The SUN Project Tray as Mitochondrion

The SUN Project Tray as Mitochondrion Ann Batiza, Ph.D. and Mary Gruhl, Ph.D, MATRIX Outer membrane (brown tray) Carbon compound INTERMEMBRANE SPACE Inner membrane (gray tray) Proton Electron ATP ADP P i NAD ATP Synthase Proton Pump (Complex I) Quinone Proton Pump (Complex III) Cytochrome c Proton Pump (Complex IV) Molecular oxygen Description of this Instructional Tool The two trays correspond to the inner and outer membranes of the mitochondrion. The brown space = intermembrane space. The gray space = matrix. The orange components correspond to the three proton pumps. The pink model is quinone and the blue model is cytochrome c. The wooden lobed component is the ATP synthase. All components can be positioned independently along the inner membrane. Small gold magnets represent electrons Small gray balls represent protons. Molecular models of NAD, ATP made of ADP and inorganic phosphate, and molecular oxygen are present. The large grey balls represent carbon atoms within a carbon compound. 1 P age

Four Key Ideas that can be Demonstrated with this Instructional Tool 1. The membrane structure of the mitochondrion 2. The process of Cellular Respiration The detailed path of electrons during cellular respiration Reactants and Products (Law of Conservation of Matter) and why organisms like us require both food and oxygen to live. Poisons that inhibit this process The necessary positioning of the ATP synthase 3. Energy Transfer in Cellular Respiration How and why energy from glucose is moved to ATP (including the 2 nd Law of Thermodynamics). How electrons move from a donor to an acceptor and how they do work in cellular respiration How energy from glucose is moved to ATP during cellular respiration Why having three pumps rather than just one is important. That without glucose, we could not live 4. Evolution and Biological Diversity in Cellular Respiration The minimal structures required for a sluggish production of ATP How other products would be produced if there were alternative electron acceptors on Complex IV. How organisms that use alternative electron acceptors are able to live in environments without oxygen. Why some living things require neither food nor oxygen to live and how this can increase biodiversity. 2 P age

Notice that the proton pumps and the ATP sythase shown on the tray above correspond to the protein complexes whose structures are shown below. Complex I Complex III Complex IV ATP Synthase NADH dehydrogenase Cytochromee bc 1 complex Cytochrome c oxidase 3 P age

Some Suggested Uses of this Instructional Tool Key idea #1. The membrane structure of the mitochondrion Inner Membrane INTERMEMBRANE SPACE MATRIX Outer Membrane Figure 2. Mitochondrial spaces represented by the gray and brown trays. Make students aware of the balloon within a balloon membrane structure of the mitochondrion. Have them imagine that the trays represent a slice through a threedimensional object. The mitochondrion has two membranes. The outer membrane is represented by the brown tray and the inner membrane is represented by the gray tray. Note that the inner membrane of the mitochondrion is actually very convoluted like the diagram above, but the matrix space is all contiguous. Ask them to in their minds complete the surface of each balloon so as to approximate the structure of a mitochondrion. Ask them how the mitochondrion looks on the outside. (A: like a solid oblong structure) Examine images of mitochondria from textbook diagrams. Ask the students how the model tray mitochondrion is not accurate. (A: It is flatter and the inner membrane is not convoluted like that in the images.) You might discuss the limitations of any model. Tell them that this model will be used to clarify what goes on inside the mitochondrion and why that balloon within a balloon structure is important. Students can use the supplied labels to define structures within the mitochondrion. For example, they might label: mitochondrion, inner membrane, outer membrane, matrix and intermembrane space. They might compare these structures to diagrams and microscopic images in textbooks. This should not be an end point, but rather a way to enable students to clearly discuss ideas regarding the process of cellular respiration in future lessons. 4 P age

Key idea #2. The process of Cellular Respiration The detailed path of electrons during cellular respiration. Have students use the tray components to follow the path of electrons from the electron donor (sugar) to electron acceptor (oxygen) so that in between the moving electrons can do work (pumping protons into the intermembrane space). The usable energy from those concentrated protons fuel the ATP synthase so that at least some of the energy from those moving electrons can be stored in ATP. Step Image Description Energy 1 Two electrons and one proton are moved from the carbon compound (broken down sugar or citric acid) to NAD. Note that the citric acid is made from a 2 carbon fragment of sugar plus the 4 carbon oxaloacetic acid that was waiting in the matrix. Sugar is the ultimate electron donor for cellular respiration. Sugar is energy source. NAD 2 carries two electrons and a proton to the 1 st proton pump (called Complex I). H L About 30% of the sun s energy (that hit the plant) was originally stored in glucose. (Just leave the proton in the matrix.) 3 The movement of these electrons causes the pump to pump four protons from the MATRIX into the INTERMEMBRANE SPACE.. The moving electrons do the work of causing the pump (Complex I) to pump protons. Notice that energy of moving electrons is being stored in these concentrated protons. The chemical energy of glucose energy of moving electrons. Some energy is lost with each transfer. 5 P age

4 The electrons are handed from Complex I to a quinone docked in a crevice within. Quinone also picks up two protons from the pool of protons in the MATRIX. 5 Quinone moves through the membrane to the 2 nd proton pump (called Complex III) and attaches the two electrons to the pump. The two protons on the quinone are pumped through the pump into the INTERMEMBRANE SPACE.. (Note that this pump is called Complex III. Complex II is not shown here and it does not pump protons.) 6 The movement of electrons across the pump causes two more protons to be pumped from the MATRIX to the INTERMEMBRANE SPACE. by Complex III. The moving electrons do the work of causing the pump (Complex III) to pump protons. Note that energy is stored in the concentrated protons. 7 From the 2 nd proton pump electrons are passed one at a time to cytochrome c. Energy of moving electrons energy of concentrated protons. But some energy is lost as heat. 6 P age

Cytochrome c is a mobile carrier 8 that hands the electrons to the 3 rd proton pump (Complex IV). (Notice that while two electrons were moved by quinone, only one electron can be carried at one time by cytochrome c. Therefore it must make two trips to ferry both electrons to the 3 rd proton pump.) 9 Eventually both electrons are carried by cytochrome c (one at a time) to the 3rd proton pump. 10 The moving electrons again do the work of causing the pump (Complex IV) to pump two protons into the INTERMEMBRANE SPACE.. Again, energy is being stored in the concentrated protons. Energy of moving electrons energy of concentrated protons. But again some energy is lost as heat. 11 Molecular oxygen (O 2 ) binds to the 3 rd proton pump, Complex IV. 7 P age

12 Molecular oxygen picks up the two electrons from the 3 rd proton pump, Complex IV. (Two more electrons will be added in the next step.) Oxygen is the ultimate electron acceptor for cellular respiration. 13 Two more electrons are moved from sugar to molecular oxygen along this electron transport chain to make a total of four electrons attracted to the oxygen molecule. Now four protons are added from the MATRIX. 14 Only now can these products be released as two molecules of water. 15 Finally, the protons that were concentrated in the intermembrane space fuel the ATP synthase. This nanomachine makes ATP from ADP and inorganic phosphate. (See the lessons on ATP and the ATPsynthase for details.) Now some of the energy from glucose is stored in ATP. H L Energy of concentrated protons chemical energy of ATP. Some is lost as heat. Some energy was lost with each energy transfer. 8 P age

Notice that some key points in this process of moving electrons from sugar to oxygen and the production of ATP have been highlighted. The ultimate electron donor is glucose, the ultimate electron acceptor is oxygen. While the electrons are moving from glucose to oxygen, they do work. The work they do is to cause the proton pumps to pump protons from the MATRIX into the INTERMEMBRANE SPACE.. Figure 3. This schematic image of the trays depicts the matrix (gray) and the intermembrane space (brown) of the mitochondrion. The signs represent protons which become concentrated in the intermembrane space. As the electrons move from a donor to an acceptor, they release energy. Some of that energy is released as heat, but some of that energy released along the way was stored in the concentrated protons. (Because it took energy to force them into that space, energy resides with the concentrated protons.) The only door open in the impermeable inner membrane is through the mechanism of the ATP synthase. Because the protons are concentrated about 10 times more in the intermembrane space than in the matrix, which accounts for a single ph difference in these compartments, by chance, protons will bounce into the ATP synthase, turn the crank on this nanomachine, and cause ATP to be made. Therefore at least some of the energy originally stored in glucose will ultimately resides in ATP. Students can carry out all these processes. They can: Strip electrons and protons from the carbons Move electrons from pump to pump Pump protons into the intermembrane space, Make water molecules as oxygen atoms accept electrons and protons. Make ATP using the small ATP synthase and small ATP models. Use the energy meter to record what happens to energy as energy is transferred. It would probably be good to use the electron donor, electron acceptor, and Moving electrons do work labels to emphasize these aspects of the process. In addition, it would be good for 9 P age

students to move the Energy label from the carbons, to the moving electrons, to the concentrated protons, to the ATP synthase and eventually to ATP to reemphasize how energy is transferred from glucose to ATP in cellular respiration. 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 One might remind students that the net chemical equation of cellular respiration is the opposite of that for photosynthesis. Students can use the supplied labels Reactant and Product (two of each) to label where the reactants and products of cellular respiration are used and created. They may also use the labels they cut out: Glucose is used here, Oxygen is used here, Carbon dioxide is made here and Water is made here to coincide with the reactant and product labels and reinforce the idea that reactants are used up in the process as products are made from the reactant atoms. Glucose is Used Here (Reactant) Carbon Dioxide is Made Here (Product) Oxygen is Used Here (Reactant) Water 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 a break down product of glucose in the MATRIX, carbon dioxide (CO 2 ) is released. 10 P age

Similarly at the 3 rd proton pump, a reactant oxygen, in gaining hydrogen atoms and electrons, gives rise to a product water. Actual Equation for Cellular Respiration C 6 H 12 O 6 6H 2 O 6O 2 12H 2 O 6 CO 2 You might discuss the fact that water is both a reactant (during the breakdown of glucose in the Krebs cycle in the MATRIX) and a product (at the 3 rd proton pump) but that there is a net production of water. The need for more reactant and product waters becomes evident if one tries to account for the production of water from 6 reactant O 2 molecules. It would be good to emphasize the rearrangements that balance the equation so that no atoms are either lost or destroyed during this process the law of conservation of matter. Poisons that inhibit the process Cyanide and carbon monoxide bind on Complex IV and block the hand off of electrons from Complex IV to oxygen. Refer students to the ABC lesson that dealt with what is required in order for electrons to move (a continuous source of electron donors and acceptors). Discuss with them what part of the process these poisons interfere with (A: the moving of electrons to the electron acceptor). Ask them what they think happens to electrons that are already 11 P age

in the queue on the other pumps (A: The electrons just stay there, but because they aren t moving, no more protons are being pumped and therefore no more ATPs are being made.) The necessary positioning of the ATP synthase Ask how the ATP synthase must be positioned in order to make ATP. One can approach this in two ways. One way is to say that ATP is made in the MATRIX and to ask students to position the ATP synthase correctly so that the bulbs are in the MATRIX. Alternatively one can say that protons are concentrated in the INTERMEMBRANE SPACE. and ask how the ATP synthase must be positioned. Since the ATP synthase is fueled by protons the ATP synthase must be positioned so as to allow protons to enter by chance from the INTERMEMBRANE SPACE.. Key idea #3: Energy Transfer in Cellular Respiration How and why energy from glucose moves electrons during cellular respiration (includes the 2 nd Law of Thermodynamics) The hydrogen fuel cell in an earlier lesson demonstrated that when electrons move, they release energy. We saw that in the case of the fuel cell at least some of that energy from moving electrons was captured to do the useful work of spinning a propeller. Nonetheless, every time electrons move they will invariably increase the random motion of matter, i.e., heat. (Remember the exploding hydrogen oxygen filled balloon!) However, 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! The figure below is based on Peter Atkin s Four Laws that Drive the Universe. Work Heat Figure 4. Energy used to do work can make matter move in a particular direction. Heat increases the random motion of matter. 12 P age

Whenever atoms are forced to move in a particular direction, useful work has been done. We can often determine that work has been done after the fact by noticing that something has happened that is very unlikely. In the case of the fuel cell, the moving electrons were able to spin the motor (a lot of matter in concerted movement!) and ultimately the attached propeller. How unlikely would it have been for the propeller to have turned on its own? But the Second Law of Thermodynamics says that with each energy transfer, even when work is done, some energy must be released as heat. Let s see how the 2 nd law applies to cellular respiration. Electrons in glucose are willing to be handed off. Once these energetic electrons leave glucose they are always moving downhill energetically. They always move from something more willing to release them to something less willing to release them. As they move and lose energy, at least some of that energy is released as heat. However, because of the path they follow first to NAD and then through a series of proton pumps (via quinone and cytochrome c) to oxygen they are also able to do some useful work. They focus the movement of protons through those proton pumps. As the electrons move along a set path within the proton pumps, they pull protons through the pump to the other side. At least some of the energy the electrons release as they move through each pump pulls the protons through. In the diagram below, the movement of electrons is indicated with a thin black arrow. A Greatest Glucose Tendency to Give Away Electrons B C D E Least 1 st proton pump 2 nd proton pump 3 rd proton pump Oxygen Figure 5. Glucose releases electrons to NAD (not shown) which carries electrons to the 1 st proton pump. The electron is handed off from there to a quinone (not shown) and eventually travels through another proton pump, to cytochrome c (not shown) and eventually to oxygen. At every step, electrons must travel energetically downhill, giving off a set amount of energy. Some energy is released as heat and some can be used to do work. The initial work done is to compel protons to move through the pumps from the MATRIX to the INTERMEMBRANE SPACE., even though they become concentrated. Therefore some of the energy released as electrons move from glucose to oxygen is stored in the concentrated protons. 13 P age

Notice how the electrons move from the ultimate electron donor, glucose, to the ultimate electron acceptor, oxygen. Glucose is much more willing to give electrons away than oxygen. We see this when we apply some heat to encourage electrons to move from glucose to oxygen. This happens when ancient carbon compounds condensed by heat and pressure into oil or coal is burned to release carbon dioxide and water. However, once water is made, the electrons cannot be enticed to leave. If we try to burn water, it hangs onto its electrons and the molecules of water merely fly off into the air as a gas. How electrons move from a donor to an acceptor and do work in cellular respiration Let s take a closer look at when electrons move from a donor to an acceptor in cellular respiration. You might ask students to consider, What is the ultimate electron donor in cellular respiration? In cellular respiration, electrons move from one entity to another, always energetically downhill, always with the loss of some heat. The major steps are diagrammed below. The letters are all relative, but are used to 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 cellular respiration. Donor Acceptor Glucose (A) 1 st Proton Pump (B) 1 st proton Pump (B) 2 nd Proton Pump (C) 2 nd Proton Pump (C) 3 rd Proton Pump (D) 3 rd Proton Pump (D) Oxygen (E) The ultimate electron donor is glucose (A), whose electrons are moved to the 1 st proton pump and so on. The ultimate electron acceptor is oxygen (E), since the electrons handed from pump to pump are eventually handed off to oxygen at the 3 rd proton pump. Notice that we have said that oxygen in this relative world is an E. The electrons used to make water are very stable and unwilling to move without a very tempting target. We will see in the lesson about the chloroplast that even water can be induced under the right circumstances to lose its electrons. 14 P age

What work do the moving electrons do? Moving electrons power that series of pumps in each cycle to concentrate protons. Those protons ultimately fuel the ATP synthase so as to make ATP. So the work done by moving electrons in photosynthesis is: Pumping protons into the intermembrane space. How energy from glucose is moved to ATP during cellular respiration Ask students to use the supplied Energy label to trace the path of energy in cellular respiration along the tray components. They will find that energy takes a two pronged path. As electrons move from glucose to oxygen they lose energy at each step. However some of their energy is used to concentrate the protons. Ultimately the concentrated protons power the turning of the ATP synthase and combining of ADP and P i to make ATP. The Path of Energy in Cellular Respiration. The arrows give the sequence through which the Energy label will pass on the tray. Notice the branching path. Ultimately some useable energy is stored in ATP as chemical energy. Heat is released during each transition. Electrons move from glucose Electrons move to NAD Electrons move through the 1 st proton pump Electrons move to quinone Electrons move through the 2 nd proton pump Electrons move to cytochrome c Electrons move through the 3 nd proton pump The turning of the ATP synthase ATP (chemical energy is stored here.) Electrons move to oxygen 15 P age

So far, we have not discussed the movement of electrons from FADH 2 generated along with NADH during the Krebs Cycle to the electron transport chain. You may want to save these details for your AP biology class. We have already seen that NADH deposits its electrons at Complex I. FADH 2 is part of Complex II which does not itself pump protons. Complex II only abuts the membrane; it is not an integral membrane protein. However FAD H 2 initially gives electrons to quinone, not to Complex I. Therefore electrons donated by FADH 2 are able to stimulate pumping by only Complexes III and IV; its electrons do not pass through Complex I and stimulate its proton pumping. This is why each FADH 2 stimulates the production of only 2 ATP while each NADH stimulates the production of 3ATP. Links to Supplementary Material A You Tube animation of cellular respiration is called Cellular Respiration (Electron transport chain). This video shows the movement of electrons and reinforces terms such as electron donor and electron acceptor. However, it introduces other names for the proton pumps. Complex I is called NADH Dehydrogenase, Complex III is called Cytochrome b c1 and Complex IV is called Cytocrhome c oxidase. It introduces details about the numbers of protons pumped that may be inaccurate, but the animation is useful nonetheless. One might ask students to see what they learn from it and what is still confusing. A more advanced and detailed version of the electron transport chain can be seen at www.johnkyrk.com/mitochondrion.html. This site was recommended by the SUN Teacher Advisory Board. It might be more appropriate to show students after they have used the membrane mat to see how electrons move from carrier to carrier. This site does an excellent job of showing how the accumulated proton gradient fuels the ATP synthase. 16 P age