1. Practice Questions

2. Oxidation-Reduction Reactions The NADH dehydrogenase complex of the mitochondrial respiratory chain promotes the following series of oxidation-reduction reactions, in which Fe +3 and Fe +2 represent the iron in iron-sulfur centers,Q is ubiquinone, QH2 is ubiquinol, and E is the enzyme: (1)NADH + H + + E-FMN → NAD + + E-FMNH2 (2) E-FMNH2 + 2Fe +3 → E-FMN + 2Fe +2 + 2H + (3) 2Fe +2 + 2H + + Q → 2Fe +3 + QH2 Sum: NADH + H + + Q → NAD + + QH2 For each of the three reactions catalyzed by the NADH dehydrogenase complex, identify (a) the electron donor, (b) the electron acceptor, (c) the conjugate redox pair, (d) the reducing agent, and (e) the oxidizing agent.

3. 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?

4. Use of FAD Rather Than NAD + in Succinate Oxidation All the dehydrogenases of glycolysis and the citric acid cycle use NAD (E’ o for NAD/NADH is -0.32 V) as electron acceptor except succinate dehydrogenase, which uses covalently bound FAD (E’ o for FAD/FADH2 in this enzyme is 0.050 V). Suggest why FAD is a more appropriate electron acceptor than NAD in the dehydrogenation of succinate, based on the E’ o values of fumarate/succinate (E ’o =0.031),NAD + /NADH, and the succinate dehydrogenase FAD/FADH2.

5. Degree of Reduction of Electron Carriers in the Respiratory Chain The degree of reduction of each carrier in the respiratory chain is determined by conditions in the mitochondrion. For example, when NADH and O2 are 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 and those beyond the block become more oxidized. For each of the conditions below, predict the state of oxidation of ubiquinone and cytochromes b, c1, c, and a +a3. (a) Abundant NADH and O2, but cyanide added (b) Abundant NADH, but O2 exhausted (c) Abundant O2, but NADH exhausted (d) Abundant NADH and O2

6. 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) 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.

7. Uncouplers of Oxidative Phosphorylation In normal mitochondria the rate of electron transfer is tightly coupled to the demand for ATP. When the rate of use of ATP is relatively low, the rate of electron transfer is low; when demand for ATP increases, 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 ratio—is about 2.5. (a) 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 ratio. (b) 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 could this agent, in principle, serve as a weight-reducing aid? Uncoupling agents are no longer prescribed, because some deaths occurred following their use. Why might 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 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. Cellular ADP Concentration Controls ATP Formation Although ADP and Pi are required for the synthesis of ATP, the rate of synthesis depends mainly on the concentration of ADP, not Pi. Why?

10. For the following reaction, [FAD]+ 2 cyt c (Fe +2 ) + 2 H +  [FADH2] + 2 cyt c (Fe +3 ) determine which of the redox couples is the electron acceptor and which is the electron donor under standard-state conditions, calculate the value of ∆E’ °, and determine the free energy change for the reaction.

11. A wealthy investor has come to you for advice. She has been approached by a biochemist who seeks financial backing for a company that would market initrophenol and dicumarol as weight-loss medications. The biochemist has explained to her that these agents are uncouplers and that they would dissipate metabolic energy as heat. The investor wants to know if you think she should invest in the biochemist’s company. How do you respond?

12. Potent poisons. What is the effect of each of the following inhibitors on e transport and ATP formation by the respiratorty chain? a) Azide: It blocks e transport and proton pumping at Complex IV. b) Atractyloside: It block e transport and ATP synthesis by inhibiting the exchange of ATP and ADP across the inner mitochondrial membrane. c) Rotenone: It blocks e transport and proton pumping at Complex I. d) DNP: It blocks ATP synthesis without inhibiting e transport by dissipating the proton gradient. e) CO: It blocks e transport and proton pumping at Complex IV. f) Antimycin A: It blocks e transport and proton pumping at Complex III.

13. A question of coupling. What is the mechanistic basis for the observation that the inhibitors of ATPase also lead to an inhibition of the e-transport chain? A: If the proton gradient is not dissipated by the influx of protons into a mitochondrion with the generation of ATP, eventually the outside of the mitochondrion develops such a large positive charge that the electron-transport chain can no longer pump protons against the gradient. Cyanide antidote. The immediate administration of nitrite is a highly effective treatment for cyanide poisoning. What is the basis for the treatment? A: --Cyanide is lethal since it binds to ferric (Fe 3+ ) form of cytochrome oxidase thus inhibits oxid. phos. --Nitrite oxidizes ferrohemoglobin (Fe 2+ ) to ferrihemoglobin (Fe 3+ ), which also binds to cyanide. ---Thus, ferrihemoglobin competes with cytochrome oxidase for cyanide. ---The competition is therapeutically effective because the amount of ferrihemoglobin that can be formed without impairing O 2 transport is much greater than the amount of cytochrome oxidase.

14. Identifying the inhibition. How do you design an experiment to determine whether a chemical is an e-transport-chain inhibitor or an inhibitor of ATP synthase? A: ----Add the inhibitor with and without an uncoupler, and monitor the rate of O2 consumption. ----If the O2 consumption increases again in the presence of inhibitor and uncoupler, the inhibitor must be inhibiting ATP synthase. ---If the O2 consumption is not affected in the presence of inhibitor and uncoupler, the inhibitor is inhibiting the e-transport chain. ---Remember that DNP can rescue the effect of oligomycin, an inhibitor of ATP synthase, but can not rescue the effect of cyanide, an inhibitor of Complex IV.

15. --The sequence of e carriers can be determined by the effects of inhibitors of e transfer on the oxid. state of each carrier. --In the presence of e donor and O2, Each inhibitor causes a characteristic Pattern of oxidized/reduced carriers. --Those before the block become reduced (blue) and those after the Block become oxidized (red). Reduced Oxidized Reduced Degree of reduction of e carriers in the respiratory chain. The Degree of reduction of e carriers in the respiratory chain is determined by the conditions existing in the mitochond. For example, when the supply of NADH and O2 is abundant, the steady-state degree of reduction of the carriers decreases as e pass from the substrate to O2. When e transfer is blocked, the carriers before the block become more reduced while those beyond the block become more oxidized. For each of the conditions below, predict the state of oxidation of each carrier in the respiratory chain (ubiquinone and cytochromes b, c1, c and a+a3).

16. a) Abundant supply of NADH and O2 A: Early carriers more reduced; later carriers more oxidized. b) Abundant supply of NADH but O2 exhausted A: All carries reduced; in the absence of O2, the reduced carries are not re-oxidized. c) Abundant supply of O2 but NADH exhausted A: All carriers oxidized. d) Abundant supply of NADH and O2 but rotenone added A: NADH is reduced, all others are oxidized. e) Abundant supply of NADH and O2 but antimycin A added A: NADH, ubiquinone and cytochrome b are reduced. Cyto c1, cyt c, cyt (a +a3) are oxidized. f) Abundant supply of NADH and O2 but cyanide added A: All carriers reduced. CN - blocks the reduction of O2 catalyzed by cytochrome oxidase.

17. The effect of Rotenone and Antimycin A on e transfer. Rotenone, a toxic nature 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 A: The inhibition of NADH dehydrogenase by rotenone decreases the rate of e flow through the respiratory chain, which in turn decreases the rate of ATP synthesis. If this reduced rate is unable to meet the organism’s ATP requirements, the organism dies. b) Explain why antimycin A is a poison A: Antimycin A strongly inhibits the oxidation of UQ in the respiratory chain, reducing the rate of e transfer and leading to the consequences described in a. c) Assuming that rotenone and antimycin A are equally effective in blocking their respective sites in the e transfer chain, which would be a more potent poison? Explain. A: Because antimycin A blocks all e flow to oxygen, it is a more potent poison than rotenone, which blocks e flow from NADH but not from FADH2.

18. Uncouplers of Oxidative Phosphorylation. In normal mitochondria the rate of e transfer is tightly coupled to the demand for ATP. Thus when ATP is demanded at a high rate, e transfer is rapid. Under such conditions of tight coupling, the no of ATP produced per atom of oxygen consumed when NADH is the e donor---know as the P/O ratio---is close to 3. a) Predict the effect of a relatively low concentration of an uncoupling agent on the rate of e transfer and the P/O ratio. A: The rate of e transfer necessary to meet the ATP demand increases, and thus the P/O ratio decreases. b) The ingestion of uncouplers (high concentration) causes profuse sweating and an increase in body temperature. Explain this phenomenon in molecular terms. What happened to P/O ratio under this condition? A: High concentrations of uncoupler produce P/O ratios near zero. The P/O ratio decreases, and more fuel must therefore be oxidized to generate the same amount of ATP. The extra heat released by this oxidation raises the body temperature. c) The uncoupler 2, 4-dinitrophenol was once prescribed as a weight-reducing drug. How can this agent sever as a reducing aid? DNP is no longer prescribed because some deaths occurred following its use. Why? A: Increased activity of the respiratory chain in the presence of DNP requires the degradation of additional fuel. By oxidizing more fuel (including fat reserves) to produce the same amount of ATP, the body loses weight. When the P/O ratio approaches zero, the lack of ATP results in death.

19. Mode of action of DCCD. When DCCD (dicyclohexylcarbodiimide) is added to a suspension of tightly coupled, actively respiring mitochondria, the rate of e transfer (measured by O2 consumption) and the rate of ATP production dramatically decrease. If a solution of 2,4-dinitrophenol (DNP) is now added to the inhibited mitochondrial preparation. O2 consumption returns to normal but ATP production remains inhibited. a) What process in e transfer or oxidative phosphorylation is affected by DCCD? A: The formation of ATP is inhibited. b) Why does DCCD affect the O2 consumption of mitochondria? Explain the effect of DNP on the inhibited mitochondrial preparation. A: The formation of ATP is tightly coupled to e transfer. DNP is an uncoupler of oxidative phosphorylation. c) Which of the following inhibitors does DCCD most resemble in its action: antimycin A, rotenone, or oligomycin? A: Oligomycin.

20. Mode of Demerol. When the widely prescribed painkiller Demerol is added to a suspension of respiring mitochondria, the ratios NADH/NAD+ and Q/QH2 increase. Which e- transport complex is inhibited by Demerol? A: Demerol interacts with Complex I and prevents e transfer from NADH to Q. The concentration of NADH increases since it cannot be reoxidized to NAD+. The concentration of Q increases since e from QH2 are transferred to O2 but Q is not reduced back to QH2. When the antibiotic myxothiazole is added to respiring mitochondria, the ratio cytochrome c1 (Fe 3+ ) / cytochrome c1 (Fe 2+ ) increases. Where does the antibiotic inhibit the e-transport chain? A: The drug inhibits e transfer from QH2 to cytochrome c1 in Complex III. The oxidized form of cytochrome c1 predominates since Fe 3+ cannot be reduced by e from QH2.

21. Mode of atractyloside. Atractyloside is a toxic glycoside from a Mediterranean plant that specifically inhibits the ADP/ATP carrier. Why does the drug cause e transport to be inhibited as well? A: ---Oxid. phos. Is normally tightly coupled to e transport. ---Unless ADP can continue to be translocated into the mitochondria matrix for the ATP synthase reaction, oxidative phosphorylation is not possible and respiration will cease. (i.e., e transport will stop and no oxygen will be reduced to water). Effects of inhibitors. The O2-consumption curve of a dilute, well-buffered suspension of mitochondria containing an excess of ADP and Pi takes the form as below. Sketch the curves obtained under the following conditions.

22. a) Amytal is added at time t=1 A: O 2 consumption ceases because amytal blocks e transport in Complex I. b) Amytal is added at t=1 and succinate is added at t=2 A: Electrons from succinate bypass the amytal block by entering the e-transport chain at Complex II and thereby restore e transport through Complexes III and IV. Rescue. c) CN- is added at t=1 and succinate is added at t=2 A: CN- blocks e transport in Complex IV, after the point of entry of succinate. No rescue. d) Oligomycin is added at t=1 and DNP is added at t=2 A: Oligomycin blocks oxid. phos. and hence O 2 consumption. DNP uncouples e transport from oxid. phos, and thereby permits O 2 consumption to resume. Rescue.