PROBLEMS

Question 21.1

1.  Reclaim resources. Human beings have only about 250 g of ATP, but even a couch potato needs about 83 kg of ATP to open that bag of chips and use the remote. How is this discrepancy between requirements and resources reconciled?

Question 21.2

2.  Like Barbie and Ken. Match each term with its description.

ATP synthase
Proton-motive force
Electron-transport chain
Glycerol 3-phosphate shuttle
Malate–aspartate shuttle
Respiratory (acceptor) control
Uncoupling protein
F1 subunit
F0 subunit
c ring
Results in heat instead of ATP
Generates the proton gradient
A proton merry-go-round
ADP controls the rate of respiration
Cytoplasmic NADH to mitochondrial NADH
Composed of a chemical gradient and a charge gradient
Proton channel
Catalytic subunit
Converts the proton-motive force into ATP
Cytoplasmic NADH to mitochondrial FADH2

Question 21.3

3.  Energy harvest. What is the yield of ATP when each of the following substrates is completely oxidized to CO2 by a mammalian cell homogenate? Assume that glycolysis, the citric acid cycle, and oxidative phosphorylation are fully active.

(a) Pyruvate

(b) Lactate

(c) Fructose 1,6-bisphosphate

(d) Phosphoenolpyruvate

(e) Galactose

(f) Dihydroxyacetone phosphate

Question 21.4

4.  Potent poisons. What is the effect of each of the following inhibitors on electron transport and ATP formation by the respiratory chain?

(a) Azide

(b) Atractyloside

(c) Rotenone

(d) DNP

(e) Carbon monoxide

(f) Antimycin A

Question 21.5

5.  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? ✓ 3

Question 21.6

6.  O2 consumption. Oxidative phosphorylation in mitochondria is often monitored by measuring oxygen consumption. When oxidative phosphorylation is proceeding rapidly, the mitochondria will rapidly consume oxygen. If there is little oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of isolated mitochondria and directed to add the following compounds in the order from a to h. With the addition of each compound, all of the previously added compounds remain present. Predict the effect of each addition on oxygen consumption by the isolated mitochondria.

(a) Glucose

(b) ADP + Pi

(c) Citrate

(d) Oligomycin

(e) Succinate

(f) 2,4-Dinitrophenol

(g) Rotenone

(h) Cyanide

Question 21.7

7.  Runaway mitochondria 1. The number of molecules of inorganic phosphate incorporated into organic form per atom of oxygen consumed, termed the P : O ratio, was frequently used as an index of oxidative phosphorylation. Suppose that the mitochondria of a patient oxidize NADH irrespective of whether ADP is present. The P : O ratio for oxidative phosphorylation by these mitochondria is less than normal. Predict the likely symptoms of this disorder.

Question 21.8

8.  An essential residue. The conduction of protons by the F0 unit of ATP synthase is blocked by the modification of a single side chain by dicyclohexylcarbodiimide, which reacts readily with carboxyl groups. What are the most likely targets of action of this reagent? How might you use site-specific mutagenesis to determine whether this residue is essential for proton conduction? ✓ 3

Question 21.9

9.  Runaway mitochondria 2. Years ago, it was suggested that uncouplers would 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? ✓ 3

Question 21.10

10.  Coupled processes. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate. Why?

Question 21.11

11.  Gone with the flow. What is the actual role of protons in the synthesis of ATP by F0F1 ATP synthase? ✓ 3 ✓ 4

Question 21.12

12.  Opposites attract. An arginine residue (Arg 210) in the a subunit of E. coli ATP synthase is near the aspartate residue (Asp 61) in the matrix-side proton channel. How might Arg 210 assist proton flow? ✓ 3

Question 21.13

13.  Variable c subunits. Recall that the number of c subunits in the c ring appears to range between 8 and 15. This number is significant because it determines the number of protons that must be transported to generate a molecule of ATP. Each 360-degree rotation of the γ subunit leads to the synthesis and release of three molecules of ATP. Thus, if there are 10c subunits in the ring (as was observed in a 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 8c subunits? 12? 15? ✓ 3

Question 21.14

14.  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. ✓ 4

Question 21.15

15.  Everything is connected. If actively respiring mitochondria are exposed to an inhibitor of ATP-ADP translocase, the electron-transport chain ceases to operate. Why? ✓ 4

Question 21.16

16.  Exaggerating the difference. Why must ATP-ADP translocase use Mg2+-free forms of ATP and ADP? ✓ 4

Question 21.17

17.  A Brownian ratchet wrench. What causes the c subunits of ATP synthase to rotate? What determines the direction of rotation? ✓ 3

Question 21.18

18.  Multiple uses. Give an example of the use of the proton-motive force in ways other than for the synthesis of ATP.

Question 21.19

19.  Connections. How does the inhibition of ATP-ADP translocase affect the citric acid cycle? Glycolysis? ✓ 4

Question 21.20

20.  Respiratory control. The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then decreases to its initial value when the added ADP has been converted into ATP (Figure 21.16). Why does the rate decrease? ✓ 4

Question 21.21

21.  The same, but different. Why is the electroneutral exchange of H2PO4− for OH− indistinguishable from the electroneutral symport of H2PO4− and H+?

Question 21.22

22.  Counterintuitive. Under some conditions, mitochondrial ATP synthase has been observed to run in reverse. How would that situation affect the proton-motive force?

Question 21.23

23.  Not hearsay, but real evidence. Describe the evidence supporting the chemiosmotic hypothesis. ✓ 3

Question 21.24

24.  Imposing a gradient. Mitoplasts are mitochondria that lack the outer membrane but are still capable of oxidative phosphorylation. Suppose that you were to soak mitoplasts in a pH 7 buffer for several hours. Then, you rapidly isolated the mitoplasts and mixed them in a pH 4 buffer containing ADP and Pi. Would ATP synthesis take place? Explain. ✓ 3

Question 21.25

25.  With sympathy. Predict the effect on ATP synthesis if the b and δ subunits of the ATPase were absent.

Chapter Integration Problems

Question 21.26

26.  Obeying the laws of thermodynamics. Why will isolated F1 subunits display ATPase activity but not ATP synthase activity? How can the enzyme then function as ATP synthase in mitochondria?

Question 21.27

27.  Etiology? What does that mean? What does the fact that rotenone increases the susceptibility to Parkinson disease indicate about the etiology of this disease?

Question 21.28

28.  The right location. Some cytoplasmic kinases, enzymes that phosphorylate substrates at the expense of ATP, bind to voltage-dependent anion channels (VDACs). What might the advantage of this binding be?

Question 21.29

29.  No exchange. Mice that are completely lacking ATP-ADP translocase (ANT−/ANT−) can be made by 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 6- to 8-fold increase in the level of mitochondrial H2O2 compared with that in normal mice. Provide a possible biochemical explanation for each of these conditions. ✓ 4

Question 21.30

30.  Alternative routes. The most common metabolic sign of mitochondrial disorders is lactic acidosis. Why? ✓ 4

Data Interpretation and Challenge Problem

Question 21.31

31.  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 (uncoordinated movement), and retinitis pigmentosa. A tissue biopsy was performed on each of three patients having this mutation, and submitochondrial particles were isolated that were capable of succinate-sustained ATP synthesis. First, the activity of the ATP synthase was measured on the addition of succinate and the below results were obtained.

ATP synthase activity (nmol of ATP formed min−1 mg−1)

Controls

3.0

Patient 1

0.25

Patient 2

0.11

Patient 3

0.17

(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 by incubating the submitochondrial particles with ATP in the absence of succinate.

ATP hydrolysis (nmol of ATP hydrolyzed min−1 mg−1)

Controls

33

Patient 1

30

Patient 2

25

Patient 3

31

(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?

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 (uncoordinated movement), and retinitis pigmentosa. A tissue biopsy was performed on each of three patients having this mutation, and submitochondrial particles were isolated that were capable of succinate-sustained ATP synthesis. First, the activity of the ATP synthase was measured on the addition of succinate and the below results were obtained. (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 by incubating the submitochondrial particles with ATP in the absence of succinate. (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?
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Challenge Problems

Question 21.32

32.  P : O ratios. The P : O ratio can be used to monitor oxidative phosphorylation (problem 7).

(a) What is the relation of the P : O ratio to the ratio of the number 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?

Question 21.33

33.  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.)

Question 21.34

34.  Currency exchange. For a proton-motive force of 0.2 V (matrix negative), what is the maximum [ATP]/[ADP][Pi] ratio compatible with ATP synthesis? Calculate this ratio three times, assuming that the number of protons translocated per ATP formed is two, three, and four and that the temperature is 25°C. ✓ 3

Question 21.35

35.  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.

Selected Readings for this chapter can be found online at www.whfreeman.com/tymoczko3e.

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