Answers to Problems

Chapter 21

The ATP is recycled by ATP-generating processes, most notably oxidative phosphorylation.
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(a) 12.5; (b) 14; (c) 32; (d) 13.5; (e) 30; (f) 16
1. Azide blocks electron transport and proton pumping at Complex IV.
2. Atractyloside blocks electron transport and ATP synthesis by inhibiting the exchange of ATP and ADP across the inner mitochondrial membrane.
3. Rotenone blocks electron transport and proton pumping at Complex I.
4. DNP blocks ATP synthesis without inhibiting electron transport by dissipating the proton gradient.
5. Carbon monoxide blocks electron transport and proton pumping at Complex IV.
6. Antimycin A blocks electron transport and proton pumping at Complex III.
If the proton gradient is not dissipated by the influx of protons into a mitochondrion with the generation of ATP, the outside of the mitochondrion eventually develops such a large positive charge that the electron-transport chain can no longer pump protons against the gradient.
1. No effect; mitochondria cannot metabolize glucose.
2. No effect; no fuel is present to power the synthesis of ATP from ADP and P_i.
3. The [O₂] falls because citrate is a fuel and ATP can be formed from ADP and P_i.
4. Oxygen consumption stops because oligomycin inhibits ATP synthesis, which is coupled to the activity of the electron-transport chain.
5. No effect, for the reasons given in part d
6. [O₂] falls rapidly because the system is uncoupled and does not require ATP synthesis to lower the proton-motive force.
7. [O₂] falls, though at a lower rate. Rotenone inhibits Complex I, but the presence of succinate will enable electrons to enter at Complex II.
8. Oxygen consumption ceases because Complex IV is inhibited and the entire chain backs up.
Such a defect (called Luft syndrome) was found in a 38-year-old woman who was incapable of performing prolonged physical work. Her basal metabolic rate was more than twice normal, but her thyroid function was normal. A muscle biopsy showed that her mitochondria were highly variable and atypical in structure. Biochemical studies then revealed that oxidation and phosphorylation were not tightly coupled in these mitochondria. In this patient, much of the energy of fuel molecules was converted into heat rather than ATP.
Dicyclohexylcarbodiimide reacts readily with carboxyl groups. Hence, the most likely targets are aspartate and glutamate side chains. In fact, Asp 61 of subunit c of E. coli F₀ is specifically modified by this reagent. The conversion of Asp 61 into asparagine by site-specific mutagenesis eliminates proton conduction, showing that the acid is required for proton conduction.
If oxidative phosphorylation were uncoupled, no ATP could be produced. In a futile attempt to generate ATP, much fuel would be consumed. The danger lies in the dose. Too much uncoupling would lead to tissue damage in highly aerobic organs such as the brain and heart, which would have severe consequences for the organism as a whole. The energy that is normally transformed into ATP would be released as heat. To maintain body temperature, sweating might increase, although the very process of sweating itself depends on ATP.
If the proton gradient cannot be dissipated by flow through ATP synthase, the proton gradient will eventually become so large that the energy released by the electron-transport chain will not be great enough to pump protons against the larger-than-normal gradient.
The proton gradient is necessary for ATP synthesis because proton flow through the enzyme causes conformational changes that convert a T subunit into an O subunit with the subsequent release of ATP. The role of the proton gradient is not to form ATP but to release it from the synthase.
Arg 210, with its positive charge, will facilitate proton release from aspartic acid by stabilizing the negatively charged Asp 61.
2.7; 4; 5
Presumably, because the muscle has greater energy needs, especially during exercise, it will require more ATP. This requirement means that more sites of oxidative phosphorylation are called for, and these sites can be provided by an increase in the amount of cristae.
If ATP and ADP cannot exchange between the matrix and the mitochondria, ATP synthase will cease to function because its substrate ADP is absent. The proton gradient will eventually become so large that the energy released by the electron-transport chain will not be great enough to pump protons against the larger-than-normal gradient.
Remember that the extra negative charge on ATP relative to that on ADP accounts for ATP’s more rapid translocation out of the mitochondrial matrix. If the charge differences between ATP and ADP were lessened by the binding of the Mg²⁺, ADP might more readily compete with ATP for transport to the cytoplasm.
The subunits are jostled by background thermal energy (Brownian motion). The proton gradient makes clockwise rotation more likely because that direction results in protons flowing down their concentration gradient.
ATP export from the matrix. Phosphate import into the matrix.
If ADP cannot enter the mitochondria, the electron-transport chain will cease to function because there will be no acceptor for the energy. NADH will build up in the matrix. Recall that NADH inhibits some citric acid cycle enzymes, and NAD⁺ is required by several citric acid cycle enzymes. Glycolysis will stop functioning aerobically but will switch to anaerobic glycolysis so that the NADH can be reoxidized to NAD⁺ by lactate dehydrogenase.
When all of the available ADP has been converted into ATP, ATP synthase can no longer function. The proton gradient becomes large enough that the energy of the electron-transport chain is not enough to pump against the gradient, and electron transport and, hence, oxygen consumption falls.

C22
The effect on the proton gradient is the same in each case.
The ATP synthase would pump protons at the expense of ATP hydrolysis, thus maintaining the proton-motive force. The synthase would function as an ATPase. There is some evidence that damaged mitochondria use this tactic to maintain, at least temporarily, the proton-motive force.
First, a closed compartment, intrinsically impermeable to protons, is required to obtain ATP synthesis. Second, electron transport does generate a proton gradient across the inner mitochondrial membrane. Third, an artificial system representing the cellular respiration system demonstrates the basic principle of the chemiosmotic hypothesis (Figure 21.2). Synthetic vesicles containing bacteriorhodopsin and mitochondrial ATP synthase purified from beef heart were created. A proton gradient is generated by bacteriorhodopsin, a purple membrane protein from halobacteria that pumps protons when illuminated. When the vesicles were exposed to light, ATP was formed.
The inside of the mitoplasts would be more basic (pH 7) relative to the outside (pH 4) after the mixing. Thus, an artificial pH gradient would have been imposed. ATP synthesis would indeed be seen under these circumstances.
If b and δ subunits were absent, the γ subunit would simply rotate the α₃β₃ ring rather than power the structural changes (O→L→T) that result in ATP synthesis. In other words, the proton-motive force would power rotation.
Recall that enzymes catalyze reactions in both directions. The hydrolysis of ATP is exergonic. Consequently, ATP synthase will catalyze the conversion of ATP into its more stable products. ATP synthase works as a synthase in vivo because the energy of the proton gradient overcomes the tendency toward ATP hydrolysis.
It suggests that malfunctioning mitochondria may play a role in the development of Parkinson disease. Specifically, it implicates Complex I.
The cytoplasmic kinases will thereby obtain preferential access to the exported ATP.
The organic acids in the blood are indications that the mice are deriving a large part of their energy needs through aerobic glycolysis. Lactate is the end product of aerobic glycolysis. Alanine is an aminated transport form of lactate. Alanine formation plays a role in succinate formation, which is caused by the reduced state of the mitochondria.
In the presence of poorly functioning mitochondria, the only means of generating ATP is by anaerobic glycolysis, which will lead to an accumulation of lactic acid in blood.
1. Succinate is oxidized by Complex II, and the electrons are used to establish a proton-motive force that powers ATP synthesis.
2. The ability to synthesize ATP is greatly reduced.
3. Because the goal was to measure ATP hydrolysis. If succinate had been added in the presence of ATP, no reaction would have taken place, because of respiratory control.
4. The mutation has little effect on the ability of the enzyme to catalyze the hydrolysis of ATP.
5. They suggest two things: (1) the mutation did not affect the catalytic site on the enzyme, because ATP synthase is still capable of catalyzing the reverse reaction, and (2) the mutation did not affect the amount of enzyme present, given that the controls and patients had similar amounts of activity.
1. The P : O ratio is equal to the product of (H⁺/2 e⁻) and (P/H⁺). Note that the P : O ratio is identical with the P : 2 e⁻ ratio.
2. 2.5 and 1.5, respectively
Cyanide can be lethal because it binds to the ferric form of cytochrome c oxidase and thereby inhibits oxidative phosphorylation. Nitrite converts ferrohemoglobin into ferrihemoglobin, which also binds cyanide. Thus, ferrihemoglobin competes with cytochrome c oxidase for cyanide. This competition is therapeutically effective because the amount of ferrihemoglobin that can be formed without impairing oxygen transport is much greater than the amount of cytochrome c oxidase.
The available free energy from the translocation of two, three, and four protons is −38.5, −57.7, and −77.4 kJ mol⁻¹ (−9.2, −13.8, and −18.5 kcal mol⁻¹), respectively. The free energy consumed in synthesizing a mole of ATP under standard conditions is 30.5 kJ (7.3 kcal). Hence, the residual free energy of −8.1, −27.2, and −46.7 kJ mol⁻¹ (−1.93, −6.5, and −11.2 kcal mol⁻¹) can drive the synthesis of ATP until the [ATP]/[ADP][P_i] ratio is 26.2, 6.5 × 10⁴, and 1.6 × 10⁸, respectively. Suspensions of isolated mitochondria synthesize ATP until this ratio is greater than 10⁴, which shows that the number of protons translocated per ATP synthesized is at least three.
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 uncoupler has no effect on the inhibition, the inhibitor is inhibiting the electron-transport chain.