The SUN Project -Tray as Chloroplast

Transcription

1 The SUN Project -Tray as Chloroplast Ann Batiza,Ph.D., Mary Gruhl, Ph.D., Tom Harrington and Donna LaFlamme, M.S. Milwaukee School of Engineering THYLAKOID LUMEN STROMA Proton Electron NADP + ADP + P i Photosystem II Quinone Proton Pump Plastocyanin Photosystem I ATP Synthase Description of Instructional Tool The two trays correspond to the thylakoid and inner membranes of the chloroplast. Therefore, the brown area is the stroma and the gray area is the thylakoid lumen. The orange component is a proton pump, the quinone is pink and plastocyanin is yellow. The wooden-lobed component is the ATP synthase. All components contain magnets making it possible to position all parts independently and anywhere along the edge of the inner membrane. Small gold magnets represent electrons and small gray balls represent protons. Carbon models that can stick together, ADP and phosphate models are also present. Molecular models of NADP + (electron acceptor) and water made of oxygen plus electrons and protons (electron donor) are also present. Flashlight represents the sun. 1 P a g e

2 Four Key Ideas that can be Demonstrated with this Instructional tool. 1. The membrane structure of the chloroplast 2. The process of Photosynthesis The detailed path of electrons during photosynthesis Reactants and Products (Law of conservation of matter) Pesticides that inhibit the process 3. Energy Transfer in Photosynthesis How and why energy from light moves electrons during photosynthesis (includes the 2 nd Law of Thermodynamics) How electrons move from a donor to an acceptor and how they do work in photosynthesis The role of light in supplying energy at two different points in photosynthesis. That without light, there would ultimately be no life on earth. 4. Biological Diversity in Photosynthesis. Some photosynthetic organisms use PSI alone, PSII alone or both photosystems. Some use an electron donor in photosynthesis that is different from water. 2 P a g e

3 Some Suggested Uses of this Instructional Tool Key idea #1: The membrane structure of the chloroplast Make students aware of the balloon-within-a-balloon membrane structure of the chloroplast. Have them imagine that the trays represent a slice through a three-dimensional object. The chloroplast has three membranes: an outer membrane, an inner membrane and essentially a very convoluted and interconnected thylakoid membrane. Although one can distinguish the small disc-shaped thylakoids and the bridges that connect them, the internal space is largely contiguous so that this creates essentially one or a few large, membrane-bound spaces. The trays represent only the two inside membranes and the spaces therein. The brown tray represents a section of the inner membrane and the gray tray represents a section of the thylakoid membrane. Ask the students in their minds to complete the surface of each balloon so as to approximate the balloon-within-a-balloon structure of a chloroplast. Q: What does the chloroplast look like on the outside? (A: It looks like a three-dimensional oblong structure) Ask students to examine images of chloroplasts from textbook diagrams. Q: How the model tray chloroplast not accurate? (A: The interconnected thylakoid disc structure, the bridges between them and the outer membrane are missing.) You might discuss the limitations of any model. Tell them that this model will be used to clarify what goes on inside the chloroplast and that they will see why the balloon-within-a-balloon structure emphasized by the trays is important. Key idea #2: The process of Photosynthesis Figure 2. The tray configured as a chloroplast. Add two waters to the lumen and 5 carbons in the stroma. The detailed path of electrons during photosynthesis Photosynthesis involves the input of light energy that gets electrons moving. One can use the tray to explore the path of electrons and the effect of these moving electrons on the production of sugar. It is important for students to know that the moving of electrons will eventually cause the production of two products, ATP and NADPH. NADPH and ATP are both required in the Calvin Cycle to make sugar. 3 P a g e

4 The tray should be prepared so that two electrons are placed on PSII and two electrons are placed on PSI. Protons should be evenly distributed between the gray area (THYLAKOID LUMEN) and the brown area (the STROMA). ADP and inorganic phosphate models should be placed in the STROMA. The model of NADP + should have a proton attached to the magnetic nitrogen, which accounts for its positive charge. Two molecules of water should be readied near PSII. Six carbons should be placed individually on the 6-CO 2 strip outside the tray and 5 carbons should be linked together in the stroma. Step Image Description Energy 1 2 Shine the flashlight on PSII. In PSII, light makes electrons start moving. Move the two electrons to the pink quinone. Add two protons from the STROMA to the quinone. H L Light here and at step 5 provide all the energy for photosynthesis. 3 Move the quinone along the membrane to the adjacent proton pump. Move the electrons to the proton pump. Pass the protons that were carried by quinone plus two more protons from the STROMA through the pump to the THYLAKOID LUMEN. 4 P a g e

5 4 Move one electron to the yellow plastocyanin. However, there is a bottleneck. Because there are electrons on PSI, there is no way for electrons to move from plastocyanin to PSI. 5 Therefore, shine the light on PSI. 6 This causes the PSI electrons to move eventually to the carrier NADP +. 7 As the NADP + picks up two electrons from PSI, it also picks up a proton from the STROMA. Therefore, NADP + becomes NADPH. 5 P a g e

6 8 Now move the electrons one-byone to plastocyanin and then to PSI. 9 Move two electrons from water to PSII and place the protons in the THYLAKOID LUMEN. 10 Pass the electrons along the electron transport chain and then add two more electrons from water. Place the protons in the THYLAKOID LUMEN. This forms molecular oxygen, one of the products of photosynthesis. 11 Pass protons from the lumen through the ATP synthase. Then assemble ATP from ADP and P i. Now you have created the two products needed (NADPH and ATP) to power the Calvin cycle. 6 P a g e

7 12 Attach one of the carbon (dioxides) to the 5-carbon compound in the stroma. 13 Move hydrogens from NADPH to the carbon compound and split ATP into ADP and Pi. The series of reactions is more complicated, but eventually all the carbon dioxides can be assembled into a 6-carbon compound in the stroma in addition to a 5-carbon compound so the cycle can begin again. H L Under optimal conditions, 27% of the energy from light is stored in glucose. The transformations required for the Calvin cycle are more complicated. However, the key idea is that the light reactions allow for the formation of NADPH and ATP. Then the NADPH and ATP are used to create carbon-carbon bonds as carbon dioxide from the air is attached to an existing carbon skeleton. About 27% of the original energy from light is now captured in the electrons of these fixed carbon compounds. During cellular respiration, the energy stored in glucose is further transformed in all living things into ATP. However, one can release the energy trapped in those carbon compounds by burning. This is why fossil fuels created from organic matter buried long ago can provide energy. However, once the fuels are removed from the ground and used up, they cannot be replaced. Reactants and Products (Law of conservation of matter) Net equation for Photosynthesis 6H 2 O + 6CO 2 C 6 H 12 O 6 + 6O 2 Net Equation for Cellular Respiration C 6 H 12 O 6 + 6O 2 6H 2 O + 6 CO 2 7 P a g e

8 One might remind students that the net chemical equation that represents photosynthesis is the opposite of that which represents cellular respiration. Students can use the supplied labels Reactant and Product (two of each) to label where the reactants and products of photosynthesis are used and assembled. They may also create labels Water is used here, Carbon dioxide is used here, Oxygen is made here, and Glucose is made here to coincide with the reactant and product labels and reinforce the idea that reactants are used up in the process and products are made. Water produces the product molecular oxygen at a site within PSII. Carbon dioxide is assemble into sugar within the stroma. Water is Used Here (Reactant) Oxygen is Made Here (Product) Carbon Dioxide is Used Here (Reactant) Glucose is Made Here (Product) While our models do not show all the atomic rearrangements required, students can see that as electrons and protons are stripped from two waters at PSII, molecular oxygen (O 2 ) is released. A reactant, water, in losing hydrogen atoms, has given rise to a product molecular oxygen. In addition, a reactant carbon dioxide although we show only the carbon - is transformed through addition of other reactant atoms into the product glucose. 8 P a g e

9 You might discuss the fact that water is actually both a reactant (supplying the electrons to PSII) and a product (during the Calvin cycle) but that there is a net use of water. The need for more reactant waters becomes evident if one tries to account for the evolution of 6O 2 from only 6 H 2 O. It would be good to emphasize the rearrangements that balance the equation so that atoms are neither lost or destroyed during this process the law of conservation of matter. Pesticides that inhibit the process (Interacting with PSII) Atrazine is a pesticide widely used for corn in the United States but banned in the European Union in 2004 because of the likelihood of ground-water contamination. It binds to the site of one of the electron carriers within PSII and prevents the passage of electrons. (1) Evidently, atrazine affects non-photosynthesizing organisms as well, since it is associated with the discovery of male frogs that developed female characteristics when swimming in contaminated water. (2) Figure 3. The map above from the US Department of the Interior (1997) shows the distribution of atrazine across the US at that time. 9 P a g e

10 (Interacting with PSI) The pesticide paraquat takes electrons from PSI and gives them to oxygen. This can create active oxygen species such as superoxide (O2 - ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (OH*) that can damage DNA, proteins, lipids, chlorophyll and membranes. [(3). Also see p. 148 of Bob Blankenship s chapter in the upcoming 5 th edition of Plant Physisology in section 12 of this workshop manual.] Key idea #3: Energy Transfer in Photosynthesis How and why energy from light moves electrons during photosynthesis (includes the 2 nd Law of Thermodynamics) Light is the ultimate source of energy for life on earth. The hydrogen fuel cell shows that when electrons move, they can do work. In the fuel cell, at least some of the energy from moving electrons was captured to do the useful work of spinning a propeller. Sometimes the energy from moving electrons can focus the movement of matter in a particular direction, a very improbable occurrence without the input of energy! Nonetheless, every time electrons move they invariably increase the random motion of matter, i.e. heat. Whenever atoms are forced to move in a particular direction, we can tell that useful work has been done. We can often determine after the fact that work has been done by noticing that something very unlikely has happened. In the case of the fuel cell, the moving electrons were able to spin the motor (a lot of matter in concerted movement!), a very unlikely event in the absence of energy being expended as useful work! Let s see how this idea applies to photosynthesis. When light shines on a chloroplast, the energy captured by an antenna system is funneled to a pair of chlorophyll molecules within PSII. At the proper wavelength (680 nm in green plants) this light will cause electrons in those chlorophylls to become so excited that they are willing to be handed off. In other words, light changes an Unexcited PSII that did not want to part with its electrons (an E in our ABC lesson) into an Excited PSII that easily gives them away (an A in that lesson). In the diagram the energy captured by PSII is shown in green. 10 P a g e

11 Initial Energy Input in Photosynthesis A Greatest Excited PSII (A) Tendency to Give Away Electrons B C D E Least Unexcited PSII (E) Figure 4. Light energy, absorbed by an unexcited PSII that is unwilling to release its electrons excites a central pair of chloropylls so they are willing to release an electron. Once these energetic electrons leave the safe confines of those chlorophylls they are always moving downhill energetically. As they move and lose energy, at least some of that energy is released as heat (the 2 nd Law of Thermodynamics). However, because they have a fruitful path to follow they are able to focus the movement of protons through a proton pump (do useful work). 11 P a g e

12 In the diagram below, the movement of electrons is indicated with a thin black arrow. Light Makes Electrons Start Moving Tendency to Give Away Electrons A B C D Greatest Excited PSII (A) Q (B) Proton Pump (C) E Least Unexcited PSII (E) Figure 5. PSII, excited by light, releases an electron from the central pair of chlorophylls. The electron is handed off within PSII to a quinone and eventually travels through a proton pump. At every step, electrons must travel energetically downhill, giving off a set amount of energy at each step. Some energy is released as heat and some can be used to do work In other words, the release of energy as the electrons exit Excited PSII(A), move to quinone (B) and then from carrier to carrier within the proton pump is accompanied by at least some useful work being done. The protons are pulled so effectively that they nonetheless enter a thylakoid lumen that is becoming increasingly crowded with this positively charged matter. Just imagine how unlikely it is to find protons being added (an indicator that useful work is being done) to all those positive charges jammed into that small space. Once the electrons leave the pump (C) they can move one at a time, still energetically downhill, to plastocyanin (D). Just like cytochrome c in cellular respiration, plastocyanin can dock on the pump. Once it has accepted its electron cargo, it is released to ferry electrons to something even more energetically downhill, Unexcited PSI (E). 12 P a g e

13 However, there is a problem! Unexcited PSI likes to hold onto its electrons and there is no room for an incoming one. The image below shows the path of electrons so far after energy from light is captured. The Path of Energy Input and Electron Movement in Photosynthesis Tendency to Give Away Electrons A B C D Greatest Excited PSII (A) Q (B) Proton Pump (C) Plastocyanin (D) E Least Unexcited PSII (E) Unexcited PSI (E) Figure 6. The path of electrons in photosynthesis is determined to some extent by the willingness of some key players to give away electrons. Here the green arrows indicate energy captured by the sun. This energy changes the Unexcited PSII (E) that hangs onto its electrons tightly into an Excited PSII (A) that is willing to eject an electron. The movement of electrons is indicated by thin black arrows. Notice that the absorption of sunlight increases the tendency to give away electrons and notice that once moving, electrons must always move from something willing to hand them off to something less willing to give them away. Electrons must always move energetically downhill. The dotted line indicates that an electron is not able to move from plastocyanin until one is removed from PSI. At this time, if light of 700nm wavelength is funneled to the reactive pair of chlorophylls in PSI, it can convert the Unexcited PSI (E) into an Excited PSI (A). PSI is now very willing to hand off its electrons. This converts PSI from an E into an A and it willingly hands off electrons from the reactive chlorophylls within it. 13 P a g e

14 The moving electrons pass energetically downhill through a few more proteins and are finally handed off to NADP + which becomes NADPH. Notice the naked proton already present on NADP+. This is why it has a +. NADP + + 2e - + H + NADPH Notice how the original extra positive charge is neutralized by addition of an electron and the extra electron and proton create a hydrogen covalently attached. e 14 P a g e

15 Because these electrons are still quite energetic, one can think of NADPH as being very willing to give them away. It is still a B. (See Figure 7 below.) The Path of Energy Input and Electron Movement in Photosynthesis A Greatest Excited PSII (A) Excited PSI (A) Tendency to Give Away Electrons B C D Q (B) Proton Pump (C) Plastocyanin (D) NADP + (B) E Least Unexcited PSII (E) Unexcited PSI (E) Figure 7. The path of electrons in photosynthesis is determined to some extent by the willingness of some key players to give away electrons. Here the green arrows indicate energy captured by the sun. This energy changes the unexcited photosystems that hang onto their electrons tightly into photosystems that are willing to eject an electron. The movement of electrons is indicated by thin black arrows. The dotted arrow from plastocyanin to the unexcited PSI indicates that this electron can be handed off by plastocyanin only when the excited PSI has ejected its own electron. Notice that the absorption of sunlight increases the tendency to give away electrons and notice that once moving, electrons must always move from something willing to hand them off to something less willing to give them away. Electrons must always move energetically downhill. 15 P a g e

16 We have described the path of electrons using only levels of energy from A to E. In reality, the z-scheme, which refers to a z flipped on its side to describe the input of energy and path of electrons, looks like the diagram below. PSI stimulated by light reaches a level even higher than PSII stimulated by light and so the z-scheme appears to be a careening zig-zag about to take off. Notice how in the diagram below, because we have provided this additional detail, the y-axis Tendency to give away electrons now extends from A to G The Z-Scheme of Energy Input and Electron Movement in Photosynthesis A Greatest Excited PSI (A) B Excited PSII (B) NADP + (B) Tendency to Give Away Electrons C D E F Q (B-) Glucose (C) Proton Pump (C) Plastocyanin (D) Unexcited PSI (E) H 2 O (F) G Least Unexcited PSII (G) Electron #1 Electron #2 Electron #3 Figure 8. Movement across the electron path from water to glucose. Energy is put into the system twice and three different electrons must travel to complete a single electron path during electron transport of photosynthesis. The ultimate electron donor, water (F) can donate electron #1 only after light has ejected electron #2 from PSII. Electron #2 can be handed off to PSI only after light has ejected electron #3 from it. Notice that electron #3 is ultimately handed to glucose. Now we need to do some electron bookkeeping. Let s refer to the z-scheme above. The electron from plastocyanin (D) can now move into Unexcited PSI,which has again become an E. (Once PSI ejects its energetic electrons, it falls back down from being an A to an E ). 16 P a g e

17 In addition, an electron from water (F), normally thought of as a very stable molecule that can be boiled and still not lose its electrons, can nonetheless give its electrons, again energetically downhill, from water (F) to Unexcited PSII (G). The electrons carried by NADPH (B) are ultimately handed off to a carbon compound that will eventually become glucose (C), the storage molecule for energy derived from the sun. Because some energy is lost as heat with each electron movement, only about 30% of the incoming light energy initially captured is actually stored in glucose under ideal conditions and about 5% under field conditions. (4) By comparison, the efficiency of solar photovoltaic cells rarely exceeds 10% (p. 35, 4). Therefore the photosynthetic machinery honed over a few billion years of evolution represents a remarkable conversion of incoming light energy into energy stored in the electrons of glucose. 17 P a g e

18 How electrons move from a donor to an acceptor and do work in photosynthesis Let s take a look at when electrons move from a donor to an acceptor in photosynthesis. You might ask students to consider, What is the ultimate electron donor in photosynthesis? There are several paths by which electrons move from one substance to another, always energetically downhill, always with the loss of some heat. Several major steps are shown below. The letters are all relative, but they show that when electrons move, they always move downhill. This suggests a classroom activity where students play the roles of these various donors and acceptors (labeled as each player and the coded letter) and enact the movement of electrons in photosynthesis. Someone playing the role of Light changes Unexcited PSII(E) into Excited PSII(A) and Unexcited PSI(E) into Excited PSI(A). Donor Acceptor Water (F) Unexcited PSII (G) Excited PSII (B) Plastocyanin (D) Plastocyanin (D) Unexcited PSI (E) Excited PSI (A) NADP + (B) NADPH (B) Glucose (C) The ultimate electron donor is water (F), which must replenish the electron from the spent PSII (G) that has lost one. Two waters are held in place at the bottom of PSII. These donate 4 electrons one-at-a-time to PSII before one molecule of oxygen (O 2 ) is released. The ultimate electron acceptor is glucose (C), since the electrons handed to NADP + and carried as NADPH are eventually added to the glucose precursors constructed during the Calvin cycle. Notice that we have said that glucose in this relative world is a C. Those electrons are still able to move and we will see in the mitochondrion that they continue to do so. What work do the moving electrons do? Moving electrons power that single pump in each cycle to concentrate protons that will ultimately fuel the ATP synthase to make ATP. But the work of photosynthesis is not over there. ATP, which ultimately derives its energy from moving electrons, and NADPH, which is ready to donate electrons of its own, are used to power the reactions of the Calvin cycle and give those energetic electrons to glucose. So the sequential work done by moving electrons in photosynthesis is: 18 P a g e

19 Pumping protons and eventually making ATP Making NADPH Using ATP and NADPH to make glucose 19 P a g e

20 How energy from light is moved to glucose during photosynthesis Ask students to use the supplied Energy label to trace the path of energy in photosynthesis along the tray components. They will find that energy takes a two-pronged path that converges at the sticky carbons that represent glucose. Figure 9. The Path of Energy in Photosynthesis (The arrows give the sequence through which the Energy label will pass on the tray. They do not indicate the relative energy at each step. ) Flashlight Moving electrons from PSII Moving electrons through the pump Light (again!) Concentrated Protons Moving electrons from PSI Turning ATP synthase NADPH ATP Glucose If the class has already charted the path of energy on the tray in cellular respiration, they could compare the two diagrams. They will see that photosynthesis is much more complicated! 20 P a g e

21 The role of light in supplying energy at two different points in photosynthesis At PSII, light supplies energy to excite and eject an electron so it can move energetically downhill. The moving electrons cause the pump to pump protons into the lumen. Ultimately these concentrated protons fuel the ATP synthase which makes ATP. At PSI, light supplies energy to excite and eject an electron so it can be passed energetically downhill to NADP + to make NADPH. Ulimately these electrons are used to fix carbon. Some bacteria have only one or the other system. That without light, there would ultimately be no life on earth Ask students to put down the flashlight and imagine the impact of the absence of light on what goes on in the chloroplast. As a class they could brainstorm a list of effects as they examine the tray. Examples might be: No electron is popped out of the chlorophylls in PSII. No electrons move. They just stay where they are. No protons are concentrated. The ATP synthase central mechanism does not turn. No ATP is made. No more electrons are handed off to NADP+ to make NADPH. No glucose is made because there is no NADPH to supply hydrogens and no ATP to supply energy to fix carbon. The plant that depends on the glucose as a source of carbon building blocks cannot produce any new plant material including leaves or shoots. The plant that depends on the glucose as a source of energy for making ATP cannot supply ATP for any life processes such as o maintaining a membrane potential that is used for the transport of materials in and out of the cell; o gating ion channels that depend upon ATP to open; o supplying energy for synthetic reactions; o supplying energy for glycolysis. Therefore, not only will plants not produce new shoots, eventually they will wither away and die. 21 P a g e

22 The diagram below introduces terms such as producers, primary consumers and secondary consumers to illustrate that all living things ultimately depend upon light as a source of energy. Producer Primary Consumer Secondary Consumer Figure 10. All living things ultimately depend upon the sun for energy. Energy from the sun is captured in glucose made by green plants and some pigmented microbes (producers). Some living things (primary consumers) get their energy from glucose and other carbon compounds containing high energy electrons directly from plants. Other living things (secondary consumers) get energy by consuming the carbon compounds contained in the primary consumer that ultimately came from producers. One might next ask students to imagine the impact of the absence of light on the livelihood of cows, horses, deer, etc. that eat these plants. Similarly one might ask students to imagine the impact on humans that depend upon glucose from plants and glucose derived from the organic compounds in animals. Students might also have mentioned that without light, oxygen (the ultimate electron acceptor for us and many other living things) will not be produced. But it might be important to point out that independent of this fact, the absence of light will stop the flow of electrons and storing of those energetic electrons in those carbon-carbon bonds. This will kill off all living things, regardless of the impact on the oxygen environment. 22 P a g e

23 Key Idea # 4. Biological Diversity in Photosynthesis The chart below shows six different types of photosynthetic bacteria. Note that some have only photosystem I, some have only photosystem II and only cyanobacteria has both PSI Photosystem Types Present in Photosynthetic Bacteria with an Example Species PS Heliobacteria I Heliobacillus I Green Sulfur Chlorobium (H 2 S is the electron donor) Cyanobacteria II I Cyanobacteria (H 2 O is the electron donor) Purple Nonsulfur Bacteria II Rhodospirillum rubrum Rhodopseudomonas palustris Green FAPs II Red FAPs II and PSII like green plants. An excellent paper about these variations and their possible evolution is in Section 4 of your workshop manual reprinted with permission from Nature. It is called Out of Thin Air and is by John F. Allen and William Martin from February 8, P a g e

24 References (1) North Carolina State University s crop science Web site at accessed December 4, 2008, as well as Wikipedia and references therein. (2) BBC News at accessed December 4, (3) online ScienceDirect College Edition Pesticide Biochemistry, from Elsevier at accessed (4) Blankenship, Robert (2002) Molecular Mechanisms of Photosynthesis, Blackwell Science Limited, Oxford. Link to Supplementary Material One of the best links to information on photosynthesis is at the University of Arizona at 24 P a g e