light-harvesting complexes of photosystems contain accessory pigments such as chlorophyll b and carotenoids, which absorb different wavelengths and pass the energy to chlorophyll a, broadening the spectrum of light usable for photosynthesis. 3. For Calvin cycle, draw where ATP and NADPH are used and where rubisco functions. Describe these steps. In the reduction phase of the Calvin cycle, ATP phosphorylates a three carbon compound, and NADPH then reduces this compound to G3P. ATP is also used in the regeneration phase, when five molecules of G3P are converted to three molecules of the five-carbon compound RuBP. Rubisco catalyzes the first step of carbon fixation—the addition of CO2 to RuBP. 4. How do the CO2 molecules used in photosynthesis reach and enter the chloroplasts inside leaf cells? CO2 enters the leaves via stomata, and being a nonpolar molecule, can cross the leaf cell membrane and the chloroplast membranes to reach the stroma of the chloroplast. 5. Explain how the use of an oxygen isotope helped elucidate the chemistry of photosynthesis. Using 18O, a heavy isotope of oxygen, as a label, researchers were able to confirm van Niel’s hypothesis that the oxygen produced during photosynthesis comes from water, not from carbon dioxide. 6. The Calvin cycle requires ATP and NADPH, products of the light reactions. If a classmate asserted that the light reactions don’t depend on the Calvin cycle and, with continual light, could just keep on producing ATP and NADPH, how would you respond? The light reactions could not keep producing NADPH and ATP without the NADP+ , ADP, and ~P i that the Calvin cycle generates. The two cycles are interdependent. 7. What color of light is least effective in driving photosynthesis? Explain. Green, because green light is mostly transmitted and reflected—not absorbed— by photosynthetic pigments 8. In the light reactions, what is the initial electron donor? Where do the electrons finally end up? Water (H2O) is the initial electron donor; NADP+ accepts electrons at the end of the electron transport chain, becoming reduced to NADPH. 9. In an experiment, isolated chloroplasts placed in an illuminated solution with the appropriate chemicals can carry out ATP synthesis. Predict what would happen to the rate of synthesis if a compound is added to the solution that makes membranes freely permeable to hydrogen ions. In this experiment, the rate of ATP synthesis would slow and eventually stop. Because the added compound would not allow a proton gradient to build up across the membrane, ATP synthase could not catalyze ATP production. 10. To synthesize one glucose molecule, the Calvin cycle uses __________ molecules of CO2, __________ molecules of ATP, and __________ molecules of NADPH. [6, 18, 12] 11. How are the large numbers of ATP and NADPH molecules used during the Calvin cycle consistent with the high value of glucose as an energy source? The more potential energy and reducing power a molecule stores, the more energy and reducing power are required for the formation of that molecule. Glucose is a valuable energy source because it is highly reduced (lots of C¬H bonds), storing lots of potential energy in its electrons. To reduce CO2 to glucose, much energy and reducing power are required in the form of large numbers of ATP and NADPH molecules, respectively. 12. Explain why a poison that inhibits an enzyme of the Calvin cycle will also inhibit the light reactions. The light reactions require ADP and NADP+, which would not be formed in sufficient quantities from ATP and NADPH if the Calvin cycle stopped. 13. Redraw the cycle in Figure 10.19 using numerals to indicate the numbers of carbons instead of gray balls, multiplying at each step to ensure that you have accounted for all the carbons. In what forms do the carbon atoms enter and leave the cycle? Three carbon atoms enter the cycle, one by one, as individual CO2 molecules, and leave the cycle in one three-carbon molecule (G3P) per three turns of the cycle. 14. Review Figures 9.9 and 10.19. Discuss the roles of intermediate and product played by glyceraldehyde 3-phosphate (G3P) in the two processes shown in these figures. In glycolysis, G3P acts as an intermediate. The 6-carbon sugar fructose 1,6bisphosphate is cleaved into two 3-carbon sugars, one of which is G3P. The other is an isomer called dihydroxyacetone phosphate (DHAP), which can be converted to G3P by an isomerase. Because G3P is the substrate for the next enzyme, it is constantly removed, and the reaction equilibrium is pulled in the direction of conversion of DHAP to more G3P. In the Calvin cycle, G3P acts as both an intermediate and a product. For every three CO2 molecules that enter the cycle, six G3P molecules are formed, five of which must remain in the cycle and become rearranged to regenerate three 5-carbon RuBP molecules. The one remaining G3P is a product, which can be thought of as the result of “reducing” the three CO2 molecules that entered the cycle into a 3-carbon sugar that can later be used to generate energy Figure 10.16: The person at the top of the photosystem I tower would not turn to his left and throw his electron into the NADPH bucket. Instead, he would throw it onto the top of the ramp at his right, next to the photosystem II tower. The electron would then roll down the ramp, get energized by a photon, and return to him. This cycle would continue as long as light was available. (This is why it’s called cyclic electron flow.) 15. How is photosynthesis similar in C4 plants and CAM plants? a) In both cases, only photosystem I is used. b) Both types of plants make sugar without the Calvin cycle. c) In both cases, rubisco is not used to fix carbon initially. d) Both types of plants make most of their sugar in the dark. 16. Which of the following statements is a correct distinction between autotrophs and heterotrophs? a) Autotrophs, but not heterotrophs, can nourish themselves beginning with CO2 and other nutrients that are inorganic. b) Only heterotrophs require chemical compounds from the environment. c) Cellular respiration is unique to heterotrophs. d) Only heterotrophs have mitochondria. 17. Which of the following sequences correctly represents the flow of electrons during photosynthesis? a) NADPH -> O2 -> CO2 b) H2O -> NADPH -> Calvin cycle c) H2O -> photosystem I -> photosystem II d) NADPH -> electron transport chain -> O2 18. The following diagram represents an experiment with isolated thylakoids. The thylakoids were first made acidic by soaking them in a solution at pH 4. After the thylakoid space reached pH 4, the thylakoids were transferred to a basic solution at pH 8. The thylakoids then made ATP in the dark. Draw an enlargement of part of the thylakoid membrane in the beaker with the solution at pH 8. Draw ATP synthase. Label the areas of high H+ concentration and low H+ concentration. Show the direction protons flow through the enzyme, and show the reaction where ATP is synthesized. Would ATP end up in the thylakoid or outside of it? Explain why the thylakoids in the experiment were able to make ATP in the dark. The ATP would end up outside the thylakoid. The thylakoids were able to make ATP in the dark because the researchers set up an artificial proton concentration gradient across the thylakoid membrane; thus, the light reactions were not necessary to establish the H+ gradient required for ATP synthesis by ATP synthase. • • In a rosebush, chlorophyll is located in thylakoids, which are in chloroplasts in the mesophyll cells of a leaf. (Thylakoids are arranged in stacks within the 30–40 chloroplasts per typical mesophyll cell. The chlorophyll molecules are embedded in the thylakoid membranes of the chloroplasts.) The reactions of the Calvin cycle are NOT directly dependent on light, but they usually do NOT occur at night as the Calvin cycle requires products only produced when the photosystems are illuminated. (The ATP and NADPH produced during the light reactions are necessary for the Calvin cycle.) • • • 19. a) b) c) d) e) 20. 21. The splitting of water molecules provides the source of electrons for photosystem II. Rubisco is the enzyme in plants that captures CO2 to begin the Calvin cycle. In the Calvin cycle, CO2 is combined with a five-carbon compound to form an unstable six-carbon compound, which decomposes into two three-carbon compounds. Rubisco catalyzes the first step of the Calvin cycle—the incorporation of a molecule of carbon dioxide with a five-carbon sugar. The six-carbon product is so unstable that it decomposes immediately into two three-carbon phosphoglycerates. Glyceraldehyde-3-phosphate (G3P) is produced in the stroma of chloroplasts. For every three molecules of CO2, six molecules of G3P are formed but five molecules must be recycled to regenerate three molecules of RuBP. It is a three-carbon sugar. For every three molecules of CO2 reduced in the Calvin cycle, six molecules of G3P are formed but only one of these molecules exits the cycle to be used by the plant cell. It is produced from glucose during glycolysis. In photosynthesis, what is the fate of the oxygen atoms present in CO2? They end up __________. as molecular oxygen and in sugar molecules The oxygen atoms present in carbon in water dioxide appear in sugar and water. The in sugar molecules and in water splitting of water produces O2. as molecular oxygen in sugar molecules A photon of which of these colors would carry the most energy? a) red b) yellow c) green d) blue (The shorter the wavelength, the greater the energy content.) e) orange During photosynthesis in chloroplasts, O2 is produced from __________ via a series of reactions associated with __________. When photosystem II absorbs light, the electron a) CO2 ... photosystem II that is excited to a higher energy level in the b) H2O ... photosystem I special chlorophyll leaves a "hole" that is filled c) CO2 ... the Calvin cycle when an enzyme extracts electrons from the d) H2O ... photosystem II hydrogen of water, releasing oxygen. e) CO2 ... both photosystem I and the Calvin cycle 22. The light reactions of photosynthesis generate high-energy electrons, which end up in __________ through linear electron flow. The light reactions also produce __________ and __________. a) NADPH ... ATP ... oxygen b) oxygen ... sugar ... ATP The electrons are transferred to NADP+, forming NADPH. ATP is c) chlorophyll ... ATP ... NADPH formed via photophosphorylation, and oxygen is produced when d) water ... sugar ... oxygen water molecules are split. Electrons do not end up in oxygen, and e) ATP ... NADPH ... oxygen sugar is not a product of the light reactions. The high-energy electrons are removed from chlorophyll, not added to it. 23. a) b) c) d) e) f) g) h) i) Which of the following occurs during the Calvin cycle? Excited electrons are conveyed from chlorophyll to an electron acceptor. Light energy is converted to chemical energy. ATP and NADPH are synthesized. CO2 is reduced. Photons are absorbed. ATP is oxidized and NADPH is reduced. (The hydrolysis of ATP releases energy to ATP is reduced and NADPH is oxidized. drive the endergonic reactions of the Calvin Linear electron flow produces the materials required for cycle, and the oxidation of NADPH provides the fixation of carbon from carbon dioxide. high-energy electrons for the reduction of ATP is hydrolyzed and NADPH is oxidized. the carbon from carbon dioxide.)