Problem Set (10) 1. Electron-transfer reactions in mitochondria The standard reduction potential for ubiquinone (Q or coenzyme Q) is 0.

045 V, and the standard reduction potential (E') for FAD is 0.219 V. Using these values, show that the oxidation of FADH2 by ubiquinone theoretically liberates enough energy to drive the synthesis of ATP. The Faraday constant, , is 96.48 kJ/Vmol. G' for ATP synthesis is +30.5 kJ/mol. Ans: E' = E'(oxidant) E'(reductant) = 0.045 (0.219) = +0.264 V G' = n E' = (2)(96.48 kJ/V.mol)(0.264 V) = 51.0 kJ/mol. Passage of two electrons from FADH2 to Q can, in principle, provide sufficient energy for synthesizing 1 ATP.

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.

3. ATP synthesis Describe, in a few words, the chemiosmotic theory for coupling oxidation to phosphorylation in mitochondria. Ans: There are three central elements in the chemiosmotic model: (1) Electron flow through asymmetrically arranged membrane-bound carriers causes transmembrane flow of H+, creating a proton gradient (a proton motive force). (2) The proton motive force drives protons back across the membrane via specific proton channels (composed of Fo). (3) The energy released by downhill movement of protons is captured when ADP and Pi are condensed by ATP synthase (FoF1). (See Fig. 19-19, p. 723.)

4. Uncouplers stop formation of ATP while allowing electron transfer to continue. Inhibitors of respiration block both electron transfer and phosphorylation. Effects of cyanide ion (CN) and 2,4-dinitrophenol (DNP) on phosphorylation (ATP synthesis) and oxygen consumption are shown below. Each of these compounds were added separately to a suspension of mitochondria supplied with O2, succinate, ADP, and Pi. Arrow indicates time of drug addition. Which one of the two is uncoupler? Which one is electron transfer blocker? Explain.

The data show that the addition of DNP inhibits only ATP synthesis, while cyanide inhibits both respiration and ATP synthesis. Cyanide ion (CN) blocks electron transfer in mitochondria at the level of cytochrome a + a3 in Complex IV. When electron transfer is blocked, proton-motive force is not established, and hence no ATP synthesis. Weak acids with hydrophobic properties such as DNP can diffuse readily across IMM. After entering the matrix in the protonated form, they can release a proton, thus dissipating the proton gradient. This will uncouple electron transfer with ATP synthesis, resulting in inhibition of ATP synthesis but no effects on electron transfer or oxygen consumption. 5. 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 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. Answer The addition of oxygen to an anaerobic suspension allows cells to convert from fermentation to oxidative phosphorylation as a mechanism for reoxidizing NADH and making ATP. Because ATP synthesis is much more efficient under aerobic conditions, the amount of glucose needed will decrease (the Pasteur effect). This decreased utilization of glucose in the presence of oxygen can be demonstrated in any tissue that is capable of aerobic and anaerobic glycolysis. (a) Oxygen allows the tissue to convert from lactic acid fermentation to respiratory electron transfer and oxidative phosphorylation as the mechanism for NADH oxidation. (b) Cells produce much more ATP per glucose molecule oxidized aerobically, so less glucose is needed. (c) As [ATP] rises, phosphofructokinase-1 is inhibited, thus slowing the rate of glucose entry into the glycolytic pathway.

6. Regulation of oxidative phosphorylation Consider a liver cell carrying out the oxidation of glucose under aerobic conditions. Suppose that we added a very potent and specific inhibitor of the mitochondrial ATP synthase, completely inhibiting this enzyme (e.g., oligomycin; see p.724). Indicate whether each of the following statements about the effect of this inhibitor is true or false; if false, explain in a sentence or two why it is false. ____ (a) ATP production in the cell will quickly drop to zero. ____ (b) The rate of glucose consumption by this cell will decrease sharply. ____ (c) The rate of oxygen consumption will increase. ____ (d) The citric acid cycle will speed up to compensate. ____ (e) The cell will switch to fatty acid oxidation as an alternative to glucose oxidation, and the inhibitor will therefore have no effect on ATP production. Ans: (a) False. Mitochondrial ATP synthesis will cease, but to compensate, cells will accelerate the production of ATP by glycolysis, preventing ATP levels from dropping to zero. (b) False. The acceleration of glycolysis noted above will actually increase the rate of glucose consumption. (c) False. Because electron transfer through the respiratory chain is tightly coupled to ATP synthesis, blocking ATP synthase blocks electron flow and oxygen consumption. (d) False. The citric acid cycle is an oxidative pathway, producing NADH. When + electron flow from NADH to O2 is blocked, NADH accumulates, NAD is depleted, and + the citric acid cycle slows for lack of an electron acceptor (NAD ). (e) False. Oxidation of fats produces NADH, FADH2, and acetyl-CoA, which is further oxidized via the citric acid cycle. For the reasons noted above, blocking electron flow through the respiratory chain prevents ATP synthesis with energy from fatty acid oxidation. 7. Cyanide poisoning Fill the blanks. Inhalation of hydrogen cyanide gas or ingestion of potassium cyanide (KCN) causes a rapid and extensive inhibition of the mitochondrial electron transport chain at the cytochrome oxidase step. Cyanide is one of the most potent and rapidly acting poisons known. It binds to the ( Fe3+ ) ion in the heme a3 of cytochrome c oxidase that catalyzes the terminal step in the electron transport chain. Cyanide thus prevents the binding of ( oxygen ) to the binuclear center and the role of ( oxygen ) as the final electron acceptor. Mitochondrial respiration and energy production cease, and cell death occurs rapidly. Death due to cyanide poisoning occurs from tissue asphyxia, most notably of the central nervous system. An antidote to cyanide poisoning, if the poisoning is diagnosed rapidly, is the administration of various nitrites that convert oxyhemoglobin to methemoglobin by oxidizing ( Fe2+ ) of hemoglobin to Fe3+. Methemoglobin (Fe3+) competes with cytochrome a3 (Fe3+) for cyanide, forming a methemoglobincyanide complex.