Principles of Biochemistry Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Electron Transport, Oxidative Phosphorylation, and Regulation of ATP Synthesis
Chapter Summary

In electron-transfer reactions, i.e., oxidation-reduction (redox) reactions, the capacity of an electron donor (reductant) to release electrons is characterized by the standard reduction potential E'₀. Redox systems with more electronegative E'₀ values tend to transfer their electrons to systems with more electropositive E'₀ values. The change in Standard Free energy in redox reactions is determined from the equation ∆G⁰' = -nF∆E'₀. In Cell/35.html">Mitochondria, hydrogen atoms split from substrates by dehydrogenases pass their electrons into the Electron Transport Chain. Moving along this chain from one carrier to another, the electrons ultimately reach molecular oxygen and reduce it to H₂O. The energy released during electron transfer is utilized for the Oxidative Phosphorylation of ADP to ATP. Oxidative phosphorylation takes place in The inner mitochondrial membrane.

From all NAD-dependent dehydrogenation reactions, reducing equivalents are transferred to mitochondrial NADH dehydrogenase, which contains FMN as a prosthetic group. Then, via a series of iron-sulfur centers, they are passed to ubiquinone, which transfers electrons to cytochrome b. Subsequently, the electrons pass sequentially to Cytochromes c₁ and c, and then to cytochrome aa₃ (cytochrome c oxidase), which contains copper. Cytochrome c oxidase transfers electrons to O₂. Complete reduction of O₂ to form two molecules of H₂O requires four electrons and four H⁺ ions. Electron transport is blocked at specific sites by rotenone, antimycin A, and cyanide. The electron transport process is accompanied by a significant drop in free energy. At three sites of the Respiratory Chain, energy is conserved through the synthesis of ATP from ADP and P_i. OXIDATIVE PHOSPHORYLATION AND electron transport can be uncoupled using uncoupling agents or ionophores such as valinomycin. For oxidative phosphorylation to occur, the inner mitochondrial membrane must remain intact and be impermeable to H⁺ and certain other ions. Electron transport is accompanied by the outward pumping of H⁺ ions from the mitochondria. According to the chemiosmotic hypothesis (one of three hypotheses proposed to explain the Mechanism of Oxidative phosphorylation), electron transport creates a concentration gradient of H⁺ ions across the inner mitochondrial membrane, with a higher concentration outside than inside. This gradient is thought to serve as the driving force for ATP synthesis as H⁺ ions return from the Cytosol to the matrix by passing through the F₀F₁-ATPase molecules in the membrane. The inner mitochondrial membrane contains transport systems for adenine NUCLEOTIDES, phosphate, and A number of metabolites. Electron transport is inhibited when ADP concentration is low and accelerates when ADP concentration rises due to various cellular processes involving ATP utilization. The rates of Glycolysis, The Citric Acid Cycle, and oxidative phosphorylation are coordinated. This coordination is ensured by interrelated regulatory mechanisms that respond to the [ATP]/[ADP][P_i] ratio and the levels of certain key metabolites reflecting the energy status of the Cells.

Cells also undergo oxidative reactions in which oxygen atoms are incorporated into organic molecules—primarily into relatively hydrophobic molecules of various foreign substances and drugs—yielding hydroxylated and carboxylated products.

Introduction/47.html">Further Reading

General Overviews

Dickerson R. E. Cytochrome c and the Evolution of Energy METABOLISM, Sci. Am., 242, 137-153, March (1980).

Hinkle P., McCarty R. E. How Cells Make ATP.

Sci. Am., 238, 104-123, March (1978). Whittaker D. A., Danks S. M. Mitochondria: Structure, Function and Assembly, Longman, London, 1978. A concise, up-to-date review. The material is clearly presented and well illustrated. An excellent guide for a general Introduction to the subject.

Historical Background and General Information

Keilin D. The history of Cell Respiration and Cytochromes, Cambridge University Press, London, 1966.

Lehninger A. L. The Mitochondrion: Molecular Basis of Structure and function, Benjamin, New York, 1965. Mitochondria as the powerhouse of The Cell.

Racker E. A New Look at Mechanisms in Bioenergetics, Academic, New York, 1976. An account of personal research with entertaining commentary.

Mechanism of Oxidative Phosphorylation

Boyer P. D., Chance B., Ernster L., Mitchell P., Racker E., Slater E. C. Oxidative Phosphorylation and Photophosphorylation, Ann. Rev. Biochem., 46, 955-1026 (1977). Diverse views on The Mechanism of oxidative phosphorylation presented by leading researchers in the field.

Mitchell P. Keilin's Respiratory Chain Concept and Its Chemiosmotic Consequences, Science, 206, 1148-1159 (1979). Nobel lecture outlining The Development of the chemiosmotic hypothesis.

Tedeschi H. Mitochondria: Structure, Biogenesis, and Transducing Functions, Springer-Verlag, New York, 1976. A more detailed survey.

Special Topics

Lehninger A. L. Mitochondria and Biological Mineralization Processes: an Exploration. In: E. Quagliariello, F. Palmieri, and T. Singer (eds), Horizons in Biochemistry and Biophysics, vol. 4, pp. 1-30, Addison-Wesley, Reading, Mass., 1977. Since mitochondria accumulate Ca²⁺ along with phosphate, they may serve as the site where the Cytology/cytology/16.html">Early stages of biological calcification take place.

Luft R., Ikkos D., Palmieri G., Ernster L., Afzelius B. A Case of Severe Hypermetabolism of Non-Thyroid Origin with a Defect in Mitochondrial Respiratory Control: A Correlated Clinical, Biochemical, and Morphological Study, J. Clin. Invest., 41, 1776-1804 (1962). The first studied case of impaired electron transport regulation in humans.

Questions and Problems

1. Redox reactions. The NADH dehydrogenase complex of the Mitochondrial Electron Transport chain catalyzes the following redox reactions (where Fe³⁺ and Fe²⁺ denote iron atoms of iron-sulfur centers, Q is ubiquinone, QH₂ is ubiquinol, and E is the enzyme):

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For each of these three Reactions Catalyzed by the NADH dehydrogenase complex, identify: (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reductant, and (e) the oxidant.

2. Standard reduction potentials. The standard reduction potential of any redox pair is defined by the half-reaction:

Oxidant + (n)Electrons = Reductant.

The standard reduction potentials of the two conjugate redox pairs NAD⁺/NADH and Pyruvate/lactate are -0.32 V and -0.19 V, respectively.

a) Which of these pairs has a greater tendency to donate electrons? Explain your reasoning.

b) Which of them is a stronger oxidizing agent? Why?

c) In which direction will the reaction proceed

Pyruvate + NADH + H⁺ = Lactate + NAD⁺,

if the initial concentrations of reactants and products are each 1 M at pH 7?

d) What is the change in standard free energy, ∆G⁰', for this reaction at 25°C?

e) What is the Equilibrium Constant for this reaction at 25°C?

3. Arrangement of carriers in an electron transport chain of a plant. A Study of the electron transport chain in spinach leaf cells revealed several substances capable of reversibly accepting and donating electrons. Their standard reduction potentials are listed below.

Based on their standard reduction potentials, deduce the probable sequence of these electron carriers in the respiratory chain. Construct an energy diagram similar to the one shown in Fig. 17-4. At which steps of the transfer is the release of free energy (under standard conditions) insufficient to drive the synthesis of one ATP molecule per pair of transferred electrons?

4. ATP yield coupled to substrate oxidation. Four substrates are listed below. Calculate the number of ATP molecules generated by the complete oxidative breakdown of one molecule of each substrate to CO₂ and H₂O.

a) Fructose-6-phosphate

b) Acetyl-CoA

c) Glyceraldehyde-3-phosphate

d) Sucrose

5. Energy span of the respiratory chain. Electron transport in the mitochondrial respiratory chain is described by the following overall equation:

a) Calculate ∆E₀' for this overall reaction of mitochondrial electron transport.

b) Calculate the standard free energy change, ∆G⁰', for this reaction.

c) How many ATP molecules can theoretically be synthesized by this reaction if the standard Free energy of ATP formation is +7.3 kcal/mol?

6. FAD rather than NAD⁺ serves as the electron acceptor in The oxidation of succinate. In all dehydrogenation steps of glycolysis and The Citric Acid cycle, NAD⁺ acts as the electron acceptor (E₀' = -0.32 V). The only exception is the reaction catalyzed by succinate dehydrogenase, which utilizes a covalently bound FAD as the electron acceptor (E₀' = +0.05 V). Why is FAD a more suitable electron acceptor than NAD⁺ for the dehydrogenation of succinate? Suggest a possible explanation by comparing the E'₀ values of the succinate-fumarate system, the NAD⁺/NADH conjugate pair, and the FAD/FADH₂ pair.

7. Reduction state of electron carriers in the respiratory chain. The reduction state of each electron carrier in the respiratory chain is determined by the conditions prevailing within the mitochondria. When adequate amounts of NADH and molecular oxygen are present, the steady-state reduction level of the carriers decreases as electrons flow from substrate to oxygen. If electron transport is blocked, carriers located before the block become more reduced, whereas those located after the block become more oxidized, as illustrated by the hydraulic model of the respiratory chain in Fig. 17-14. How would these models look in the following four cases:

a) Sufficient NADH and O₂ are present, but cyanide is added.

b) Sufficient NADH is present, but O₂ is depleted.

c) Sufficient O₂, but the NADH pool is depleted.

d) Sufficient amounts of both NADH and O₂.

8. Effects of rotenone and antimycin A on electron transport. Rotenone (a toxic compound produced by certain plant species) strongly inhibits The activity of mitochondrial NADH dehydrogenase. The toxic antibiotic antimycin A strongly inhibits ubiquinol oxidation.

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

b) Why does antimycin A act as a poison in animal Tissues?

c) Assuming that both substances block their respective sites in the respiratory chain with equal efficiency, which of them would be a more potent poison? Provide a reasoned argument.

9. Uncoupling agents in oxidative phosphorylation. In normal mitochondria, The rate of electron transport is tightly coupled to the demand for ATP. Therefore, if the rate of ATP utilization is relatively low, the rate of electron transport is correspondingly low. Conversely, if ATP is consumed at a high rate, the electron transport rate is also high. Under such conditions (with these two processes tightly coupled), the P/O ratio—i.e., the number of ATP molecules formed per atom of oxygen consumed when NADH serves as the electron donor—is approximately 3.

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

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

c) 2,4-Dinitrophenol, an uncoupling agent, was at one time used for weight loss management. What is the fundamental basis for this effect? Such uncoupling agents are no longer used as Pharmaceuticals because of known cases where their administration proved fatal. Why can the intake of uncoupling agents cause death?

10. MECHANISM OF ACTION of dicyclohexylcarbodiimide (DCCD). If DCCD is added to a suspension of actively respiring mitochondria in which respiration is tightly coupled to phosphorylation, a sharp decrease is observed in both the rate of electron transport (estimated by oxygen consumption) and the rate of phosphorylation (estimated by ATP formation). Subsequent addition of 2,4-dinitrophenol to these inhibited mitochondrial preparations shows that oxygen consumption returns to normal levels, whereas ATP synthesis remains inhibited.

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

b) Why does DCCD impair O₂ consumption in mitochondria? What is the mechanism of action of 2,4-dinitrophenol on the DCCD-inhibited mitochondrial preparation?

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

11. Oxidative phosphorylation in inverted submitochondrial vesicles. According to the chemiosmotic hypothesis, during electron transport in intact mitochondria, H⁺ ions are "pumped" out, generating a pH gradient across the mitochondrial membrane. This pH gradient stores energy that drives H⁺ ions back from the surrounding medium into the mitochondrial matrix. As H⁺ ions pass through F₀F₁-ATPase molecules, ATP is synthesized from ADP and P_i. It has been demonstrated that inverted vesicles derived from the inner mitochondrial membrane, with their F₀F₁-ATPase heads facing outward (Fig. 17-15), are also capable of oxidative phosphorylation.

a) Draw a diagram showing the direction of H⁺ ion pumping during electron transport in submitochondrial vesicles.

b) Indicate on this diagram the direction of the H⁺ ion flow through the F₀F₁-ATPase molecules during ATP synthesis.

c) How would oligomycin and atractyloside affect electron transport and ATP Synthesis in such submitochondrial vesicles?

12. Brown adipose tissue mitochondria. Newborn infants possess a specialized adipose tissue in the neck and upper back region that is virtually absent in adults—so-called brown fat. The brown color is imparted by mitochondria, which are extremely abundant in this tissue. Certain animals that undergo hibernation or are adapted to cold environments also possess brown fat. Whereas in Liver mitochondria the oxidation of NADH typically yields three ATP molecules per atom of oxygen consumed, in brown fat mitochondria the ATP yield per atom of oxygen consumed is less than one.

a) What physiological function might be served by this low P/O ratio in newborn brown fat?

b) Suggest possible mechanisms that could account for such a low P/O ratio characteristic of brown fat mitochondria.

13. Mitochondrial dicarboxylate transport system. The inner mitochondrial membrane contains a dicarboxylate transport system that mediates The transport of malate and a-ketoglutarate across the membrane. This transport system is inhibited by n-butylmalonate. Suppose n-butylmalonate is added to a suspension of aerobic Kidney cells utilizing glucose as their sole fuel source. How would n-butylmalonate affect a) glycolysis, b) oxygen consumption, c) lactate production, and d) ATP synthesis?

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

a) Why does the introduction of oxygen into the cell suspension halt lactate accumulation?

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

c) How does the rate of glucose consumption decrease after oxygen consumption has begun? Explain this in terms of the specific action of Enzymes.

15. Changes in the cellular energy charge. When the physiological activity of Skeletal Muscle cells changes, their energy charge, which is normally 0.89, initially drops sharply to approximately 0.70 before gradually returning to its baseline level.

a) What specific change in activity causes this sudden drop in The energy charge? Explain your answer.

b) How should this sudden change affect the rates of glycolysis and respiration?

c) In what way can the energy charge influence glycolysis and respiration?

16. How many H⁺ ions are contained in a single mitochondrion? The chemiosmotic hypothesis postulates that As a result of electron transport, H⁺ ions are "pumped" out of the mitochondrial matrix, thereby generating a pH gradient across the mitochondrial membrane where the external phase becomes more acidic than the internal one. According to this hypothesis, the tendency 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 F₀F₁-ATPase. In mitochondria suspended in a medium at pH 7.4, oxidative phosphorylation takes place. The pH of the mitochondrial matrix under these conditions has been found to be 7.7.

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

b) Determine The ratio of outside-to-inside H⁺ concentrations, which reflects the energy stored in this concentration difference (see Ch. 14).

c) Determine the number of H⁺ ions present in a single respiring liver mitochondrion. For this calculation, assume that the internal space of the mitochondrion is a sphere with a diameter of 1.5 µm.

d) Given your findings, can this pH gradient alone be considered a sufficient energy source for ATP synthesis?

e) If you believe that this pH gradient alone is insufficient, what other source of energy required for ATP synthesis would you suggest?