Cell biology 3410.001 chapter 14

3. When the drug dinitrophenol (DNP) is added to mitochondria, the inner membrane becomes permeable to protons (H+). In contrast, when the drug nigericin is added to mitochondria, the inner membrane becomes permeable to K+. (A) How does the electrochemical proton gradient change in response to DNP? (B) How does it change in response to nigericin?

A. . The DNP collapses the electrochemical proton gradient completely. H+ ions that are pumped to one side of the membrane flow back freely, and therefore no energy to drive ATP synthesis can be stored across the membrane. B. An electrochemical gradient is made up of two components: a concentration gradient and an electrical potential. If the membrane is made permeable to K+ with nigericin, K+ will be driven into the matrix by the electrical potential of the inner membrane (negative inside, positive outside). The influx of positively charged K+ will abolish the membrane's electrical potential.+ In contrast, the concentration component of the H gradient (the pH difference) is unaffected by nigericin. Therefore, only part of the driving force that makes it + energetically favorable for H ions to flow back into the matrix is lost.

12. Which of the following statements are correct? Explain your answers. A. Many, but not all, electron-transfer reactions involve metal ions. B. The electron-transport chain generates an electrical potential across the membrane because it moves electrons from the intermembrane space into the matrix. C. The electrochemical proton gradient consists of two components: a pH difference and an electrical potential. D. Ubiquinone and cytochrome c are both diffusible electron carriers. E. Plants have chloroplasts and therefore can live without mitochondria.

A. True. NAD+ and quinones are examples of compounds that do not have metal ions but can participate in electron transfer. B. False. The potential is due to protons (H+) that are pumped across the membrane from the matrix to the intermembrane space. Electrons remain bound to electron carriers in the inner mitochondrial membrane. C. True. Both components add to the driving force that makes it energetically favorable for H+ to flow back into the matrix. D. True. Both move rapidly in the plane of the membrane. E. False. Not only do plants need mitochondria to make ATP in cells that do not have chloroplasts, such as root cells, but mitochondria make most of the cytosolic ATP in all plant cells.

18. As your first experiment in the laboratory, your adviser asks you to reconstitute purified bacteriorhodopsin, a light-driven H+ pump from the plasma membrane of photosynthetic bacteria, and purified ATP synthase from ox-heart mitochondria together into the same membrane vesicles—as shown in figure Q14-18. You are then asked to add ADP and Pi to the external medium and shine light into the suspension of vesicles. A. What do you observe? B. What do you observe if not all the detergent is removed and the vesicle membrane therefore remains leaky to ions? C. You tell a friend over dinner about your new experiments, and he questions the validity of an approach that utilizes components from so widely divergent, unrelated organisms: "Why would anybody want to mix vanilla pudding with brake fluid?" Defend your approach against his critique.

A. When the vesicles are exposed to light, H+ ions (derived from H2O) pumped into the vesicles by the bacteriorhodopsin flow back out through the ATP synthase, causing ATP to be made in the solution surrounding the vesicles in response to light. B. If the vesicles are leaky, no H+ gradient can form and thus ATP synthase cannot work. C. Using components from widely divergent organisms can be a very powerful experimental tool. Because the two proteins come from such different sources, it is very unlikely that they form a direct functional interaction. The experiment therefore strongly suggests that electron transport and ATP synthesis are separate events. This approach is therefore a valid one.

15. Assume that the conversion of oxidized ubiquinone to reduced ubiquinone by NADH dehydrogenase occurs on the matrix side of the inner mitochondrial membrane and that its oxidation by cytochrome c reductase occurs on the intermembrane space side of the membrane (see Figures 14-14 and 14-23). What are the consequences of this arrangement for the generation of the H+ gradient across the membrane?

As oxidized ubiquinone becomes reduced, it picks up two electrons but also two protons from water NADH (Figure 14-23). Upon oxidation, these protons are released. If reduction occurs on one side of the membrane and oxidation at the other side, a proton is pumped across the membrane for each electron transported. Electron transport by ubiquinone thereby contributes directly to the generation of the H+ gradient.

1. Dinitrophenol (DNP) is a small molecule that renders membranes permeable to protons. In the 1940s, small amounts of this highly toxic compound were given to patients to induce weight loss. DNP was effective in melting away the pounds, especially promoting the loss of fat reserves. Can you explain how it might cause such loss? As an unpleasant side reaction, however, patients had an elevated temperature and sweated profusely during the treatment. Provide an explanation for these symptoms.

By making membranes permeable to protons, DNP collapses—or at very small concentrations diminishes—the proton gradient across the inner mitochondrial membrane. Cells continue to oxidize food molecules to feed high-energy electrons into the electron- transport chain, but H+ ions pumped across the membrane flow back into the mitochondria in a futile cycle. As a result, the energy of the electrons cannot be tapped to drive ATP synthesis, and instead is released as heat. Patients who have been given small doses of DNP lose weight because their fat reserves are used more rapidly to feed the electron- transport chain, and the whole process simply "wastes" energy as heat. A similar mechanism of heat production is used naturally in a specialized tissue composed of brown fat cells, which is abundant in newborn humans and in hibernating animals. These cells are packed with mitochondria that leak part of their H+ gradient futilely back across the membrane for the sole purpose of warming up the organism. These cells are brown because they are packed with mitochondria, which contain high concentrations of pigmented proteins, such as cytochromes.

14. In the following statement, choose the correct one of the alternatives in italics and justify your answer. "If no O2 is available, all components of the mitochondrial electron- transport chain will accumulate in their reduced/oxidized form. If O2 is suddenly added again, the electron carriers in cytochrome c oxidase will become reduced/oxidized before/after those in NADH dehydrogenase."

If no O2 is available, all components of the mitochondrial electron-transport chain will accumulate in their reduced form. This is the case because electrons derived from NADH enter the chain but cannot be transferred to O2. The electron-transport chain therefore stalls with all of its components in the reduced form. If O2 is suddenly added again, the electron carriers in cytochrome c oxidase will become oxidized before those in NADH dehydrogenase. This is true because, after O2 addition, cytochrome c oxidase will donate its electrons directly to O2, thereby becoming oxidized. A wave of increasing oxidation then passes backward with time from cytochrome c oxidase through the components of the electron-transport chain, as each component regains the opportunity to pass on its electrons to downstream components.

20. Some bacteria have become specialized to live in an environment of high pH (pH ~10). Do you suppose that these bacteria use a proton gradient across their plasma membrane to produce their ATP? (Hint: all cells must maintain their cytoplasm at a pH close to neutrality.)

If these bacteria used a proton gradient to make their ATP in a fashion analogous to that in other bacteria (that is, fewer protons inside than outside), they ess A13.19 would need to raise their cytoplasmic pH even higher than that of their environment (pH 10). Cells with a cytoplasmic pH greater than 10 would not be viable. These bacteria must therefore use gradients of ions other than H+, such as Na+ gradients, in the chemiosmotic coupling between electron transport and an ATP synthase.

13. A single proton moving down its electrochemical gradient into the mitochondrial matrix space liberates 4.6 kcal/ mole of free energy (∆G). How many protons have to flow across the inner mitochondrial membrane to synthesize one molecule of ATP if the ∆G for ATP synthesis under intracellular conditions is between 11 and 13 kcal/mole? (∆G is discussed in Chapter 3, pp. 90-100.) Why is a range given for this latter value, and not a precise number? Under which conditions would the lower value apply?

It takes three protons. The precise value of the ΔG for ATP synthesis depends on the concentrations of ATP, ADP, and Pi (as described in Chapter 3). The higher the ratio of the concentration of ATP to ADP, the more energy it takes to make additional ATP. The lower value of 11 kcal/ mole therefore applies to conditions where cells have expended a lot of energy and have therefore decreased the normal ATP/ADP ratio.

7. Two different diffusible electron carriers, ubiquinone and cytochrome c, shuttle electrons between the three protein complexes of the electron-transport chain. Could the same diffusible carrier, in principle, be used for both steps? Explain your answer.

It would not be productive to use the same carrier in two steps. If ubiquinone, for example, could transfer electrons directly to the cytochrome c oxidase, the cytochrome c reductase complex would often be skipped when electrons are collected from NADH dehydrogenase. Given the large difference in redox potential between ubiquinone and cytochrome c oxidase, a large amount of energy would be released as heat and thus be wasted. Electron transfer directly between NADH dehydrogenase and cytochrome c would similarly allow the cytochrome c reductase complex to be bypassed.

6. At many steps in the electron- transport chain, Fe ions are used as part of heme or FeS clusters to bind the electrons in transit. Why do these functional groups that carry out the chemistry of electron transfer need to be bound to proteins? Provide several different reasons why this is necessary.

One can describe four essential roles for the proteins in the process. First, the chemical environment provided by a protein's amino acid side chains sets the redox potential of each Fe ion such that electrons can be passed in a defined order from one component to the next, giving up their energy in small steps and becoming more firmly bound as they proceed. Second, the proteins position the Fe ions so that the electrons can move efficiently between them. Third, the proteins prevent electrons from skipping an intermediate step; thus, as we have learned for other enzymes (discussed in Chapter 4), they channel the electron flow along a defined path. Fourth, the proteins couple the movement of the electrons down their energy ladder to the pumping of protons across the membrane, thereby harnessing the energy that is released and storing it in a proton gradient that is then used for ATP production.

2. Electron micrographs show that mitochondria in heart muscle have a much higher density of cristae than mitochondria in skin cells. Suggest an explanation for this observation.

The inner mitochondrial membrane is the site of oxidative phosphorylation, and it produces most of the cell's ATP. Cristae are portions of the mitochondrial inner membrane that are folded inward. Mitochondria that have a higher density of cristae have a larger area of inner membrane and therefore a greater capacity to carry out oxidative phosphorylation. Heart muscle expends a lot of energy during its continuous contractions, whereas skin cells have a smaller energy demand. An increased density of cristae therefore increases the ATP-production capacity of the heart muscle cell. This is a remarkable example of how cells adjust the abundance of their individual components according to need.

22. A manuscript has been submitted for publication to a prestigious scientific journal. In the paper, the authors describe an experiment in which they have succeeded in trapping an individual ATP synthase molecule and then mechanically rotating its head by applying a force to it. The authors show that upon rotating the head of the ATP synthase, ATP is produced, in the absence of an H+ gradient. What might this mean about the mechanism whereby ATP synthase functions? Should this manuscript be considered for publication in one of the best journals?

The redox potential of FADH2 is too low to transfer electrons to the NADH dehydrogenase complex, but high enough to transfer electrons to ubiquinone (Figure 14-24). Therefore, electrons from FADH2 can enter the electron-transport chain only at this step (Figure A14-19). Because the NADH dehydrogenase complex is bypassed, fewer H+ ions are pumped across the membrane and less ATP is made. This example shows the versatility of the electron-transport chain. The ability to use vastly different sources of electrons from the environment to feed electron transport is thought to have been an essential feature in the early evolution of life.

17a. In an insightful experiment performed in the 1960s, chloroplasts were first soaked in an acidic solution at pH 4, so that the stroma and thylakoid space became acidified (figure Q14-17). They were then transferred to a basic solution (pH 8). This quickly increased the pH of the stroma to 8, while the thylakoid space temporarily remained at pH 4. A burst of ATP synthesis was observed, and the pH difference between the thylakoid and the stroma then disappeared. A. Explain why these conditions lead to ATP synthesis.

The switch in solutions creates a pH gradient across the thylakoid membrane. The flow of H+ ions down its electrochemical potential drives ATP synthase, which converts ADP to ATP.

22. A manuscript has been submitted for publication to a prestigious scientific journal. In the paper, the authors describe an experiment in which they have succeeded in trapping an individual ATP synthase molecule and then mechanically rotating its head by applying a force to it. The authors show that upon rotating the head of the ATP synthase, ATP is produced, in the absence of an H+ gradient. What might this mean about the mechanism whereby ATP synthase functions? Should this manuscript be considered for publication in one of the best journals?

This experiment would suggest a two-step model for ATP synthase function. According to this model, the flow of protons through the base of the synthase drives rotation of the head, which in turn causes ATP synthesis. In their experiment, the authors have succeeded in uncoupling these two steps. If rotating the head mechanically is sufficient to produce ATP in the absence of any applied proton gradient, the ATP synthase is a protein machine that indeed functions like a "molecular turbine." This would be a very exciting experiment indeed, because it would directly demonstrate the relationship between mechanical movement and enzymatic activity. There is no doubt that it should be published and that it would become a "classic."

4. The remarkable properties that allow ATP synthase to run in either direction allow the interconversion of energy stored in the H+ gradient and energy stored in ATP to proceed in either direction. (A) If ATP synthase making ATP can be likened to a water-driven turbine producing electricity, what would be an appropriate analogy when it works in the opposite direction? (B) Under what conditions would one expect the ATP synthase to stall, running neither forward nor backward? (C) What determines the direction in which the ATP synthase operates?

A. Such a turbine running in reverse is an electrically driven water pump, which is analogous to what the ATP synthase becomes when it uses the energy of ATP hydrolysis to pump protons against their electrochemical gradient across the inner mitochondrial membrane. B. The ATP synthase should stall when the energy that it can draw from the proton gradient is just equal to the ΔG required to make ATP; at this equilibrium point there will be neither net ATP synthesis nor net ATP consumption. C. As the cell uses up ATP, the ATP/ADP ratio in the matrix falls below the equilibrium point just described, and ATP synthase uses the energy stored in the proton gradient to synthesize ATP in order to restore the original ATP/ ADP ratio. Conversely, when the electrochemical proton gradient drops below that at the equilibrium point, ATP synthase uses ATP in the matrix to restore this gradient