Oxidative Phosphorylation and Photophosphorylation

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OxidativePhosphorylation andPhotophosphorylation

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<p>2608T_ch19sm_S223-S237</p> <p>02/22/2008</p> <p>2:47 pm</p> <p>Page 223 pinnacle 111:WHQY028:Solutions Manual:Ch-19:</p> <p>Oxidative Phosphorylation and Photophosphorylation</p> <p>chapter</p> <p>19</p> <p>1. 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, Q is ubiquinone, QH2 is ubiquinol, and E is the enzyme: (1) NADH H E-FMN 88n NAD E-FMNH2 (2) E-FMNH2 2Fe3 88n E-FMN 2Fe2 2H (3) 2Fe2 2H Q 88n 2Fe3 QH2 Sum: NADH H Q 88n 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. Answer Oxidation-reduction reactions require an electron donor and an electron acceptor. Recall that electron donors are reducing agents; electron acceptors are oxidizing agents. (1) NADH is the electron donor (a) and the reducing agent (d); E-FMN is the electron acceptor (b) and the oxidizing agent (e); NAD/NADH and E-FMN/E-FMNH2 are conjugate redox pairs (c). (2) E-FMNH2 is the electron donor (a) and reducing agent (d); Fe3 is the electron acceptor (b) and oxidizing agent (e); E-FMN/E-FMNH2 and Fe3/Fe2 are redox pairs (c). (3) Fe2 is the electron donor (a) and reducing agent (d); Q is the electron acceptor (b) and oxidizing agent (e); and Fe3/Fe2 and Q/QH2 are redox pairs (c). 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? Answer The long isoprenoid side chain makes ubiquinone very soluble in lipids and allows it to diffuse in the semifluid membrane. This is important because ubiquinone transfers electrons from Complexes I and II to Complex III, all of which are embedded in the inner mitochondrial membrane.</p> <p>S-223</p> <p>2608T_ch19sm_S223-S237</p> <p>02/22/2008</p> <p>2:47 pm</p> <p>Page 224 pinnacle 111:WHQY028:Solutions Manual:Ch-19:</p> <p>S-224</p> <p>Chapter 19 Oxidative Phosphorylation and Photophosphorylation</p> <p>3. Use of FAD Rather Than NAD in Succinate Oxidation All the dehydrogenases of glycolysis and the citric acid cycle use NAD (E for NAD/NADH is 0.32 V) as electron acceptor except succinate dehydrogenase, which uses covalently bound FAD (E 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 values of fumarate/succinate (E 0.031), NAD/NADH, and the succinate dehydrogenase FAD/FADH2. Answer From the difference in standard reduction potential (E) for each pair of halfreactions, we can calculate the G values for the oxidation of succinate using NAD and oxidation using E-FAD. For NAD: G n E 2(96.5 kJ/V mol)(0.32 V 0.031 V ) 68 kJ/mol For E-FAD: G 2(96.5 kJ/V mol)(0.050 V 0.031 V ) 3.7 kJ/mol The oxidation of succinate by E-FAD is favored by the negative standard free-energy change, which is consistent with a K eq of 1. Oxidation by NAD would require a large, positive, standard free-energy change and have a K eq favoring the synthesis of succinate. 4. 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 (see Fig. 196). 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 Answer As shown in Figure 196, the oxidation-reduction state of the carriers in the electron-transfer system varies with the conditions. (a) Cyanide inhibits cytochrome oxidase (a a3); all carriers become reduced. (b) In the absence of O2, no terminal electron acceptor is present; all carriers become reduced. (c) In the absence of NADH, no carrier can be reduced; all carriers become oxidized. (d) These are the usual conditions for an aerobic, actively metabolizing cell; the early carriers (e.g., Q) are somewhat reduced, while the late ones (e.g., cytochrome c) are oxidized. 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. (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.</p> <p>2608T_ch19sm_S223-S237</p> <p>02/22/2008</p> <p>2:47 pm</p> <p>Page 225 pinnacle 111:WHQY028:Solutions Manual:Ch-19:</p> <p>Chapter 19 Oxidative Phosphorylation and Photophosphorylation</p> <p>S-225</p> <p>Answer (a) The inhibition of NADH dehydrogenase by rotenone decreases the rate of electron flow through the respiratory chain, which in turn decreases the rate of ATP production. If this reduced rate is unable to meet its ATP requirements, the organism dies. (b) Antimycin A strongly inhibits the oxidation of reduced Q in the respiratory chain, severely limiting the rate of electron transfer and ATP production. (c) Electrons flow into the system at Complex I from the NAD-linked reactions and at Complex II from succinate and fatty acylCoA through FAD (see Figs. 198 and 1916). Antimycin A inhibits electron flow (through Q) from all these sources, whereas rotenone inhibits flow only through Complex I. Thus, antimycin A is a more potent poison. 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 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 donorthe P/O ratiois 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? Answer Uncouplers of oxidative phosphorylation stimulate the rate of electron flow but not ATP synthesis. (a) At relatively low levels of an uncoupling agent, P/O ratios drop somewhat, but the cell can compensate for this by increasing the rate of electron flow; ATP levels can be kept relatively normal. At high levels of uncoupler, P/O ratios approach zero and the cell cannot maintain ATP levels. (b) As amounts of an uncoupler increase, the P/O ratio decreases and the body struggles to make sufficient ATP by oxidizing more fuel. The heat produced by this increased rate of oxidation raises the body temperature. The P/O ratio is affected as noted in (a). (c) Increased activity of the respiratory chain in the presence of an uncoupler requires the degradation of additional energy stores (glycogen and fat). By oxidizing more fuel in an attempt to produce the same amount of ATP, the organism loses weight. If the P/O ratio nears zero, the lack of ATP will be lethal. 7. Effects of Valinomycin on Oxidative Phosphorylation When the antibiotic valinomycin is added to actively respiring mitochondria, several things happen: the yield of ATP decreases, the rate of O2 consumption increases, heat is released, and the pH gradient across the inner mitochondrial membrane increases. Does valinomycin act as an uncoupler or an inhibitor of oxidative phosphorylation? Explain the experimental observations in terms of the antibiotics ability to transfer K ions across the inner mitochondrial membrane.</p> <p>2608T_ch19sm_S223-S237</p> <p>02/22/2008</p> <p>2:47 pm</p> <p>Page 226 pinnacle 111:WHQY028:Solutions Manual:Ch-19:</p> <p>S-226</p> <p>Chapter 19 Oxidative Phosphorylation and Photophosphorylation</p> <p>Answer The observed effects are consistent with the action of an uncouplerthat is, an agent that causes the free energy released in electron transfer to appear as heat rather than in ATP. In respiring mitochondria, H ions are translocated out of the matrix during electron transfer, creating a proton gradient and an electrical potential across the membrane. A significant portion of the free energy used to synthesize ATP originates from this electric potential. Valinomycin combines with K ions to form a complex that passes through the inner mitochondrial membrane. So, as a proton is translocated out by electron transfer, a K ion moves in, and the potential across the membrane is lost. This reduces the yield of ATP per mole of protons flowing through ATP synthase (FoF1). In other words, electron transfer and phosphorylation become uncoupled. In response to the decreased efficiency of ATP synthesis, the rate of electron transfer increases markedly. This results in an increase in the H gradient, in oxygen consumption, and in the amount of heat released. 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? Answer (a) DCCD inhibits ATP synthesis. In tightly coupled mitochondria, this inhibition leads to inhibition of electron transfer also. (b) A decrease in electron transfer causes a decrease in O2 consumption. 2,4-Dinitrophenol uncouples electron transfer from ATP synthesis, allowing respiration to increase. No ATP is synthesized and the P/O ratio decreases. (c) DCCD and oligomycin inhibit ATP synthesis (see Table 194). 9. Compartmentalization of Citric Acid Cycle Components Isocitrate dehydrogenase is found only in the mitochondrion, but malate dehydrogenase is found in both the cytosol and mitochondrion. What is the role of cytosolic malate dehydrogenase? Answer Malate dehydrogenase catalyzes the conversion of malate to oxaloacetate in the citric acid cycle, which takes place in the mitochondrion, and also plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle (Fig. 1929). This shuttle requires the presence of malate dehydrogenase in the cytosol and the mitochondrial matrix. 10. The Malatea-Ketoglutarate Transport System The transport system that conveys malate and -ketoglutarate across the inner mitochondrial membrane (see Fig. 1929) is inhibited by nbutylmalonate. 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, and (d) ATP synthesis. Answer NADH produced in the cytosol cannot cross the inner mitochondrial membrane, but must be oxidized if glycolysis is to continue. Reducing equivalents from NADH enter the mitochondrion by way of the malate-aspartate shuttle. NADH reduces oxaloacetate to form malate and NAD, and the malate is transported into the mitochondrion. Cytosolic oxidation of glucose can continue, and the malate is converted back to oxaloacetate and NADH in the mitochondrion (see Fig. 1929).</p> <p>2608T_ch19sm_S223-S237</p> <p>02/22/2008</p> <p>2:47 pm</p> <p>Page 227 pinnacle 111:WHQY028:Solutions Manual:Ch-19:</p> <p>Chapter 19 Oxidative Phosphorylation and Photophosphorylation</p> <p>S-227</p> <p>(a) If n-butylmalonate, an inhibitor of the malate-ketoglutarate transporter, is added to cells, NADH accumulates in the cytosol. This forces glycolysis to operate anaerobically, with reoxidation of NADH in the lactate dehydrogenase reaction. (b) Because reducing equivalents from the oxidation reactions of glycolysis do not enter the mitochondrion, oxygen consumption slows and eventually ceases. (c) The end product of anaerobic glycolysis, lactate, accumulates. (d) ATP is not formed aerobically because the cells have converted to anaerobic glycolysis. Overall, ATP synthesis decreases drastically, to 2 ATP per glucose molecule. 11. 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? Answer The steady-state concentration of Pi in the cell is much higher than that of ADP. As the ADP concentration rises as a result of ATP consumption, there is little change in [Pi], so Pi cannot serve as a regulator. 12. Time Scales of Regulatory Events in Mitochondria Compare the likely time scales for the adjustments in respiratory rate caused by (a) increased [ADP] and (b) reduced pO2. What accounts for the difference? Answer In (a), respiratory control by ADP, the increase in respiratory rate is limited by the rate of diffusion of ADP, and the response would be expected to occur in fractions of a millisecond. The adjustment to (b), hypoxia mediated by HIF-1, requires a change in concentration of several proteins, the result of increased synthesis or degradation. The time scale for protein synthesis or degradation is typically many seconds to hoursmuch longer than the time required for changes in substrate concentration. 13. The Pasteur Effect When O2 is added to an anaerobic suspension of cells consuming glucose at a high rate, the rate of glucose consumption declines greatly as the O2 is used up, and accumulation of lactate ceases. This effect, first observed by Louis Pasteur in the 1860s, is characteristic of most cells capable of aerobic and anaerobic glucose catabolism. (a) Why does the accumulation of lactate...</p>