Key Terms vc acid (ticarboxsic acid, TC citrate synthase (p. 304) Krebs) cycle (p. $97) iron-sulfur (nonheme in acyl COA (497) 306) xidative phosphorylation (p. 498) pyruvate dehydrogenase complex (p. 499) np. 502) flavoprot metabolon (p. 512) isocitrate dehydrogenase (p. 506) ecketoglutarate dehydrogenase (p. 507) 521 Problems anaplerotic reaction (p. 516) beriberi(p. 517) elyoxylate cycle (p. 518) isocitrate lyase (p. 518) alate synthase (p. 519) elyoxysome (p. 519) ‘on) protein Problems. 1, Naming names. What are the five enzymes (including regulatory enzymes) that constitute the pyruvate dehydro- genase complex? Which reactions do they catalyze? 2. Coenzymes. What coenzymes are required by the pyruvate dehydrogenase complex? What are their roles? 3, More coenzymes. Distinguish between catalytic coen- zymes and stoichiometric coenzymes in the pyruvate dehy- drogenase complex. 4, Joined at the hip. List some of the advantages of organiz- ing the enzymes that catalyze the formation of acetyl CoA from pyruvate into a single large complex. 5. Flow of carbon atoms, What isthe fate ofthe radioactive label when each of the following compounds is added to a cell extract containing the enzymes and cofactors of the glycolytic pathway, the citric acid cycle, and the pyruvate dehydrogenase complex? (The 'C label is printed in red.) (a) ; (b) e He co0- Hye c00" ro) ; (d) q eco 30S scon (¢) Glucose 6-phosphate labeled at C-1 6 C+ C)9Cs (@) Which enzymes are required to get net synthesis of ‘oxaloacetate from acetyl CoA? (b) Write a balanced equation for the net synthesis. (c) Do mammalian cells contain the requisite enzymes? 7. Driving force. What is the AG” for the complete oxida- tion of the acetyl unit of acetyl CoA by the citric acid cycle? 8. Acting catalytically. The citric acid cycle itself, which is composed of enzymatically catalyzed steps, can be thought of essentially as a supramolecular enzyme. Explain. 9. A potent inhibitor. Thiamine thiazolone pyrophosphate binds to pyruvate dehydrogenase about 20,000 times as strongly as does thiamine pyrophosphate, and it competi- tively inhibits the enzyme. Why? 8 nc | cf TL ws ve 10. Lactic acidosis, Patients in shock often suffer from lactic acidosis owing to a deficiency of Os. Why does a lack of O3 lead to lactic acid accumulation? One treatment for shock is to administer dichloroacetate, which inhibits the kinase associated with the pyruvate dehydrogenase complex. What is the biochemical rationale for this treatment? 11. Energy rich. What are the thioesters in the reaction catalyzed by PDH complex? 12. Alternative fates. Compare the regulation of the pyru- vate dehydrogenase complex in muscle and in liver 13. Mutations. (a) Predict the effect of a mutation that enhances the activity of the kinase associated with the PDH complex. (b) Predict the effect of a mutation that reduces the activity of the phosphatase associated with the PDH complex. 14. Flaking paint, green wallpaper. Clare Boothe Luce, ambassador to Italy in the 1950s (and Connecticut con- gressperson, playwright, editor of Vanity Fair, and the wife of Henry Luce, founder of Time magazine and Sports Illustrated) became ill when she was staying at the ambas~ sadorial residence in Rome. The paint on the dining room ceiling, an arsenic-based paint, was flaking; the wallpaper of her bedroom in the ambassadorial residence was colored ‘a mellow green owing to the presence of cupric arsenite in the pigment. Suggest a possible cause of Ambassador Luce's illness 15. A hoax, perhaps? The citric acid cycle is part of aerobic respiration, but no Op is required for the cycle. Explain this paradox,522 CHAPTER 17 The Citric Acid Cycle 16. Coupling reactions. The oxidation of malate by NAD. to form oxaloacetate is a highly endergonic reaction under standard conditions |AG"" = 29 kJ mol | (7 keal mol!) The reaction proceeds readily under physiological conditions (a) Why? (b) Assuming an [NAD*]/[ NADH ratio of 8 and a pH of 7, what is the lowest [malate]/ [oxaloacetate] ratio at which oxaloacetate can be formed from malate? 17. Synthesizing a-ketoglutarate. It is possible, with the use of the reactions and enzymes considered in this chapter, to convert pyruvate into «-ketoglutarate without depleting any of the citric acid cycle components. Write a balanced reaction scheme for this conversion, showing cofactors and identifying the required enzymes. 18. Seven o'clock roadblock. Malonate is a competitive inhibitor of succinate dehydrogenase. How will the concen- trations of citric acid cycle intermediates change immedi- ately after the addition of malonate? Why is malonate not a substrate for succinate dehydrogenase? 00" CH coo- Malonate 19. No signal, no activity. Why is acetyl CoA an especially appropriate activator for pyruvate carboxylase? 20. Power differentials. As we will see in the next chapter, when NADH reacts with oxygen 2.5 ATP are generated. When FADH) reduces oxygen only 1.5 ATP are generated. Why then does succinate dehydrogenase produce FADH) and not NADH when succinate is reduced to fumarate? 21. Back to Orgo. Before any oxidation can occur in the citric acid cycle, citrate must be isomerized into isocitrate Why is this the case? 22. A nod is as good as a wink to a blind horse. Explain why a GTP molecule, or another nucleoside triphosphate, is energetically equivalent to an ATP molecule in metabolism. 23. One from two. The synthesis of citrate from acetyl CoA and oxaloacetate is a biosynthetic reaction. What is the energy source that drives formation of citrate? Chapter Integration Problems 24. Fats into glucose? Fats are usually metabolized into acetyl CoA and then further processed through the citric acid cycle. In Chapter 16, we saw that glucose can be synthesized from oxaloacetate, a citric acid cycle intermedi: ate. Why, then, after a long bout of exercise depletes our carbohydrate stores, do we need to replenish those stores by 7 ON eating carbohydrates? Why do we not simply by converting fats into carbohydrates? "lacey, remative fuels. As we will see (Chapter 29) fa breakdown generates a large amount of acetyl Coa will be the effect of fatty acid breakdown on, pyruvan it Vogenase complex activity? On glycolysis? ch Mechanism Problems 26. ‘Theme and variation. Propose a reaction mecha the condensation of acetyl CoA and glyoxylate in en oxylate cycle of plants and bacteria. 9 27, Symmetry problems, In experiments carced out to investigate the citric acid cycle, oxaloacetate label. HC in the carboxyl carbon atom farthest from the j group was introduced to an active prepatating mitochondria. : 0. 00" Se CH coo- oxaloacetate Analysis of the a-ketoglutarate formed showed tt none of the radioactive label had been lost. Decarbonyae of a-ketoglutarate then yielded succinate devoid of rai tivity, All the label was in the released CO», Why werete early investigators of the citric acid cycle surprised thatal the label emerged in the CO2? 28. Symmetric molecules reacting asymmetrically. The nt pretation of the experiments described in Problem 27x that citrate (or any other symmetric compound) cant an intermediate in the formation of a-ketoglutarate, bes of the asymmetric fate of the label. This view seemedom pelling until Alexander Ogston incisively pointed owit 1948 that “it is possible that an asymmetric enzyme ubst attacks a symmetrical compound can distinguish btwer identical groups [italics added].” For simplicity, oms+ molecule in which two hydrogen atoms, a group X, /# different group Y are bonded to a tetrahedral carbon # as a model for citrate. Explain how a symmetric males* can react with an enzyme in an asymmetric way. Data Interpretation Problem cit 29. A little goes a long way. As will become cles Chapter 18, the activity of the citric acid cycle can b tored by measuring the amount of O, consumed greater the rate of Oz consumption, the faster the the cycle. Hans Krebs used this assay to investi cycle in 1937. He used as his experimental syst” Pigeon-breast muscle, which is rich in mitochondt * g set of experiments, Krebs measured the O2 comsu™P the presence of carbohydrate only and in the Pre — | the rte itAc" Prosthetic Enzyme ‘eoup Typet mol! heal mol! ‘CoA + oxaloacetate + HO—> Givate = “a crate CoA +H synthase Aconitase Res b +84 320 Aconitase Fes © 21 05. Tsocitrate dte 44 20 dehydrogenase 4 Ketoglutarate Lipoie acid, dte 301 dehydrogenase FAD, TPP. complex on Succinyl CoA f 33008 SEATP + CoA synthetase cinate + FAD (enzyme bound) —* Succinate FAD, Fes © 0 0 dehydrogenase Fumarase © © oxaloacetate + NADH + Ht Malate dehydrogenase 3) condensation, (bday drain: eration a) decarboxylation |e) oxnaton:F) wubsvate level phospho aonUnaIeA 1 Uxoanve Fnospnoryauion Problems 1. Breathe or ferment? Compare fermentation and respira- tion with respect to electron donors and electron acceptors, 2. Reference states. ‘The standard oxidation-reduction potential for the reduction of O» to HO is given as 0.82 V in Table 18.1, However, the value given in textbooks of chemistry is 1.23 V. Account for this difference. 3, Less energetic electrons. Why are electrons carried by FADED not as energy rich as those carried by NADH? ‘What is the consequence of this difference? 4. Now prove it. Calculate the energy released by the reduc- ion of O2 with FADHy 5. Thermodynamic constraint. Compare the AG values for the oxidation of succinate by NAD* and by FAD. Use the data given in Table 18.1 to find the Ej of the NAD* “NADH and fumarate-succinate couples, and assume that 4 for the FAD-FADH> redox couple is nearly 0.03 V. Why is FAD rather than NAD* the electron acceptor in the reaction catalyzed by succinate dehydrogenase? 6. The benefactor and beneficiary. Identify the oxidant and the reductant in the following reaction Pyruvate + NADH + H* == lactate + NAD* 7. Six of one, half dozen of the other. How is the redox potential (AE) related to the free-energy change of a reaction (AG")? 8. Location, location, location. Iron is a component of many of the electron carriers ofthe electron-transport chain. How can it participate ina series of coupled redox reactions if the Ep value is +0.77 V, as seen in Table 18.1? 9. Line up. Place the following components of the electron- transport chain in their proper order: (a) cytochrome c; (b)Q-cytochrome oxidoreductase; (c) NADH-Qreductase; €) cytochrome c oxidase; (e) ubiquinone S @eyt ) bi att wp 10. Match 'em. ee (a) Complex 1, Qeeytochrome c oxidoreductase™ (b) Complex IL_ 2. Coenzyme Q (c) Complex IL_ 3. Succinate-Q reductase (4) ComplexIV__ 4. NADH-Q oxidoreductase (©) Ubiquinone___5. Cytochrome c oxidase 11, Structural considerations. Explain why coenzyme Q is an effective mobile electron carrier in the electron-transport chain. 12. Inhibitors. Rotenone inhibits electron flow through NADH-Q oxidoreductase. Antimycin A blocks electron flow between cytochromes b and c1. Cyanide blocks elec- tron flow through cytochrome oxidase to Q2. Predict the relative oxidation-reduction state of each of the following respiratory-chain components in mitochondria that are treated with each of the inhibitors: NAD": NADH.Q, oxidoreductase; coenzyme Q; cytochrome ci eytochromec: cytochrome a 13. Rumored to be a favorite of Elvis. Amytal is a barbity. rate sedative that inhibits electron flow through Complex How would the addition of amytal to actively respiring mitochondria affect the relative oxidation-reduction states of the components of the electron-transport chain and the citric acid cycle? 14, Efficiency. What is the advantage of having Complexes I, II], and IV associated with one another in the form of a respirasome? 15. ROS, not ROUS. What are the reactive oxygen species and why are they especially dangerous to cells? 16. Reclaim resources. Humans have only about 230 ¢ of ATP, but even a couch potato needs about 83 kg of ATP to ‘open the bag of chips and use the remote, How is this ds: crepancy between requirements and resources reconciled? 17. Energy harvest. What is the yield of ATP when each of the following substrates is completely oxidized to CO, bya mammalian cell homogenate? Assume that glycolysis, the citric acid cycle, and oxidative phosphorylation are fully active (a) Pyruvate (b) Lactate (©) Fructose 1,6-bisphosphate (4) Phosphoenolpyruvate (©) Galactose (f) Dihydroxyacetone phosphate 18. Potent poisons. What is the effect of each of the follow ing inhibitors on electron transport and ATP formation by the respiratory chain? (a) Azide (4) DNP (b) Atractyloside (c) Carbon monoxide (©) Rotenone (8) Antimycia A 19. A question of coupling. What is the mechanistic basis for the observation that the inhibitors of ATP synthase also lead to an inhibition of the electron-transport chain? 20. A Brownian ratchet wrench. What causes the ¢ subunits of ATP synthase to rotate? What determines the direction of rotation? 21, Alternative routes. The most common metabolic sign of ‘mitochondrial disorders is lactic acidosis, Why? 22. Connections, How does the inhibition of ATP-AD? translocase affect the citric acid cycle? Glycolysis? 23, O2 consumption, Oxidative phosphorylation in mito chondria is often monitored by measuring oxygen ©”sumption. When oxidative phosphorylation is proceeding the mitochondria will rapidly consume oxygen. If thereislittle oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of jeolated mitochondria and directed to add the following compounds in the order from a to h. With the addition of ‘ach compound, all of the previously added compounds remain present. Predict the effect of each addition on oxy- genconsumption by the isolated mitochondria {@) Glocose (e) Succinate ib) ADP +P, (f) Dinitrophenol (o) Citrate (g) Rotenone (d) Oligomycin (h) Cyanide 24. P:O ratios. The number of molecules of inorganic phosphate incorporated into organic form per atom of oxy- gen consumed, termed the P: O ratio, was frequently used asan index of oxidative phosphorylation (a) What is the relation of the P: O ratio to the ratio of the umber of protons translocated per electron pair (H* /2e~) and the ratio of the number of protons needed to synthesize ATP and transport it to the cytoplasm (P/H*)? (b) What are the P:O ratios for electrons donated by matrix NADH and by succinate? 25. Cyanide antidote. The immediate administration of nitrite is a highly effective treatment for cyanide poisoning What is the basis for the action of this antidote? (Hint: Nitrite oxidizes ferrohemoglobin to ferrihemoglobin.) 26. Runaway mitochondria 1. Suppose that the mitochon- dria of a patient oxidize NADH irrespective of whether ADP is present. The P:O ratio for oxidative phosphoryla- tion by these mitochondria is less than normal. Predict the likely symptoms of this disorder. 27, Recycling device. The cytochrome b component of Qeytochrome ¢ oxidoreductase enables both electrons of QH; to be effectively utilized in generating a proton-motive force. Cite another recycling device in metabolism that brings a potentially dead end reaction product back into the mainstream, 28. Crossover point. The precise site of action of a ‘espiratory-chain inhibitor can be revealed by the crossover technique. Britton Chance devised elegant spectroscopic ‘methods for determining the proportions of the oxidized and reduced forms of each carrier. This determination is feasible because the forms have distinctive absorption spec tra, as illustrated in the adjoining graph for cytochrome ¢. You are given a new inhibitor and find that its addition to espiring mitochondria causes the carriers between NADH and QH; to become more reduced and those between cyto- chrome ¢ and Op to become more oxidized. Where does Your inhibitor act? Absorbance coefficient (M-! cm” x 400 300 600 Wavelength (nen) 29. Runaway mitochondria 2. Years ago, uncouplers were suggested to make wonderful diet drugs. Explain why this idea was proposed and why it was rejected. Why might the producers of antiperspirants be supportive of the idea? 30. Everything is connected. If actively respiring mitochon- dria are exposed to an inhibitor of ATP-ADP translocase, the electron-transport chain ceases to operate. Why? 31. Identifying the inhibition. You are asked to determine whether a chemical is an electron-transport-chain inhibitor ‘or an inhibitor of ATP synthase. Design an experiment to make this determination. 32, To each according to its needs. It has been noted that the mitochondria of muscle cells often have more cristae than the mitochondria of liver cells. Provide an explanation for this observation. 33. Opposites attract. An arginine residue (Arg 210) in the a subunit of ATP synthase is near the aspartate residue (Asp 61in the matrix-side proton channel. How might Arg 210 assist proton flow? 34, Variable ¢ subunits, Recall that the number of ¢ sub- units in the e ring appears to range between 10 and 14. This number is significant because it determines the number of protons that must he transported to generate a molecule of ATP. Each 360-degree rotation of the y subunit leads to the synthesis and release of three molecules of ATP. Thus, if there are 10 ¢ subunits in the ring (as was observed in @ crystal structure of yeast mitochondrial ATP synthase), each ATP generated requires the transport of 10/3 = 3.33 protons. How many protons are required to form ATP if the ring has 12 ¢ subunits? 142 35. Counterintuitive. Under some conditions, mitochon- drial ATP synthase has been observed to actually run in reverse. How would that situation affect the proton-motive force?36, Etiology? What does that mean? What does the fact that rotenone appears to increase the susceptibility to Parkinson disease indicate about the etiology of Parkinson disease? 37. Exaggerating the difference. Why must ATP-ADP translocase (also called adenine nucleotide translocase or ANT) use Mg?*-free forms of ATP and ADP? 38. Respiratory control. The rate of oxygen consumption by rkedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP (sce Figure 18.39). Why does the rate decrease? mitochondria increase: 39. Same, but different. Why is the electroneutral exchange of H)PO," for OH” indistinguishable from the electroneu- tral symport of H)PO4” and H*? 40. Multiple uses. Give an example of the use of the proton- motive force in ways other than for the synthesis of ATP? Chapter Integration Problems 41, Just obeying the laws. Why do isolated F; subunits of ATP synthase catalyze ATP hydrolysis? 42. The right location. Some cytoplasmic kinases, enzymes that phosphorylate substrates at the expense of ATP, bind to voltage-dependent anion channels. What might the advantage of this binding be? 43. No exchange. Mice that completely lack ATP-ADP translocase (ANT~/ANT™) can be made by using the knockout technique. Remarkably, these mice are viable but, have the following pathological conditions: (1) high serum. levels of lactate, alanine, and succinate; (2) little electron transport; and (3) a six- to eightfold increase in the level of mitochondrial HO, compared with that in normal mice. Provide a possible biochemical explanation for each of these conditions 44, Maybe you shouldn't take your vitamins. Exercise is known to increase insulin sensitivity and to ameliorate type 2 diabetes (Chapter 27). Recent research suggests that taking antioxidant vitamins might mitigate the beneficial effects of exercise with respect to ROS protection. (a) What are the antioxidant vitamins? (b) How does exercise protect against ROS? (6) Explain why vitamins might counteract the effects of exercise. Data Interpretation Problem 45. Mitochondrial disease. A mutation in a mitochondrial gene encoding a component of ATP synthase has been identified. People who have this mutation suffer from muscle weakness, ataxia, and retinitis pigmentosa. A tissue biopsy was performed on each of three patients having this, mutation, and submitochondral paticles were isolate, at the ativity of the ATP synthase was measured the tion of succinate and the following results were obtaineg were capable of succinate-sustained ATP synthe ATP synthase activity (nmol of ATP formed min”! mg”) Controls 30 Patient 1 025 Patient 2 ou Patient 3 047 (a) What was the purpose of the addition of succinate? {b) What is the effect of the mutation on succinate coupled ATP synthesis? Next, the ATPase activity of the enzyme was measured incubating the submitochondrial particles with ATP in the absence of succinate. ATP hydrolysis, (nmol of ATP hydrolyzed ‘min-!'mg”') Controls Patient Patiens 2 Patient 3 (c) Why was succinate omitted from the reaction? (d) What is the effect of the mutation on ATP hydrolysis? (e) What do these results, in conjunction with those obtained in the first experiment, tell you about the nature of the mutation? Mechanism Problem 46. Chiral clue. ATP4S, a slowly hydrolyzed analog of ATP, can be used to probe the mechanism of phosphor transfer reactions. Chiral ATPyS has been synthesized containing 'O in a specific + position and ordinary "O elsewhere in the molecule. The hydrolysis of this chil molecule by ATP synthase in '7O-enriched water yields inorganic ('60,0,'8O]thiophosphate having the follow ing absolute configuration. In contrast, the hydrolysis this chiral ATPyS by a calcium-pumping ATPase from muscle gives thiophosphate of the opposite configuration What is the simplest interpretation of these data? AG pis s ANS % advo “Thiophosphate adProblems 1. Complementary powers. Photosystem I produces a pow erful reductant, whereas photosystem I produces a power ful oxidant. Identify the reductant and oxidant and describe their roles. 2, Ifa little is good. What is the advantage of having an extensive set of thylakoid membranes in the chloroplasts? 3. Cooperation. Explain how light-harvesting complexes enhance the efficiency of photosynthesis. 4. One thing leads to another. What is the ultimate electron acceptor in photosynthesis? The ultimate electron donor? What powers the electron flow between the donor and the acceptor? 5. Neutralization compensation. In chloroplasts, a greater pH gradient across the thylakoid membrane is required to power the synthesis of ATP than is required across the mitochondrial inner membrane. Explain this difference. 6. Environmentally appropriate. Chlorophyll isa hydropho bic molecule, Why is this property crucial for the function of chlorophyll? 7. Proton origins. What are the various sources of protons that contribute to the generation of a proton gradient in chloroplast 8. Efficiency matters. What fraction of the energy of 700-nm light absorbed by photosystem I is trapped as high-enerey electrons? 9. That's not right. Explain the defect or defects in te hypothetical scheme for the light reactions of photosyie. sis depicted here. NK 3 \ e \. 8 oo MP § Xt Photosystem | g z HO. “Nx Photosystem II 10. Electron transfer. Calculate the AE} and AG* for he reduction of NADP* by ferredoxin. Use data given ° Table 18.1 11. To boldly go. (a) It can be argued that, if life wereto elsewhere in the universe, it would require some eT like photosynthesis. Why is this argument reasonable? (0) the Enterprise were to land on a distant plant and find measurable oxygen in the atmosphere, could the crew ©” clude that photosynthesis is not taking place?12, Weed killer 1. Dichlorophenyldimethylurea (CMU), 4 herbicide, interferes with photophosphorylation and Oy ‘olution. However, it does not block O, evolution in the presence of an artificial election acceptor such as fetes Fide, Propose a site for the inhibitory aetion of DCMU. 14, Weed killer 2. Predict the effect of the herbicide dichlo- rophenyldimethylurea (DCMU) on a plant’ ability to per form exelic photophosphorylation. 14, Infrared-harvest, Consider the relation between the ‘energy of a photon and its wavelength, (@) Some bacteria are able to harvest 1000-nm light. What js the energy (in kilojoules or kilocalories) of a mole (also called an einstein) of 1000-nm photons? {b) What is the maximum increase in redox potential that canbe induced by a 1000-nm photon? {c) What is the minimum number of 1000-nm photons needed to form ATP from ADP and P? Assume a AG of 30k] mol | (12 kcal mol” ') for the phosphorylation reaction. 15. Missing acceptors. Suppose that a bacterial reaction center containing only the special pair and the quinones has been prepared. Given the separation of 22 A between the special pair and the closest quinone, estimate the rate of electron transfer between the excited special pair and this quinone. 16, Close approach. Suppose that energy transfer between two chlorophyll a molecules separated by 10 A takes place in 10 picoseconds. Suppose that this distance is increased to 20 A with all other factors remaining the same. How long would energy transfer take? Chapter Integration Problems 17. Functional equivalents. What structural feature of mito- chondria corresponds to the thylakoid membranes? 18. Compare and contrast. Compare and contrast oxidative Phosphorylation and photosynthes 19. Energy accounts. On page 580, the balance sheet for the «oxt of the photosynthetically powered synthesis of glucose is presented. Eighteen molecules of ATP are required. Yet, when glucose undergoes combustion in cellular respira- tion, 30 molecules of ATP are produced. Account for the difference, 20. Looking for a place to rest. Albert Szent-Gyorgyi, Nobel Prize-winning biochemist, once said something to the effect: Life is nothing more than an electron looking for place to rest. Explain how this pithy statement applies to Photosynthesis and cellular respiration. Problems Mechanism Problem 21, Hill eaction, In 1939, Robert Lill discovered that chlo- roplasts evolve Oy when they are illuminated in the pres ence of an artificial electron acceptor such as errieyanide [eS (CN)! Ferticyanide is reduced to ferrocyanide [Fe""(CN),]" ia this process. No NADPH or reduced plastocyanin is produced. Propose a mechanism for the ill reaction. Data Interpretation and Chapter integration Problem 22, The same, but different, The Bry complex of mito: chondrial or chloroplast ATP synthase will function as an ATPase in vitro. The chloroplast enzyme (both synthase and ATPase activity) is sensitive to redox control, whereas the mitochondrial enzyme is not. To determine where the enzymes differ, a segment of the mitochondrial -y subunit ‘was removed and replaced with the equivalent segment from the chloroplast y subunit. The AT Pase activity of the modified enzyme was then measured as a function of redox conditions (a) What is the redox regulator of the ATP synthase in vivo? The adjoining graph shows the ATPase activity of modified and control enzymes under various redox conditions. Modiied enayme and thioredoxin | ore gay S00 FE so ++ DTT + thioredoxin 22 Modified eayme__ + + DTT only Be olf ++ DIT + thioredoxin BE 00 ‘ es : go 5 i 15, Dithiothretol (OTT), mit [Data for 0. Bald eta. Biol Cher, 275:12757-12762, 2000} (b) What is the effect of inereasing the reducing power of the reaction mixture for the control and the modified enzymes? (c) What is the effect of the addition of thioredoxin? How do these results differ from those in the presence of DTT alone? Suggest a possible explanation for the difference. (d) Did the researchers succeed in identifying the region of the + subunit responsible for redox regulation? (c) What is the biological rationale of regulation by high concentrations of reducing agents? (£) What amino acids in they subunit are most likely affected by the reducing conditions? (g) What experiments might confirm your answer to part e?