LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

19. OXIDATIVE PHOSPHORYLATION AND PHOTOPHOSPHORYLATION

Problems and Solutions

1. Oxidation-reduction reactions.

The NADH dehydrogenase complex of the mitochondrial Respiratory Chain catalyzes the following oxidation-reduction reactions:

(1) NADH + Н⁺ + E-FMN —> NAD⁺+ E-FMNH₂

(2) E-FMNH₂ +2 Fe³⁺—> E-FMN + 2 Fe²⁺ + 2 H⁺

(3) 2 Fe²⁺ 2 H⁺ + Q —> 2 Fe³⁺ + QH₂

Overall reaction:

NADH + H⁺ + Q —> NAD⁺ + QH₂

Here, Fe³⁺ and Fe²⁺ represent the iron atoms of iron-sulfur centers, Q is ubiquinone, QH₂ is ubiquinol, and E denotes the enzyme.

For each of the following Reactions Catalyzed by the NADH dehydrogenase complex, specify: a) the electron donor, b) the electron acceptor, c) the conjugated redox pair, d) the reducing agent, and e) the oxidizing agent.

2. Every part of the ubiquinone molecule performs a specific function.

During electron transfer along the mitochondrial respiratory chain, only the quinone moiety of ubiquinone undergoes redox transformations, while the lateral isoprenoid chain remains unchanged. What is The Role of this chain in electron transfer processes?

3. Succinate oxidation uses FAD rather than NAD⁺ as an electron acceptor.

Для всех дегидрогеназ, участвующих в процессах гликолиза и в цикле лимонной кислоты, акцептором электронов служит NAD⁺, Е'° (NAD⁺ / NADH) = -0,32 В. Единственное исключение — реакция, катализируемая сукцинатдегидрогеназой, использующей в качестве акцептора электронов ковалентно связанный с ней FAD; в комплексе с ферментом Е'° (FAD/ FADH₂) = 0,050 В. Сравните Е'° для NAD⁺ / NADH и для FAD / FADH₂ в комплексе с ферментом со стандартным восстановительным потенциалом сопряженной пары фумарат / сукцинат (Е'° = 0,031 В), и объясните, почему при дегидрировании сукцината FAD — более подходящий акцептор электронов, чем NAD⁺.

4. The degree of reduction of electron carriers in the respiratory chain.

The reduction state of each electron carrier in the mitochondrial respiratory chain is determined by the conditions prevailing within the Cell/35.html">Mitochondria. When NADH and molecular oxygen are abundant, the reduction state of the electron carriers decreases as electrons flow from the substrate to oxygen. When Electron transport is blocked, carriers situated before the block in the respiratory chain become more reduced, whereas those located after the block become more oxidized, as illustrated in Fig. 19-6. Indicate the oxidation states of ubiquinone, Cytochromes b, c₁, c, and a + a₃ for the following four conditions:

a) sufficient NADH and O₂, but cyanide is added;

b) sufficient NADH, but the O₂ supply is depleted;

c) sufficient O₂, but the NADH supply is depleted;

d) sufficient NADH and O₂.

5. Effect of rotenone and antimycin A on electron transport.

Rotenone, a toxic compound produced by certain plant species, strongly inhibits mitochondrial NADH dehydrogenase activity in insects and fish. The toxic antibiotic antimycin A severely inhibits The oxidation of ubiquinol.

a) Why is rotenone a lethal poison for certain insects and fish?

b) Why is antimycin a poison?

c) Suppose both of these substances block their respective sites in the respiratory chain with equal efficiency. Which of them would be a more potent poison?

6. Uncoupling agents in Oxidative Phosphorylation.

In normal mitochondria, The rate of electron transfer is strictly regulated by ATP demands. Therefore, when ATP utilization is relatively low, the rate of electron transfer is also modest. If ATP is consumed rapidly, the electron transfer rate increases accordingly. Under such conditions (with these two processes tightly coupled), the P/O ratio—that is, the number of ATP molecules formed per atom of consumed

oxygen when NADH serves as the electron donor—is approximately 2.5.

a) How should relatively low and relatively high concentrations of an uncoupling agent affect the rate of electron transfer and the P/O ratio?

b) Ingestion of uncoupling agents causes profuse sweating and an elevated body Temperature. Explain this phenomenon at THE MOLECULAR LEVEL. How does the P/O ratio change in the presence of uncoupling agents?

c) The uncoupling agent 2,4-dinitrophenol was once tested as an anti-obesity drug. What is the pharmacological rationale behind this effect? Nowadays, uncoupling agents of this type are no longer used therapeutically because of known fatal cases. Why can the intake of uncoupling agents cause death?

7. Effect of valinomycin on oxidative phosphorylation.

When the antibiotic valinomycin is added to a suspension of actively respiring mitochondria, a decrease in ATP formation and oxygen consumption rate is observed, accompanied by heat release and an increase in the transmembrane pH gradient across the mitochondria. Explain these findings based on valinomycin's ability to transport K⁺ ions across The inner mitochondrial membrane.

8. MECHANISM OF ACTION of dicyclohexylcarbodiimide (DCCD).

When DCCD is added to a suspension of actively respiring mitochondria in which Respiration is tightly coupled to phosphorylation, There is a sharp drop in both the rate of electron transfer (measured by oxygen uptake) and the rate of phosphorylation (measured by ATP synthesis). When 2,4-dinitrophenol is added to such inhibited mitochondrial preparations, oxygen consumption returns to normal, but ATP synthesis remains suppressed.

a) Which stage of electron transfer or Oxidative phosphorylation is affected by DCCD?

b) Why does DCCD impair mitochondrial oxygen consumption? What is The Mechanism of action of 2,4-dinitrophenol on the inhibited mitochondrial preparation?

c) Which of the following inhibitors is DCCD most similar to in its mode of action: antimycin A, rotenone, oligomycin, or arsenate?

9. Compartmentation of Citric Acid Cycle components.

It has been found that in redox reactions, isocitrate dehydrogenase is localized exclusively in the mitochondria, whereas malate dehydrogenase is found in both the Cytosol and the mitochondria. What is the role of cytosolic malate dehydrogenase in The Citric Acid Cycle?

10. Malate–α-ketoglutarate transport system.

The transport system mediating the translocation of malate and α-ketoglutarate across the inner membrane (see Fig. 19-27) is inhibited by n-butylmalonate. When n-butylmalonate is added to an aerobic suspension of Kidney Cells utilizing only glucose as a metabolic fuel, how will this inhibitor affect cellular processes? Specifically, consider the following processes:

a) Glycolysis;

b) oxygen uptake;

c) lactate production;

d) ATP synthesis.

11. ATP synthesis is regulated by the intracellular ADP concentration.

The synthesis of ATP via oxidative phosphorylation requires ADP and phosphate, but the rate of synthesis depends primarily on the intracellular ADP concentration. Provide a reasoned explanation for this fact.

12. Time scale of regulatory processes in mitochondria.

Compare the time intervals required to regulate respiration rate upon (a) an increase in ADP concentration and (b) a decrease in pO₂. What is the reason for the differences?

13. The Pasteur Effect.

When oxygen is introduced into a suspension of anaerobic cells consuming glucose at a high rate, the cells begin to consume O₂ and the rate of glucose consumption drops sharply. Concurrently, lactate accumulation ceases. This phenomenon, characteristic of cells capable of both aerobic and anaerobic glucose METABOLISM, was first observed by Louis Pasteur in the 1860s and is therefore known as the Pasteur effect.

a) Why does lactate accumulation cease upon the Introduction of oxygen into The Cell suspension?

b) Why does the rate of glucose consumption decrease in the presence of oxygen?

c) How does the onset of oxygen consumption lead to a decrease in the rate of glucose consumption? Explain this based on the specific action of Enzymes.

14. Yeast mutants defective in cellular respiration and alcohol production.

Respiratory-deficient yeast mutants (rho-, "petites") are generated by treating normal yeast cells with mutagenic agents. Such mutant yeast cells are unable to synthesize the enzyme cytochrome c oxidase; As a result, their Fermentation processes are not inhibited by oxygen, meaning they do not exhibit the Pasteur effect (see Problem 13). Some ethanol producers use these mutant Yeasts to produce large amounts of alcohol from wood sawdust. Explain the advantages of using mutant yeasts over normal yeast cells for this purpose. Why is the Pasteur effect absent when the cells lack the enzyme cytochrome c oxidase?

15. Advantages of supercomplexes in electron transfer.

Emerging evidence suggests that mitochondrial complexes I, II, III, and IV are part of a larger supercomplex. What advantage might be conferred by a system in which all four complexes are assembled into a single supercomplex?

16. How many H⁺ ions are contained in a single mitochondrion.

The chemiosmotic hypothesis posits that as a result of electron transport, H⁺ ions are "pumped" out from the mitochondrial matrix, thereby establishing a pH gradient across the mitochondrial membrane where the outer side is more acidic than the inner side. The ability of H⁺ ions to diffuse back—from the surrounding medium into the mitochondrial matrix (where their concentration is lower)—serves as the driving force for ATP synthesis catalyzed by ATP synthase. In mitochondria suspended in a medium at pH 7.4, Oxidative phosphorylation takes place. The pH of the mitochondrial matrix has been shown to be 7.7.

a) Calculate the molar concentrations of H⁺ ions in the surrounding medium and in the matrix under these conditions.

b) Determine The ratio of outside-to-inside H⁺ concentrations, which helps estimate the energy potentially corresponding to this concentration difference (see Equation 11-4, Vol. 1).

c) Determine the number of H⁺ ions per respiring rat Liver mitochondrion. For your calculations, assume that the interior space of a mitochondrion is a sphere with a diameter of 1.5 µm.

d) Considering the results of your calculations, can this pH gradient alone be considered a sufficient source of energy for ATP synthesis?

e) If the pH gradient alone is insufficient, what other source of energy required for ATP synthesis can you identify?

17. Rate of ATP turnover in rat Heart Muscle.

Aerobic cells of rat heart muscle derive more than 90% of their required ATP through oxidative phosphorylation, utilizing glucose as "fuel." Each gram of rat heart tissue consumes oxygen at a rate of 10.0 µmol/min.

a) Calculate the rates of glucose consumption and ATP production in rat heart muscle.

b) Calculate how many seconds the cellular ATP pool would last if the initial ATP concentration was 5.0 µmol per gram of Muscle tissue. What does this result signify, given the necessity for precise Regulation of ATP production in cells? In your calculations, take into account that concentration is expressed per gram of muscle tissue (which consists primarily of Water).

18. Rate of ATP Cleavage in flight muscle.

In the flight Muscles of the green blowfly (Lucilia sericata), ATP is generated exclusively via oxidative phosphorylation. To maintain an ATP concentration of 7.0 µmol per gram of muscle during flight, the insect must consume oxygen at a rate of 187 mL/h per gram of body mass. Calculate the rate of ATP consumption in the flight muscles, assuming these muscles make up 20% of the insect's mass. For how long could the ATP pool be sustained without replenishment from oxidative phosphorylation? Assume that reducing equivalents are shuttled by the glycerol 3-phosphate shuttle and that the consumed oxygen is at a pressure of 101.3 kPa (1 atm) at a temperature of 25 °C.

19. Mitochondrial defects and Cancer.

Mutations in genes encoding certain mitochondrial Proteins are associated with an increased risk of developing specific types of cancer. How can mitochondrial defects lead to cancer?

20. Variability in the severity of Mitochondrial Diseases.

In individuals with disorders caused by a specific Mitochondrial Genome defect, symptoms can range from mild to severe. Explain this phenomenon.

21. Transport of reducing equivalents across the membrane.

In aerobic cells, extramitochondrial NADH must be oxidized by the mitochondrial respiratory chains. When NADH labeled with a radioactive hydrogen isotope (4-³H[NADH]) was introduced into a rat hepatocyte preparation containing mitochondria and all cytosolic enzymes, radioactivity was soon detected in the mitochondrial matrix. When 7-¹⁴C[NADH] was introduced, the matrix remained non-radioactive. What Conclusions regarding the oxidation processes of extramitochondrial NADH can be drawn from these observations?

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22. High Blood Alanine levels are associated with impaired oxidative phosphorylation.

It has been found that most individuals with impaired oxidative phosphorylation have a rather high concentration of alanine in their blood. Explain this observation.

23. Functions of stored NAD and dehydrogenase.

For Pyruvate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, NAD⁺ serves as the electron acceptor, even though both of these enzymes are localized in different cellular compartments of eukaryotes. Explain this fact.

24. Diabetes as a consequence of mitochondrial defects.

Glucokinases play a crucial role in Glucose metabolism in pancreatic β-cells. Individuals with two defective copies of the glucokinase Gene exhibit severe congenital diabetes, whereas defects in only a single copy lead to a much milder condition (maturity-onset diabetes of the young, MODY2). Explain this difference, taking into account The properties of β-cells.

25. Consequences of mutations in mitochondrial complex II.

A single-nucleotide substitution in the succinate dehydrogenase gene (complex II) is associated with the onset of mid-small intestinal carcinoma. Propose a mechanism to explain this observation.

26. Photochemical efficiency of light at different wavelengths.

When a green plant is illuminated with light at a wavelength of 680 or 700 nm, the rate of Photosynthesis, measured by oxygen O₂ evolution, is higher in the former case. However, simultaneous illumination of the plant with light of both wavelengths yields a higher photosynthetic rate than illumination with either wavelength alone. Explain the reason for this.

27. Calculation of the photosynthetic balance.

In 1804, Theodor de Saussure demonstrated that the total mass of oxygen released by a plant during photosynthesis plus the synthesized dry matter is greater than the mass of carbon dioxide absorbed. Explain this fact.

28. The role of H₂S in certain photosynthetic Bacteria.

In purple sulfur bacteria, photosynthesis can occur in the light in the presence of H₂O and ¹⁴CO₂, but only if H₂S is present and oxygen is absent. During photosynthesis (the rate of which is measured by The formation of ¹⁴C-glucose), H₂S is converted into elemental sulfur, and no oxygen is evolved. What role does The conversion of H₂S into elemental sulfur play? Why is no oxygen evolved?

29. Enhancement of Photosystem I reducing power by light energy.

Upon absorption of red light at a wavelength of 700 nm by photosystem I, the standard reduction potential of its reaction center P700 changes from 0.40 to -1.2 V. What fraction of the absorbed light is stored as reducing power?

30. Electron flow in Photosystems I and II.

Predict how an inhibitor of electron passage through pheophytin will affect electron transfer through (a) PSI and (b) PSII. Explain your reasoning.

31. Limited ATP Synthesis in the dark.

If spinach Chloroplasts are illuminated in the absence of ATP and phosphate, and then ADP and phosphate are added in the dark, ATP synthesis is observed for a short period of time. Explain the results of this experiment.

32. Mechanism of action of the herbicide diuron.

If chloroplasts are treated with a herbicide such as diuron, oxygen evolution and Photophosphorylation cease. Oxygen evolution (but not photophosphorylation) can be restored by adding an external electron acceptor, such as the Hill reagent. How does this herbicide kill weeds? At what point in the pathway shown in Fig. 19-56 does the inhibitory effect of diuron likely occur? Provide a reasoned answer.

33. Effect of venturicidin on oxygen metabolism.

Venturicidin is a potent inhibitor of ATP synthase; it interacts with the CF₀ component of the enzyme and blocks proton translocation through the CF₀CF₁ complex. How will venturicidin affect oxygen dynamics in a suspension of well-illuminated chloroplasts? Would any changes occur if the experiment were conducted in the presence of an uncoupling agent such as 2,4-dinitrophenol? Explain your answer.

34. Bioenergetics of photophosphorylation.

The steady-state concentrations of ATP, ADP, and phosphate in isolated spinach leaves at full illumination and pH 7 are 120.0, 6.0, and 700 µM, respectively.

a) How much free Energy is required to synthesize 1 mol of ATP under these conditions?

b) The energy for ATP synthesis is provided by light-driven electron transport in chloroplasts. What is the minimum potential difference required for The transfer of an electron pair under these conditions to drive ATP synthesis? (You may use Equation 13-6, p. 47.)

35. Light energy induces redox reactions.

Suppose a hypothetical photosynthetic microorganism oxidizes H₂S and transfers electrons to NAD⁺. What must be the wavelength of the absorbed light for its energy to be sufficient for removing an electron from H₂S and transferring it to NAD⁺ under standard conditions? The efficiency of the photochemical process in this case is 100%, and the standard reduction potentials for H₂S and NAD⁺ are -243 and -320 mV, respectively. Consider the scheme in Fig. 19-46.

36. Equilibrium constants of water-splitting reactions.

The coenzyme NADP⁺ is the terminal electron acceptor in chloroplasts, which can be written as the reaction equation:

2 Н₂O + 2 NADP⁺ —> 2 NADPH + 2 Н⁺ + O₂

Using the data in Table 19-2, calculate the equilibrium constants for this reaction at 25 °С. (The relationships between K'_eq and ∆G'° are discussed on p. 15.) What is the source of energy driving this reaction in chloroplasts?

37. Energetics of phototransformations.

In plant photosynthesis, the formation of a single O₂ molecule requires the absorption of eight light quanta.

2 Н₂O + 2 NADP⁺ + 8 photons —> 2 NADPH + 2 Н⁺ + O₂

Calculate the Free energy change for this reaction if the wavelength of the absorbed light is 700 nm (red) and the efficiency of the photosynthetic process is 100%.

38. Electron transfer to the Hill reagent.

If potassium ferricyanide (the Hill reagent) is added to spinach chloroplast extracts and these preparations are then illuminated, oxygen will be evolved According to the equation

2 Н₂O + 4 Fe³⁺ —> O₂ + 4 Н⁺ + 4 Fe²⁺

where Fe³⁺ is ferricyanide and Fe²⁺ is ferrocyanide. Is NADPH formed during this reaction? Justify your answer.

39. How often does a chlorophyll molecule absorb a photon?

Spinach leaves contain 20 μgg/cm² of chlorophyll (M_r = 892). During the day, under an average light intensity of 5.4 J/(cm² • min), the leaves absorb about 50% of the solar energy. How often does a single chlorophyll molecule absorb a photon? Calculate the fraction of chlorophyll molecules that are simultaneously excited, given that the average lifetime of an excited chlorophyll molecule in vivo is 1 ns.

40. Effect of Monochromatic Light on Electron Flow.

The oxidation or reduction state of an electron carrier during photosynthetic electron transfer can sometimes be determined from spectrophotometric data. When chloroplasts are illuminated with 700 nm light, cytochrome f, plastocyanin, and plastoquinone are oxidized. However, when chloroplasts are illuminated with 680 nm light, these electron carriers become reduced. Explain this observation.

41. Function of Cyclic Photophosphorylation.

When the [NADPH]/[NADP⁺] concentration ratio in a chloroplast is high, cyclic photophosphorylation predominates (see Fig. 19-56). Does oxygen participate in cyclic photophosphorylation? Is NADPH produced? Explain your reasoning. What is The primary function of cyclic photophosphorylation?

Analyzing Experimental Data

42. Photophosphorylation: Discovery, Rejection of the Hypothesis, and Rediscovery.

During the 1930s and 1940s, scientists began to understand the mechanism of photosynthesis. At the same time, the role of "energy-rich phosphate bonds" (now known as ATP) in glycolysis and cellular respiration was becoming clear. Many theories attempted to explain the mechanism of photosynthesis, particularly regarding the role of light. This problem examines a process previously referred to as the "primary photochemical process," meaning the initial conversion of absorbed light energy in a photosynthetic cell. Interestingly, a crucial component of the modern model of photosynthesis was formulated quite early, only to be rejected, forgotten for several years, and later accepted once again.

In 1944, Emerson, Stauffer, and Umbreit suggested that "the function of light energy in photosynthesis is the formation of 'energy-rich' phosphate bonds" (p. 107). According to their model (hereinafter referred to as the Emerson model), the free energy required for both the fixation and reduction of CO₂ is derived precisely from these "energy-rich" phosphate bonds (i.e., ATP) generated through the absorption of light by a chlorophyll-protein complex.

This model was refuted by Rabinowitch in 1945. Summarizing the findings of Emerson and co-workers, Rabinowitch concluded: "Until other evidence is obtained, we raise an energetic objection against this hypothesis. Photosynthesis is an energy-storing process. Why convert a light quantum (even red light, whose energy is about 43 kcal/einstein) into a 'phosphate quantum' with an energy of only 10 kcal/mol? That would be a step in the wrong direction—toward energy dissipation rather than storage" (vol. 1, p. 228). This argument, along with other observations, led to the rejection of the Emerson model. It was not until the 1950s that the Emerson model was recognized as correct, albeit with certain modifications.

Review the information presented in the paper by Emerson and colleagues (items a–g below). Answer the following three questions:

1. How does this information support the Emerson model, which proposes that light energy is used directly by chlorophyll to synthesize ATP, which in turn provides the energy required for CO₂ fixation and reduction?

2. How might Rabinowitch have explained this information based on his own model (and most models of that time), in which light energy was used directly by chlorophyll to synthesize reducing equivalents? Rabinowitch wrote: "Theoretically, there is no reason why the total electron energy contained in molecules excited by Light absorption should not be available for oxidation-reduction processes" (vol. 1, p. 152). In this model, the reducing substances were subsequently used for CO₂ fixation and reduction, and the energy required for this accounted for only a fraction of the large amount of free energy generated by the reduction reactions.

3. How is this information explained in light of our modern understanding of photosynthesis?

a) Chlorophyll contains a Mg²⁺ ion, which is known to be an essential cofactor in many enzymes catalyzing phosphorylation and dephosphorylation reactions.

b) A crude "chlorophyll-protein" preparation isolated from photosynthetic cells exhibited phosphorylating activity.

c) The phosphorylating activity of the "chlorophyll-protein" was inhibited by light.

d) The levels of certain phosphorylated compounds in photosynthetic cells changed dramatically in the light (Emerson and co-workers were unable to identify the specific substances).

As it turned out later, both models—those of Emerson and Rabinowitch—were correct to a certain extent.

e) Explain how these two models relate to the modern model of photosynthesis.

In refuting the Emerson model, Rabinowitch insisted: "Structure/149.html">The problem of phosphate-storage theory becomes most acute when we turn to the fact that, at low light intensities, eight or ten light quanta suffice to reduce one molecule of carbon dioxide. If each quantum were to produce one high-energy phosphate molecule, only 80 to 100 kcal/einstein would be stored, whereas photosynthesis requires at least 112 kcal/mol, and likely more due to losses in irreversible partial reactions" (vol. 1, p. 228).

f) How do Rabinowitch's calculations (8 to 10 photons per reduced CO₂ molecule) compare with the accepted value today? For additional information, refer to Chapter 20.

g) How would you refute Rabinowitch's argument based on our current knowledge of photosynthesis?