Chapter Review in Chapter 19 Oxidative Phosphorylation

Chapter Review

KEY TERMS

Terms in bold are defined in the glossary.

Problems

1. Oxidation-Reduction Reactions Complex I, the NADH dehydrogenase complex of the mitochondrial respiratory chain, promotes the following series of oxidation-reduction reactions, in which ${Fe}^{3 +}$ $Fe Superscript 3 plus$ and ${Fe}^{2 +}$ $Fe Superscript 2 plus$ represent the iron in iron-sulfur centers, Q is ubiquinone, ${QH}_{2}$ $QH Subscript 2$ is ubiquinol, and E is the enzyme:
1. $NADH + H^{+} + E-FMN \to {NAD}^{+} + {E-FMNH}_{2}$ $NADH plus upper H Superscript plus Baseline plus upper E hyphen FMN right-arrow NAD Superscript plus Baseline plus upper E hyphen FMNH Subscript 2 Baseline$
2. ${E-FMNH}_{2} + 2 {Fe}^{3 +} \to E-FMN + 2 {Fe}^{2 +} + 2 H^{+}$ $upper E hyphen FMNH Subscript 2 Baseline plus 2 Fe Superscript 3 plus Baseline right-arrow upper E hyphen FMN plus 2 Fe Superscript 2 plus Baseline plus 2 upper H Superscript plus$
3. $2 {Fe}^{2 +} + 2 H^{+} + Q \to 2 {Fe}^{3 +} + {QH}_{2}$ $2 Fe Superscript 2 plus Baseline plus 2 upper H Superscript plus Baseline plus upper Q right-arrow 2 Fe Superscript 3 plus Baseline plus QH Subscript 2 Baseline$
  - Sum: $NADH + H^{+} + Q \to {NAD}^{+} + {QH}_{2}$ $NADH plus upper H Superscript plus Baseline plus upper Q right-arrow NAD Superscript plus Baseline plus QH Subscript 2 Baseline$
For each of the three reactions catalyzed by Complex I, identify (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reducing agent, and (e) the oxidizing agent.
2. All Parts of Ubiquinone Have a Function In electron transfer, only the quinone portion of ubiquinone undergoes oxidation-reduction; the isoprenoid side chain remains unchanged. What is the function of this chain?
3. Use of FAD Rather Than $NA D^{+}$ $NA upper D Superscript plus$ in Succinate Oxidation All the dehydrogenases of glycolysis and the citric acid cycle use ${NAD}^{+}$ $NAD Superscript plus$ $(E^{'} °$ $left-parenthesis upper E prime degree$ for ${NAD}^{+} /NADH$ $NAD Superscript plus Baseline slash NADH$ is $− 0.32 V)$ $minus 0.32 upper V right-parenthesis$ as electron acceptor except succinate dehydrogenase, which uses covalently bound FAD ( $E^{'} °$ $upper E prime degree$ for ${FAD}^{+} {/FADH}_{2}$ $FAD Superscript plus Baseline slash FADH Subscript 2$ in this enzyme is 0.050 V). Suggest why FAD is a more appropriate electron acceptor than ${NAD}^{+}$ $NAD Superscript plus$ in the dehydrogenation of succinate, based on the $E^{'} °$ $upper E prime degree$ values of fumarate/succinate $(E^{'} ° = 0.031 V), {NAD}^{+} /NADH$ $left-parenthesis upper E Superscript prime Baseline degree equals 0.031 upper V right-parenthesis comma NAD Superscript plus Baseline slash NADH$ , and the succinate dehydrogenase ${FAD/FADH}_{2}$ $FAD slash FADH Subscript 2$ .
4. Degree of Reduction of Electron Carriers in the Respiratory Chain Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory chain. For example, when NADH and $O_{2}$ $upper O Subscript 2$ are abundant, the steady-state degree of reduction of the carriers decreases as electrons pass from the substrate to $O_{2}$ $upper O Subscript 2$ . When electron transfer is blocked, the carriers before the block become more reduced and those beyond the block become more oxidized (see Fig. 19-6). For each of the mitochondrial conditions listed, predict the state of oxidation of ubiquinone and cytochromes b, $c_{1}$ $c 1$ , c, and $a + a_{3}$ $a plus a 3$ .
1. Abundant NADH and $O_{2}$ $upper O Subscript 2$ , but cyanide added
2. Abundant NADH, but $O_{2}$ $upper O Subscript 2$ exhausted
3. Abundant $O_{2}$ $upper O Subscript 2$ , but NADH exhausted
4. Abundant NADH and $O_{2}$ $upper O Subscript 2$
5. Effect of Rotenone and Antimycin A on Electron Transfer Rotenone, a toxic natural product from plants, strongly inhibits NADH dehydrogenase of insect and fish mitochondria. Antimycin A, a toxic antibiotic, strongly inhibits the oxidation of ubiquinol.
1. Explain why rotenone ingestion is lethal to some insect and fish species.
2. Explain why antimycin A is a poison.
3. Given that rotenone and antimycin A are equally effective in blocking their respective sites in the electron-transfer chain, which would be a more potent poison? Explain.
6. Uncouplers of Oxidative Phosphorylation In normal mitochondria, the rate of electron transfer is tightly coupled to the demand for ATP. When the rate of ATP use is relatively low, the rate of electron transfer is low; when demand for ATP increases, the electron-transfer rate increases. Under these conditions of tight coupling, the number of ATP molecules produced per atom of oxygen consumed when NADH is the electron donor — the $P/O$ $upper P slash upper O$ ratio — is about 2.5.
1. Predict the effect of a relatively low and a relatively high concentration of uncoupling agent on the rate of electron transfer and the $P/O$ $upper P slash upper O$ ratio.
2. Ingestion of uncouplers causes profuse sweating and an increase in body temperature. Explain this phenomenon in molecular terms. What happens to the $P/O$ $upper P slash upper O$ ratio in the presence of uncouplers?
3. Physicians used to prescribe the uncoupler 2,4-dinitrophenol (DNP) as a weight-reducing drug. How could this agent, in principle, serve as a weight-reducing aid? Physicians no longer prescribe uncoupling agents, because some deaths occurred following their use. Why might the ingestion of uncouplers cause death?
7. Effects of Valinomycin on Oxidative Phosphorylation When investigators add the antibiotic valinomycin (see Fig. 11-43) to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of $O_{2}$ $upper O Subscript 2$ consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane increases. Does valinomycin act as an uncoupler or as an inhibitor of oxidative phosphorylation? Explain the experimental observations in terms of the antibiotic’s ability to transfer $K^{+}$ $upper K Superscript plus$ ions across the inner mitochondrial membrane.
8. Cellular ADP Concentration Controls ATP Formation Although ATP synthesis requires both ADP and $P_{i}$ $upper P Subscript i$ , the rate of synthesis depends mainly on the concentration of ADP, not $P_{i}$ $upper P Subscript i$ . Why?
9. Reactive Oxygen Species Describe the role played by superoxide dismutase in ameliorating the effects of reactive oxygen species.
10. How Many Protons in a Mitochondrion? Electron transfer translocates protons from the mitochondrial matrix to the external medium, establishing a pH gradient across the inner membrane (outside more acidic than inside). The tendency of protons to diffuse back into the matrix is the driving force for ATP synthesis by ATP synthase. During oxidative phosphorylation by a suspension of mitochondria in a medium of pH 7.4, the measured pH of the matrix is 7.7.
1. Calculate $[H^{+}]$ $left-bracket upper H Superscript plus Baseline right-bracket$ in the external medium and in the matrix under these conditions.
2. What is the outside-to-inside ratio of $[H^{+}]$ $left-bracket upper H Superscript plus Baseline right-bracket$ ? How much energy for ATP synthesis is available in this concentration difference. (Hint: See Eqn 11-4, p. 392.)
3. Calculate the number of protons in a respiring liver mitochondrion, assuming its inner matrix compartment is a sphere of diameter $1.5 μ m$ $1.5 mu m$ .
4. From these data, is the pH gradient alone sufficient to generate ATP?
5. If not, suggest how the necessary energy for synthesis of ATP arises.
11. Rate of ATP Turnover in Rat Heart Muscle Rat heart muscle operating aerobically fills more than 90% of its ATP needs by oxidative phosphorylation. Each gram of tissue consumes $O_{2}$ $upper O Subscript 2$ at the rate of $10.0 μ mol/min$ $10.0 mu mol slash min$ , with glucose as the fuel source.
1. Calculate the rate at which the heart muscle consumes glucose and produces ATP.
2. For a steady-state ATP concentration of $5.0 μ mol/g$ $5.0 mu mol slash g$ of heart muscle tissue, calculate the time required (in seconds) to completely turn over the cellular pool of ATP. What does this result indicate about the need for tight regulation of ATP production? (Note: Concentrations are expressed as micromoles per gram of muscle tissue because the tissue is mostly water.)
12. Rate of ATP Breakdown in Insect Flight Muscle ATP production in the flight muscle of the fly Lucilia sericata results almost exclusively from oxidative phosphorylation. During flight, maintaining an ATP concentration of $7.0 μ mol/g$ $7.0 mu mol slash g$ of flight muscle requires 187 mL of $O_{2} /h • g$ $upper O Subscript 2 Baseline slash h bullet g$ of body weight. Assuming that flight muscle makes up 20% of the fly’s weight, calculate the rate at which the flight-muscle ATP pool turns over. How long would the reservoir of ATP last in the absence of oxidative phosphorylation? Assume that the glycerol 3-phosphate shuttle transfers the reducing equivalents and that $O_{2}$ $upper O Subscript 2$ is at $25 ° C$ $25 degree upper C$ and $101.3 kPa (1 atm)$ $101.3 kPa left-parenthesis 1 atm right-parenthesis$ .
13. High Blood Alanine Level Associated with Defects in Oxidative Phosphorylation Most individuals with genetic defects in oxidative phosphorylation have relatively high concentrations of alanine in their blood. Explain this in biochemical terms.
14. Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenase is found only in mitochondria, but malate dehydrogenase is found in both the cytosol and mitochondria. What is the role of cytosolic malate dehydrogenase?
15. Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH must undergo oxidation by the mitochondrial respiratory chain. Consider a preparation of rat hepatocytes containing mitochondria and all the cytosolic enzymes. After the introduction of $[{4-}^{3} H] NADH$ $left-bracket 4 hyphen cubed upper H right-bracket NADH$ , radioactivity soon appears in the mitochondrial matrix. Conversely, no radioactivity appears in the matrix after the introduction of $[{7-}^{14} C] NADH$ $left-bracket 7 hyphen Superscript 14 Baseline upper C right-bracket NADH$ . What do these observations reveal about the oxidation of extramitochondrial NADH by the respiratory chain?

[4- superscript 3 end superscript H] N A D H is a six-membered ring with double bonds on the left and right sides, a top vertex bonded to two superscript 3 end superscript H, N substituted for C at the bottom vertex and further bonded to R, and an upper right vertex bonded to C double bonded to O above and bonded to N H 2 to the lower right. [7 – superscript 14 end superscript C] N A D H is similar except that the top vertex is bonded to 2 H that are not labeled and the upper right vertex is bonded to superscript 14 end superscript C double bonded to O above and bonded to N H 2 to the lower right.
16. NAD Pools and Dehydrogenase Activities Although both pyruvate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase use ${NAD}^{+}$ $NAD Superscript plus$ as their electron acceptor, the two enzymes do not compete for the same cellular NAD pool. Why?
17. The Malate–α-Ketoglutarate Transport System n-Butylmalonate inhibits the transport system that conveys malate and α-ketoglutarate across the inner mitochondrial membrane (see Fig. 19-31). Suppose you add n-butylmalonate to an aerobic suspension of kidney cells using exclusively glucose as fuel. Predict the effect of this inhibitor on
1. glycolysis,
2. oxygen consumption,
3. lactate formation, and
4. ATP synthesis.
18. Time Scales of Regulatory Events in Mitochondria Compare the likely time scales for the adjustments in respiratory rate caused by
1. increased [ADP] and
2. reduced ${pO}_{2}$ $pO Subscript 2$ . What accounts for the difference?
19. The Pasteur Effect When investigators add $O_{2}$ $upper O Subscript 2$ to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the cells consume the $O_{2}$ $upper O Subscript 2$ , and accumulation of lactate ceases. This effect, first observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of both aerobic and anaerobic glucose catabolism.
1. Why does the accumulation of lactate cease after the addition of $O_{2}$ $upper O Subscript 2$ ?
2. Why does the presence of $O_{2}$ $upper O Subscript 2$ decrease the rate of glucose consumption?
3. How does the onset of $O_{2}$ $upper O Subscript 2$ consumption slow down the rate of glucose consumption? Explain in terms of specific enzymes.
20. Respiration-Deficient Yeast Mutants and Ethanol Production Researchers can produce respiration-deficient yeast mutants ( $p^{-}$ $p Superscript minus$ ; “petites”) from wild-type parents by treatment with mutagenic agents. The mutants lack cytochrome oxidase, a deficit that markedly affects their metabolic behavior. One striking effect is that fermentation is not suppressed by $O_{2}$ $upper O Subscript 2$ — that is, the mutants do not experience the Pasteur effect (see Problem 19). Some companies are very interested in using these mutants to ferment wood chips to ethanol for energy use. Why does the absence of cytochrome oxidase eliminate the Pasteur effect? Explain the advantages of using these mutants rather than wild-type yeast for large-scale ethanol production.
21. Mitochondrial Disease and Cancer Mutations in the genes that encode certain mitochondrial proteins are associated with a high incidence of some types of cancer. How might defective mitochondria lead to cancer?
22. Variable Severity of a Mitochondrial Disease Different individuals with a disease caused by the same specific defect in the mitochondrial genome may have symptoms ranging from mild to severe. Explain why.
23. Diabetes as a Consequence of Mitochondrial Defects Glucokinase is essential in the metabolism of glucose in pancreatic β cells. Humans with two defective copies of the glucokinase gene exhibit a severe, neonatal diabetes, whereas those with only one defective copy of the gene have a much milder form of the disease (maturity onset diabetes of the young, MODY2). Explain this difference in terms of the biology of the β cell.
24. Effects of Mutations in Mitochondrial Complex II Single nucleotide changes in the gene for succinate dehydrogenase (Complex II) are associated with midgut carcinoid tumors. Suggest a mechanism to explain this observation.

DATA ANALYSIS PROBLEM

25. Membrane Fluidity and Respiration Rate The mitochondrial electron transfer complexes and the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ ATP synthase are embedded in the inner mitochondrial membrane in eukaryotes and in the inner membrane of bacteria. Electrons are shuttled between complexes in part by coenzyme Q, or ubiquinone, a factor that migrates within the membrane. Jay Keasling and coworkers explored the effect of membrane fluidity on rates of respiration in E. coli.

E. coli naturally adjusts its membrane lipid content to maintain membrane fluidity at different temperatures. Workers in the Keasling lab bioengineered an E. coli strain to allow them to control expression of the enzyme FabB, which catalyzes the limiting step in the synthesis of unsaturated fatty acids in E. coli.
1. How does the content of unsaturated fatty acids affect membrane fluidity?
2. The researchers were able to modulate the content of unsaturated fatty acids in the membrane lipid from 15% to 80%. They did not try to completely block synthesis of unsaturated fatty acids to extend the experimental range in the membrane to 0%. Why not?
3. When the cells were grown under aerobic conditions, the researchers found that bacterial growth rate increased as the concentration of unsaturated fatty acids in the membrane increased. However, when oxygen was very limited, the unsaturated fatty acid content of the membrane had no effect on growth rate. How might you explain this observation?
4. The researchers measured rates of respiration, finding a strong correlation between those rates and the fraction of membrane fatty acids that was unsaturated. When the unsaturated fatty acid content of the membranes was kept low, the cells accumulated pyruvate and lactate. Explain these observations.
5. Next, they measured rates of diffusion of membrane phospholipids and ubiquinone in vesicles derived from E. coli membranes. The diffusion rates increased as a function of the content of unsaturated fatty acids. These measured rates were consistent with simulations carried out to model the effects of ubiquinone diffusion on respiration. What overall conclusion can be drawn from this work?

Reference

Budin, I, T. de Rond, Y. Chen, L.J.G. Chan, C.J. Petzold, and J.D. Keasling. 2018. Viscous control of cellular respiration by membrane lipid composition. Science 362:1186–1189.