Vocabulary. Chapter 20: Electron Transport and Oxidative Phosphorylation

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1 Vocabulary ATP Synthase: the enzyme responsible for production of ATP in mitochondria Chemiosmotic Coupling: the mechanism for coupling electron transport to oxidative phosphorylation; it requires a proton gradient across the inner mitochondrial membrane Coenzyme Q: an oxidation reduction coenzyme in mitochondrial electron transport Conformational Coupling: a mechanism for coupling electron transport to oxidative phosphorylation that depends on a conformational change in the ATP synthetase Cytochrome: any one of a group of heme-containing proteins in the electron transport chain Glycerol Phosphate Shuttle: a mechanism for transferring electrons from NADH in the cytosol to FADH2 in the mitochondrion Malate Aspartate Shuttle: a mechanism for transferring electrons from NADH in the cytosol to NADH in the mitochondrion Q Cycle: a series of reactions in the electron transport chain that provides the link between twoelectron transfers and one-electron transfers Proton Gradient: the difference between the hydrogen ion concentrations in the mitochondrial matrix and that in the intermembrane space, which is the basis of coupling between oxidation and phosphorylation P/O Ratio: the ratio of ATP produced by oxidative phosphorylation to oxygen atoms consumed in electron transport Reduction Potential: a standard voltage that indicates the tendency of a reduction half reaction to take place Respiratory Complexes: the multi-enzyme systems in the inner mitochondrial membrane that carry out the reactions of electron transport Uncoupler: a substance that overcomes the proton gradient in mitochondria, allowing electron transport to proceed in the absence of phosphorylation

2 Chapter Summary What is the importance of mitochondrial structure in ATP production? In the final stages of aerobic metabolism, electrons are transferred from NADH to oxygen (the ultimate electron acceptor) in a series of oxidation reduction reactions known as the electron transport chain. In the process, protons are pumped across the inner mitochondrial membrane. This series of events depends on the presence of oxygen in the final step. This pathway allows for the reoxidation of the reduced electron carriers produced in glycolysis, the citric acid cycle, and several other catabolic pathways, and is the true source of the ATPs produced by catabolism. Phosphorylation depends on the compartmented structure of mitochondria. How can reduction potentials be used to predict the direction of electron transport? The overall reaction of the electron transport chain shows a very large, negative DG ' due to the large differences in reduction potentials between the reactions involving NADH and those involving oxygen. If NADH were to reduce oxygen directly, the DE ' would be more than1 V. In reality, many redox reactions are in between, and the correct order of events in the electron transport chain was predicted by comparing the reduction potentials of the individual reactions long before the order was established experimentally. What reactions take place in the respiratory complexes? Four separate respiratory complexes can be isolated from the inner mitochondrial membrane. Each can carry out the reactions of a portion of the electron transport chain. In addition to the respiratory complexes, two electron carriers, coenzyme Q and cytochrome c, are not bound to the complexes but are free to move within and along the membrane respectively. Complex I accomplishes the reoxidation of NADH and sends electrons to coenzyme Q. Complex II reoxidizes FADH2 and also sends electrons to CoQ. Complex III involves the Q cycle and shuttles electrons to cytochrome c. Complex IV takes the electrons from cytochrome c and passes them to oxygen in the final step of electron transport. What is the nature of the iron-containing proteins of electron transport? A number of iron-containing proteins are part of the electron transport chain. In the cytochrome proteins, the iron is bound to a heme group. In other proteins, the iron is bound to the protein along with sulfur.

3 What is the coupling factor in oxidative phosphorylation? A complex protein oligomer is the coupling factor that links oxidation and phosphorylation. The complete protein spans the inner mitochondrial membrane and projects into the matrix as well. The portion of the protein that spans the membrane is called F0; it consists of three different kinds of polypeptide chains (a, b, and c).the portion that projects into the matrix is called F1; it consists of five different kinds of polypeptide chains (a, b, g, d, and e, in the ratio a3b3gde). The F1 sphere is the site of ATP synthesis. The whole protein complex is called ATP synthase. It is also known as mitochondrial ATPase. During the process of electron transport, several reactions occur in which reduced carriers that have both electrons and protons to donate are linked to carriers that can only accept electrons. At these points, hydrogen ions are released to the other side of the inner mitochondrial membrane, causing the formation of a ph gradient. The energy inherent in the charge and chemical separation of the hydrogen ions is used to phosphorylate ADP to ATP when the hydrogen ions pass back into the mitochondria through ATP synthase. What is chemiosmotic coupling? Two mechanisms, the chemiosmotic mechanism and the conformational coupling mechanism, have been proposed to explain the coupling of electron transport and ATP production. Chemiosmotic coupling is the mechanism most widely used to explain the manner in which electron transport and oxidative phosphorylation are coupled to one another. In this mechanism, the proton gradient is directly linked to the phosphorylation process. The way in which the proton gradient leads to the production of ATP depends onion channels through the inner mitochondrial membrane; these channels are a feature of the structure of ATP synthase. Protons flow back into the matrix through proton channels in the F0 part of the ATP synthase. The flow of protons is accompanied by formation of ATP, which occurs in the F1 unit. What is conformational coupling? In the conformational coupling mechanism, the proton gradient is indirectly related to ATP production. Recent evidence appears to show that the effect of the proton gradient is not the formation of ATP but the release of tightly bound ATP from the synthase as a result of the conformational change.

4 Do respiratory inhibitors have a connection with respiratory complexes? Many of the workings of the electron transport chain have been elucidated by experiments using respiratory inhibitors. These inhibitors specifically block the transfer of electrons at specific points in the respiratory complexes. Examples are CO and CN2, both of which block the final step of the electron transport chain, and rotenone, which blocks the transfer of electrons from NADH reductase to coenzyme Q. When such a blockage occurs, it causes electrons to pile up behind the block, giving a reduced carrier that cannot be oxidized. By noting which carriers become trapped in a reduced state and which ones are trapped in an oxidized state, we can establish the link between carriers. How do shuttle mechanisms differ from one another? Two shuttle mechanisms the glycerol phosphate shuttle and the malate aspartate shuttle transfer the electrons, but not the NADH, produced in cytosolic reactions into the mitochondrion. In the first of the two shuttles, which is found in muscle and brain, the electrons are transferred to FAD; in the second, which is found in kidney, liver, and heart, the electrons are transferred to NAD+. With the malate aspartate shuttle, 2.5 molecules of ATP are produced for each molecule of cytosolic NADH, rather than 1.5 ATP in the glycerol phosphate shuttle, a point that affects the overall yield of ATP in these tissues. Approximately 2.5 molecules of ATP are generated for each molecule of NADH that enters the electron transport chain and approximately 1.5 molecules of ATP for each molecule of FADH2. When glucose is metabolized anaerobically,the only net ATPs that are produced are those from the substrate-level phosphorylation steps. This leads to a total of only two ATPs per glucose entering glycolysis. When the pyruvate generated from glycolysis can enter the citric acid cycle, and the resulting NADH and FADH2 molecules are reoxidized through the electron transport chain, a total of 30or 32 ATPs are produced, with the difference being due to the two possible shuttles.

5 Questions and Answers 1. Briefly summarize the steps in the electron transport chain from NADH to oxygen. Electrons are passed from NADH to a flavin-containing protein to coenzyme Q. From coenzyme Q, the electrons pass to cytochrome b, then to cytochrome c, via the Q cycle, followed by cytochromes a and a3. From the cytochrome aa3 complex, the electrons are finally passed to oxygen. 2. Are electron transport and oxidative phosphorylation the same process? Why or why not? Electron transport and oxidative phosphorylation are different processes. Electron transport requires the respiratory complexes of the inner mitochondrial membrane, whereas oxidative phosphorylation requires ATP synthase, also located on the inner mitochondrial membrane. Electron transport can take place in the absence of oxidative phosphorylation. 3. List the reactions of electron transport that liberate enough energy to drive the phosphorylation of ADP. In all reactions, electrons are passed from the reduced form of one reactant to the oxidized form of the next reactant in the chain. The notation number of iron sulfur proteins. refers to any one of a

6 4. Show how the reactions of the electron transport chain differ from those in Question 3 when FADH2 is the starting point for electron transport. Show how the reactions that liberate enough energy to drive the phosphorylation of ADP differ from the pathway when NADH is the starting point. When FADH2 is the starting point for electron transport, electrons are passed from FADH2 to coenzyme Q in a reaction carried out by Complex II that bypasses Complex I. 5. How does mitochondrial structure contribute to aerobic metabolism, particularly to the integration of the citric acid cycle and electron transport? Mitochondrial structure confines the reduced electron carriers produced by the citric acid cycle to the matrix. There they are close to the respiratory complexes of the electron transport chain that will pass the electrons from the carriers produced by the citric acid cycle to oxygen, the ultimate recipient of electrons and hydrogens. 6. Why is it reasonable to compare the electron transport process to a battery? The electron transport chain translocates charged particles by chemical means. Interconversion of chemical and electrical energy is exactly what a battery does.

7 7. Using the information in Table 20.2, calculate DG ' for the following reaction: 8. Why are all the reactions in Table 20.1 written as reduction reactions? The reactions are all written in the same direction for purposes of comparison. By convention, they are written as reduction, rather than oxidation, reactions.

8 9. Calculate E ' for the following reaction:

9 10. Calculate E ' for the following reaction: 11. Calculate E ' for the following reaction:

10 12. For the following reaction, identify the electron donor and the electron acceptor and calculate E '. 13. Which is more favorable energetically, the oxidation of succinate to fumarate by NAD+ or by FAD? Give the reason for your answer.

11 14. Comment on the fact that the reduction of pyruvate to lactate, catalyzed by lactate dehydrogenase, is strongly exergonic(recall this from Chapter 15), even though the standard free-energy change for the half reaction is positive (DG ' kj mol kcal mol21), indicating an endergonic reaction. 15. What do cytochromes have in common with hemoglobin or myoglobin? They all contain the Heme group, with minor differences in the heme side chains in most cytochromes. 16. How do the cytochromes differ from hemoglobin and myoglobin in terms of chemical activity? Cytochromes are proteins of electron transport; the heme ion alternates between the Fe(II) and Fe(III) states. The function of hemoglobin and myoglobin is oxygen transport and storage, respectively. The iron remains in the Fe(II) state. 17. Which of the following does not play a role in respiratory complexes: cytochromes, flavoproteins, iron sulfur proteins, or coenzyme Q? Coenzyme Q is not bound to any of the respiratory complexes. It moves freely in the inner mitochondrial membrane. 18. Do any of the respiratory complexes play a role in the citric acid cycle? If so, what is that role? A part of Complex II catalyzes the conversion of succinate to fumarate in the citric acid cycle. 19. Do all the respiratory complexes generate enough energy to phosphorylate ADP to ATP? Three of the four respiratory complexes generate enough energy to phosphorylate ADP to ATP. Complex II is the sole exception.

12 20. Two biochemistry students are about to use mitochondria isolated from rat liver for an experiment on oxidative phosphorylation. The directions for the experiment specify addition of purified cytochrome c from any source to the reaction mixture. Why is the added cytochrome c needed? Why does the source not have to be the same as that of the mitochondria? Cytochrome c is not tightly bound to the mitochondrial membrane and can easily be lost in the course of cell fractionation. This protein is so similar in most aerobic organisms that cytochrome c from one source can easily be substituted for that from another source. 21. Cytochrome oxidase and succinate-coq oxidoreductase are isolated from mitochondria and are incubated in the presence of oxygen, along with cytochrome c, coenzyme Q, and succinate. What is the overall oxidation reduction reaction that can be expected to take place? 22. What are two advantages of the components of the electron transport chain being embedded in the inner mitochondrial membrane? The components are in the proper orientation for the electrons to be transferred rapidly from one component to the next; if the components were in solution, speed would be limited to the rate of diffusion. A second advantage, which is actually a necessity, is that the components are properly positioned to facilitate the transport of protons from the matrix to the intermembrane space. 23. Reflect on the evolutionary implications of the structural similarities and functional differences of cytochromes on the one hand and hemoglobin and myoglobin on the other. From an evolutionary standpoint, two different functions can be performed by identical structures or by structures that are close to identical, with only minor differences in the protein moieties. The organism saves a considerable amount of energy by not having to evolve and to operate two pathways.

13 24. Experimental evidence strongly suggests that the protein portions of cytochromes have evolved more slowly (as judged by the number of changes in amino acids per million years) than the protein portions of hemoglobin and myoglobin and even more slowly than hydrolytic enzymes. Suggest a reason why. The key point here is not the active site, which has a low tolerance for mutations, but the molecules with which the proteins in question are associated. Cytochromes are membrane-bound and must associate with other members of the electron transport chain; most mutations are likely to interfere with the close fit, and thus they are not preserved (because they are lethal). Globins, although soluble, still form some associations, so more mutations can be tolerated, with some limits. Hydrolytic enzymes are soluble and not likely to associate with other polypeptides except substrates. They can tolerate a higher proportion of mutations. 25. What is the advantage of having mobile electron carriers in addition to large membrane-bound complexes of carriers? Having mobile electron carriers in addition to membrane-bound respiratory complexes allows electron transport to use the most readily available complex rather than to use the same one all the time. 26. What is the advantage of having a Q cycle in electron transport in spite of its complexity? The Q cycle allows for a smooth transition from two-electron carriers (NADH and FADH2) to one-electron carriers(cytochromes). 27. Why do the electron-transfer reactions of the cytochromes differ in standard reduction potential, even though all the reactions involve the same oxidation reduction reaction of iron? The protein environment of the iron differs in each of the cytochromes, causing differences in the reduction potential. 28. Is there a fundamental difference between the one- and two-electron reactions in the electron transport chain? All the reactions in the electron transport chain are electron transfer reactions, but some of the reactants and products inherently transfer either one or two electrons, as the case may be.

14 29. What is the underlying reason for the differences in spectroscopic properties among the cytochromes? The heme groups differ slightly in the various kinds of cytochromes. This is the main difference, with some modification due to the different protein environments. 30. What would be some of the challenges involved in removing respiratory complexes from the inner mitochondrial membrane in order to study their properties? Respiratory complexes contain a number of proteins, some of them quite large. This is the first difficulty. Like most proteins bound to membranes, the components of respiratory complexes are easily denatured on removal from their environment. 31. Describe the role of the F1 portion of ATP synthase in oxidative phosphorylation. The F1 portion of the mitochondrial ATP synthase, which projects into the matrix, is the site of ATP synthesis. 32. Is mitochondrial ATP synthase an integral membrane protein? The F0 portion of mitochondrial ATP synthase lies within the inner mitochondrial membrane, but the F1 portion projects into the matrix. 33. Define P/O ratio and indicate why it is important. 34. In what sense is mitochondrial ATP synthase a motor protein? 35. What is the approximate P/O ratio that can be expected if intact mitochondria are incubated in the presence of oxygen, along with added succinate? A P/O ratio of 1.5 can be expected because oxidation of succinate passes electrons to coenzyme Q via a flavoproteins intermediate, bypassing the first respiratory complex.

15 36. Why is it difficult to determine an exact number for P/O ratios? Exact values for P/O ratios are difficult to determine because of the complexity of the systems that pump protons and phosphorylate ADP. The number of ADP molecules phosphorylated is directly related to the number of protons pumped across the membrane. This figure has been a matter of some controversy. It has been difficult for chemists and biochemists to accept uncertain stoichiometry. 37. What are some of the difficulties in determining the exact number of protons pumped across the inner mitochondrial membrane by the respiratory complexes? The difficulties in determining the number of protons pumped across the inner mitochondrial membrane by respiratory complexes are those inherent in working with large assemblies of proteins that must be bound in a membrane environment to be active. As experimental methods improve, the task becomes less difficult. 38. Briefly summarize the main arguments of the chemiosmotic coupling hypothesis. The chemiosmotic coupling mechanism is based on the difference in hydrogen ion concentration between the intermembrane space and the matrix of actively respiring mitochondria. The hydrogen ion gradient is created by the proton pumping that accompanies the transfer of electrons. The flow of hydrogen ions back into the matrix through a channel in the ATP synthase is directly coupled to the phosphorylation of ADP. 39. Why does ATP production require an intact mitochondrial membrane? An intact mitochondrial membrane is necessary for compartmentalization, which in turn is necessary for proton pumping. 40. Briefly describe the role of uncouplers in oxidative phosphorylation. Uncouplers overcome the proton gradient on which oxidative phosphorylation depends. 41. What role does the proton gradient play in chemiosmotic coupling? In chemiosmotic coupling, the proton gradient is related to ATP production. The proton gradient leads to conformational changes in a number of proteins, releasing tightly bound ATP from the synthase as a result of the conformational change. 42. Why was dinitrophenol once used as a diet drug? Dinitrophenol is an uncoupler of oxidative phosphorylation. The rationale was to dissipate energy as heat.

16 43. Criticize the following statement: The role of the proton gradient in chemiosmosis is to provide the energy to phosphorylate ADP. The energy released as protons pass through the F particles is actually used to cause conformational changes in the F1 proteins, thereby releasing ATP. The tight conformation (one of three)provides a hydrophobic environment in which ADP is phosphorylated by adding Pi without requiring immediate energy. 44. What is the effect of each of the following substances on electron transport and production of ATP? Be specific about which reaction is affected.(a) Azide (b) Antimycin A (c) Amytal (d) Rotenone (e) Dinitrophenol (f) Gramicidin A (g) Carbon monoxide (a) Azide inhibits the transfer of electrons from cytochrome aa3to oxygen. (b) Antimycin A inhibits the transfer of electrons from cytochrome b to coenzyme Q in the Q cycle. (c) Amytal inhibits the transfer of electrons from NADH reductase to coenzyme Q. (d) Rotenone inhibits the transfer of electrons from NADH reductase to coenzyme Q. (e) Dinitrophenol is an uncoupler of oxidative phosphorylation. (f) Gramicidin A is an uncoupler of oxidative phosphorylation. (g) Carbon monoxide inhibits the transfer of electrons from cytochrome aa3 to oxygen. 45. How can respiratory inhibitors be used to indicate the order of components in the electron transport chain? Methods exist to determine the amounts of the oxidized and reduced components of the electron transport chain present in a sample. If a respiratory inhibitor is added, the reduced form of the component before the blockage point in the chain accumulates as does the oxidized form of the component immediately after the blockage point. 46. What is the fundamental difference between uncouplers and respiratory inhibitors? Uncouplers overcome the proton gradient created by electron transport, whereas respiratory inhibitors block the flow of electrons.

17 47. How does the yield of ATP from complete oxidation of one molecule of glucose in muscle and brain differ from that in liver, heart, and kidney? What is the underlying reason for this difference? The complete oxidation of glucose produces 30 molecules of ATP in muscle and brain and 32 ATP in liver, heart, and kidney. The underlying reason is the different shuttle mechanisms for transfer to mitochondria of electrons from the NADH produced in the cytosol by glycolysis. 48. The malate aspartate shuttle yields about 2.5moles of ATP for each mole of cytosolic NADH. Why does nature use the glycerol phosphate shuttle, which yields only about 1.5 moles of ATP? The transport product (in the matrix) of the malate aspartate shuttle is NADH, whereas that of the glycerol phosphate shuttle is FADH2. The latter shuttle can thus go against a transmembrane NADH concentration gradient, whereas the former cannot. 49. What yield of ATP can be expected from complete oxidation of each of the following substrates by the reactions of glycolysis, the citric acid cycle, and oxidative phosphorylation? (a) Fructose-1,6-bisphosphate (b) Glucose (c) Phosphoenolpyruvate (d) Glyceraldehyde-3- phosphate (e) NADH (f) Pyruvate (d) 17 (a) 34 (e) 2.5 (b) 32 (f) 12.5 (c) The free-energy change (DG ') for the oxidation of the cytochrome aa3 complex by molecular oxygen is kj kcal for each mole of electron pairs transferred. What is the maximum number of moles of ATP that could be produced in the process? How many moles of ATP are actually produced? What is the efficiency of the process, expressed as a percentage?