Chapter 18 : Oxidative Phosphorylation and Photophosphorylation

SUMMARY

Chemiosmotic theory provides the intellectual framework for understanding many biological energy transductions, including the processes of oxidative phosphorylation in mitochondria and photophosphorylation in chloroplasts. The mechanism of energy coupling is similar in both cases. The conservation of free energy involves the passage of electrons through a chain of membrane-bound oxidation-reduction (redox) carriers and the concomitant pumping of protons across the membrane, producing an electrochemical gradient, the protonmotive force. This force drives the synthesis of ATP by membrane-bound enzyme complexes through which protons flow back across the membrane, down their electrochemical gradient. Protonmotive force also drives other energy-requiring processes of cells.

In mitochondria, H atoms removed from substrates by the action of NAD-linked dehydrogenases donate their electrons to the respiratory (electron transfer) chain, which transfers them to molecular O₂, reducing it to H₂O. Shuttle systems convey reducing equivalents from cytosolic NADH to mitochondrial NADH. Reducing equivalents from all NAD-linked dehydrogenations are transferred to mitochondrial NADH dehydrogenase (Complex I), which contains FMN as its prosthetic group. They are then passed via a series of Fe-S centers to ubiquinone, which transfers the electrons to cytochrome b, the first carrier in Complex III. In this complex, electrons pass through two b-type cytochromes and cytochrome cl before reaching an Fe-S center. The Fe-S center passes electrons, one at a time, through cytochrome c and into Complex IV, cytochrome oxidase. This copper-containing enzyme, which also contains cytochromes a and a₃, accumulates electrons, then passes them to O₂, reducing it to H₂O.

There are alternative paths of entry of electrons into this chain of carriers. Succinate, for example, is oxidized by succinate dehydrogenase (Complex II), which contains a flavoprotein (with FAD) that passes electrons through several Fe-S centers and into the chain at the level of ubiquinone. Electrons derived from the oxidation of fatty acids pass into ubiquinone via the electron-transferring flavoprotein (ETFP).

The flow of electrons through Complexes I, III, and IV results in the pumping of protons across the mitochondrial inner membrane, making the matrix alkaline relative to the extramitochondrial space. This proton gradient provides the energy (proton-motive force) for ATP synthesis from ADP and Pi by an inner-membrane protein complex, ATP synthase, also called F₀F₁ ATPase. The details of this ATP-synthesizing mechanism are still under investigation. Bacteria carry out oxidative phosphorylation by essentially the same mechanism, using electron carriers and an ATP synthase in the plasma membrane. Oxidative phosphorylation produces most of the ATP required by aerobic cells; it is regulated by cellular energy demands. In brown fat tissue, which is specialized for the production of metabolic heat, electron transfer is uncoupled from ATP synthesis; the energy of fatty acid oxidation is therefore dissipated as heat.

Photophosphorylation in the chloroplasts of green plants and in cyanobacteria also involves electron flow through a series of membrane-bound carriers. In the light reactions of plants, the absorption of a photon excites chlorophyll molecules and other (accessory) pigments that funnel the energy into reaction centers in the thylakoid membranes of chloroplasts. At the reaction centers, photoexcitation results in a charge separation that produces one chemical species that is a good electron donor (reducing agent) and another that is a good electron acceptor. In chloroplasts there are two different photoreaction centers, which function
together. Photosystem I passes electrons from its excited reaction center, P700, through a series of carriers to ferredoxin, which then reduces NADP⁺ to NADPH. The reaction center, P680, of photosystem II passes electrons to plastoquinone, reducing it to the quinol form. The electrons lost from P680 are replaced by electrons abstracted from H₂O (hydrogen donors other than H₂O are used in other organisms). This light-driven splitting of H₂O is catalyzed by a Mn-containing protein complex; O₂ is produced. Reduced plastoquinone carries electrons from photosystem II to the cytochrome bf complex; these electrons pass to the soluble protein plastocyanin, and then to P700 to replace those lost during its photoexcitation. Electron flow through the cytochrome bf complex is accompanied by proton pumping across the thylakoid membrane, and the proton-motive force thus created drives ATP synthesis by a CF₀CF₁ complex closely similar to the F₀F₁ complex of mitochondria. This flow of electrons through photosystems II and I thus produces both NADPH and ATP. A second type of electron flow (cyclic flow) produces ATP only.

Both mitochondria and chloroplasts contain their own genomes and are believed to have originated from prokaryotic endosymbionts of early eukaryotic cells. Oxidative phosphorylation in aerobic bacteria and photophosphorylation in photosynthetic bacteria are closely similar, in machinery and mechanism, to the homologous processes in mitochondria and chloroplasts.

problems ( Answer )

l.Oxidation-Reduction Reactions The NADH dehydrogenase complex of the mitochondrial respiratory chain promotes the following series of oxidation-reduction reactions, in which Fe³⁺ and Fe²⁺ represent the iron in iron-sulfur centers, UQ is ubiquinone, UQHz is ubiquinol, and E is the enzyme:

(1) NADH + H⁺ + E-FMN NAD⁺ + E-FMNH₂
(2) E-FMNH₂ + 2Fe³⁺ E-FMN + 2Fe²⁺ + 2H⁺
(3) 2Fe²⁺ + 2H⁺ + UQ 2Fe³⁺ + UQH₂
Sum: NADH + H⁺ + UQ NAD⁺ + UQH₂

For each of the three reactions catalyzed by the NADH dehydrogenase complex, identify

(a) theelectron donor,
(b) the electron acceptor,
(c) the conjugate redox pair,
(d) the reducing agent, and
(e) the oxidizing agent.

2. Standard Reduction Potentials The standard reduction potential of any redox couple is defined for the half cell reaction (or half reaction):

Oxidizing agent + n electrons reducing agent

The standard reduction potentials of the NAD⁺ / NADH and pyruvate/lactate redox pairs are -0.320 and -0.185 V, respectively.

(a) Which redox pair has the greater tendency to lose electrons? Explain.
(b) Which is the stronger oxidizing agent? Explain.
(c) Beginning with 1 M concentrations of each reactant and product at pH 7, in which direction will the following reaction proceed?
Pyruvate + NADH + H⁺ lactate + NAD⁺
(d) What is the standard free-energy change, ΔG°', at 25 °C for this reaction?
(e) What is the equilibrium constant for this reaction at 25 °C?

3. Energy Span of the Respircztory Chain Electron transfer in the mitochondrial respiratory chain may be represented by the net reaction equation

NADH + H⁺ + ½2O₂ H₂O + NAD⁺

(a) Calculate the value of the change in standard reduction potential, ΔE'₀, for the net reaction of mitochondrial electron transfer.
(b) Calculate the standard free-energy change, ΔG°', for this reaction.
(c) How many ATP molecules could theoretically be generated per molecule of NADH oxidized by this reaction, given a standard free energy of ATP synthesis of 30.5 kJ/mol?
(d) How many ATP molecules could be synthesized under typical cellular conditions (see Box 13-2 )?

4. Use of FAD Rather Than NAD⁺ in the Oxidcztion of Succinate All the dehydrogenation steps in glycolysis and the citric acid cycle use NAD⁺ (E₀ for NAD⁺/NADH = -0.32 V) as the electron acceptor except succinate dehydrogenase, which uses covalently bound FAD (Eo for FAD/FADH₂ in this enzyme = 0.05 V). Why is FAD a more appropriate electron acceptor than NAD⁺ in the dehydrogenation of succinate? Give a possible explanation based on a comparison of the E₀ values of the fumarate/succinate pair (Eo = 0.03), the NAD⁺/NADH pair, and the succinate dehydrogenase FAD/ FADH₂ pair.

5. Degree of Reduction of Electron Carriers in the Respiratory Chain The degree of reduction of each electron carrier in the respiratory chain is determined by the conditions existing in the mitochondrion. For example, when the supply of NADH and O₂ is abundant, the steady-state degree of reduction of the carriers decreases as electrons pass from the substrate to O₂. When electron transfer is blocked, the carriers before the block become more reduced while those beyond the block become more oxidized (Fig. 18-7). For each of the conditions below, predict the state of oxidation of each carrier in the respiratory chain (ubiquinone and cytochromes b, cl, c, and a + a₃).

(a) Abundant supply of NADH and O₂ but cyanide added
(b) Abundant supply of NADH but O₂ exhausted
(c) Abundant supply of O₂ but NADH exhausted (d) Abundant supply of NADH and O₂

6. The 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.

(a) Explain why rotenone ingestion is lethal to some insect and fish species.
(b) Explain why antimycin A is a poison.
(c) Assuming 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.

7. Uncouplers of OxidcztiUe Phosphorylation In normal mitochondria the rate of electron transfer is tightly coupled to the demand for ATP. Thus when the rate of utilization of ATP is relatively low, the rate of electron transfer is also low. Conversely, when ATP is demanded at a high rate, electron transfer is rapid. Under such conditions of tight coupling, the number of ATP molecules produced per atom of oxygen consumed when NADH is the electron donor-known as the P/O ratio-is close to 3.

(a) Predict the effect of a relatively low and a relatively high concentration of an uncoupling agent on the rate of electron transfer and the P/O ratio.
(b) The ingestion of uncouplers causes profuse sweating and an increase in body temperature. Explain this phenomenon in molecular terms. What happens to the P/O ratio in the presence of uncouplers?
(c) The uncoupler 2,4-dinitrophenol was once prescribed as a weight-reducing drug. How can this agent, in principle, serve as a reducing aid? Such uncoupling agents are no longer prescribed because some deaths occurred following their use. Why can the ingestion of uncouplers lead to death?

8. Mode of Action of Dicyclohexylcarbodiimide (DCCD) When DCCD is added to a suspension of tightly coupled, actively respiring mitochondria, the rate of electron transfer (measured by O₂ consumption) and the rate of ATP production dramatically decrease. If a solution of 2,4-dinitrophenol is now added to the inhibited mitochondrial preparation, O₂ consumption returns to normal but ATP production remains inhibited.

(a) What process in electron transfer or oxidative phosphorylation is affected by DCCD?
(b) Why does DCCD affect the O₂ consumption of mitochondria? Explain the effect of 2,4-dinitrophenol on the inhibited mitochondrial preparation.
(c) Which of the following inhibitors does DCCD most resemble in its action: antimycin A, rotenone, or oligomycin?

9. The Malate-α-Ketoglutarate Transport System of Mitochondria The inner mitochondrial membrane transport system that promotes the transport of malate and α-ketoglutarate across the membrane (Fig. 18-25) is inhibited by n-butylmalonate. Suppose n-butylmalonate is added to an aerobic suspension of kidney cells using glucose exclusively as fuel. Predict the effect of this inhibitor on

(a) Glycolysis
(b) Oxygen consumption
(c) Lactate formation
(d) ATP synthesis

10. The Pasteur Effect When O₂ is added to an anaerobic suspension of cells using glucose at a high rate, the rate of glucose consumption declines dramatically as the added O₂ is consumed. In addition, the accumulation of lactate ceases. This ef fect, first observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of both aerobic and anaerobic utilization of glucose.

(a) Why does the accumulation of lactate cease after O₂ is added?
(b) Why does the presence of O₂ decrease the rate of glucose consumption?
(c) How does the onset of O₂ consumption slow down the rate of glucose consumption?

Explain in terms of specific enzymes.

11. How Many Protons in a Mitochondrion? Electron transfer functions to translocate protons from the mitochondrial matrix to the external medium to establish a pH gradient across the inner membrane, the outside more acidic than the inside. The tendency of protons to diffuse from the outside into the matrix, where [H⁺] is lower, is the driving force for ATP synthesis via the ATP synthase. During oxidative phosphorylation by a suspension of mitochondria in a medium of pH 7.4, the internal pH of the matrix has been measured as 7.7.

(a) Calculate [H⁺] in the external medium and in the matrix under these conditions.
(b) What is the outside:inside ratio of [H⁺]? Comment on the energy inherent in this concentration. (Hint: See p. 383, Eqn 13-5.)
(c) Calculate the number of protons in a respiring liver mitochondrion, assuming its inner matrix compartment is a sphere of diameter 1.5 μm.
(d) From these data would you think the pH gradient alone is sufficiently great to generate ATP?
(e) If not, can you suggest how the necessary energy for synthesis of ATP arises?

12. Rate of ATP Turnouer in Rat Heart Muscle Rat heart muscle operating aerobically fills more than 90% of its ATP needs by oxidative phosphorylation. This tissue consumes O₂ at the rate of 10 μmol/ min&•g of tissue, with glucose as the fuel source.

(a) Calculate the rate at which this tissue consumes glucose and produces ATP.
(b) If the steady-state concentration of ATP in rat heart muscle is 5 μmol/g of 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. )

13. Rate of ATP Breakdown in Flight Muscle ATP production in the flight muscles of the fly Lucilia sericata results almost exclusively from oxidative phosphorylation. During flight, 187 ml of O₂ / h•g of fly body weight is needed to maintain an ATP concentration of 7 μmol/g of flight muscle. Assuming that the flight muscles represent 20?0 of the weight of the fly, 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 reducing equivalents are transferred by the glycerol-3-phosphate shuttle and that O₂ is at 25 °C and 101.3 kPa (1 atm). (Note: Concentrations are expressed in micromoles per gram of flight muscle. )

14. Transmembrane Movement of Reducing Equivalents Under aerobic conditions, extramitochondrial NADH must be oxidized by the mitochondrial electron transfer chain. Consider a preparation of rat hepatocytes containing mitochondria and all the enzymes of the cytosol. If [4-³H]NADH is introduced, radioactivity appears quickly in the mitochondrial matrix. However, if [7-^l4C]NADH is introduced, no radioactivity appears in the matrix. What do these observations tell us about the oxidation of extramitochondrial NADH by the electron transfer chain?

15. Photochemical Efficiency of Light at Different Wavelengths The rate of photosynthesis, measured by O₂ production, is higher when a green plant is illuminated with light of wavelength 680 nm than with light of 700 nm. However, illumination by a combination of light of 680 nm and 700 nm gives a higher rate of photosynthesis than light of either wavelength alone. Explain.

16. Role of H₂S in Some Photosynthetic Bacteria Illuminated purple sulfur bacteria carry out photosynthesis in the presence of H₂O and 14CO₂, but only if H₂S is added and O₂ is absent. During the course of photosynthesis, measured by formation of [^l4C]carbohydrate, H₂S is converted into elemental sulfur, but no O₂ is evolved. What is the role of the conversion of H₂S into sulfur? Why is no O₂ evolved?

17. Boosting the Reducing Power of Photosystem I by Light Absorption When photosystem I absorbs red light at 700 nm, the standard reduction potential of P700 changes from 0.4 to about -1.2 V. What fraction of the absorbed light is trapped in the form of reducing power?

18. Mode of Action of the Herbicide DCMU When chloroplasts are treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, or Diuron), a potent herbicide, O₂ evolution and photophosphorylation cease. Oxygen evolution but not photophosphorylation can be restored by the addition of an external electron acceptor, or Hill reagent. How does this herbicide act as a weed killer? Suggest a location for the inhibitory site of this herbicide in the scheme shown in Figure 18-44. Explain.

19. Bioenergetics of Photophosphorylation The steady-state concentrations of ATP, ADP, and Pi in isolated spinach chloroplasts under full illumination at pH 7.0 are 120, 6, and 700 μm, respectively.

(a) What is the free-energy requirement for the synthesis of 1 mol of ATP under these conditions?
(b) The energy for ATP synthesis is furnished by light-induced electron transfer in the chloroplasts. What is the minimum voltage drop necessary during the transfer of a pair of electrons to synthesize ATP under these conditions? (You may need to refer to p. 389, Eqn 13-8.)

20. Equilibrium Constant for Water-Splitting Reactions The coenzyme NADP⁺ is the terminal electron acceptor in chloroplasts, according to the reaction

2H₂O + 2NADP⁺ 2NADPH + 2H⁺ + O₂

Use the information in Table 18-2 to calculate the equilibrium constant at 25 °C for this reaction. (The relationship between Keq and ΔG°' is discussed on p. 368. ) How can the chloroplast overcome this unfavorable equilibrium?

21. Energetics of Phototransduction During photosynthesis, eight photons of light must be absorbed (four by each photosystem) for every 02 molecule produced:

H₂O + 2NADP⁺ + 8 photons 2NADPH + 2H⁺ + O₂

Assuming that these photons have a wavelength of 700 nm (red) and that the absorption and utilization of light energy are 100% efficient, calculate the free-energy change for the process.

22. Electron Transfer to a Hill Reagent Isolated spinach chloroplasts evolve Oz when illuminated in the presence of potassium ferricyanide (the Hill reagent), according to the equation

2H₂O + 4Fe³⁺ O₂ + 4H⁺ + 4Fe²⁺

where Fe³⁺ represents ferricyanide and Fe²⁺, ferrocyanide. Is NADPH produced in this process? Explain.

23. How Often Does a Chlorophyll Molecule Absorb a Photon? The amount of chlorophyll a (Mr 892) in a spinach leaf is about 20 μg/cm² of leaf. In noonday sunlight (average energy 5.4 J/cm² •min), the leaf absorbs about 50% of the radiation. How often does a single chlorophyll molecule absorb a photon? If the average lifetime of an excited chlorophyll molecule in vivo is 1 ns, what fraction of chlorophyll molecules are excited at any one time?

24. Effect of Monochromatic Light on Electron Flow The extent to which an electron carrier is oxidized or reduced during photosynthetic electron transfer can sometimes be observed directly with a spectrophotometer. When chloroplasts are illuminated with 700 nm light, cytochrome f, plastocyanin, and plastoquinone are oxidized. When chloroplasts are illuminated with 680 nm light, however, these electron carriers are reduced. Explain.

25. Function of Cyclic Photophosphorylation When the (NADPH)/[NADP⁺] ratio in chloroplasts is high, photophosphorylation is predominantly cyclic (Fig. 18-44). Is O₂ evolved during cyclic photophosphorylation? Explain. Can the chloroplast produce NADPH this way? What is the main function of cyclic photophosphorylation?

SUMMARY

Further Reading

problems ( Answer )