Breathe or ferment? Compare fermentation and respiration with respect to electron donors and electron acceptors.

Reference states. The standard oxidation–reduction potential for the reduction of O₂ to H₂O is given as 0.82 V in Table 18.1. However, the value given in textbooks of chemistry is 1.23 V. Account for this difference.

Less energetic electrons. Why are electrons carried by FADH₂ not as energy rich as those carried by NADH? What is the consequence of this difference?

Now prove it. Calculate the energy released by the reduction of O₂ with FADH₂.

Thermodynamic constraint. Compare the ΔG°′ values for the oxidation of succinate by NAD⁺ and by FAD. Use the data given in Table 18.1 to find the of the NAD⁺ – NADH and fumarate-succinate couples, and assume that for the FAD – FADH₂ redox couple is nearly 0.05 V. Why is FAD rather than NAD⁺ the electron acceptor in the reaction catalyzed by succinate dehydrogenase?

Give and accept. Distinguish between an oxidizing agent and a reducing agent.

The benefactor and beneficiary. Identify the oxidant and the reductant in the following reaction.

Six of one, half dozen of the other. How is the redox potential ( ) related to the free-energy change of a reaction (ΔG°′)?

Location, location, location. Iron is a component of many of the electron carriers of the electron-transport chain. How can it participate in a series of coupled redox reactions if the value is +0.77 V, as seen in Table 18.1?

Line up. Place the following components of the electron-transport chain in their proper order:

Like mac and cheese. Match each term with its description.

Match ’em.

Structural considerations. Explain why coenzyme Q is an effective mobile electron carrier in the electron-transport chain.

Inhibitors. Rotenone inhibits electron flow through NADH-Q oxidoreductase. Antimycin A blocks electron flow between cytochromes b and c₁. Cyanide blocks electron flow through cytochrome oxidase to O₂. Predict the relative oxidation–reduction state of each of the following respiratory-chain components in mitochondria that are treated with each of the inhibitors:

Rumored to be a favorite of Elvis. Amytal is a barbiturate sedative that inhibits electron flow through Complex I. How would the addition of amytal to actively respiring mitochondria affect the relative oxidation–reduction states of the components of the electron-transport chain and the citric acid cycle?

Efficiency. What is the advantage of having Complexes I, III, and IV associated with one another in the form of a respirasome?

Linked In. What citric acid cycle enzyme is also a component of the electron-transport chain?

ROS, not ROUS. What are the reactive oxygen species and why are they especially dangerous to cells?

Reclaim resources. Humans have only about 250 g of ATP, but even a couch potato needs about 83 kg of ATP to open the bag of chips and use the remote. How is this discrepancy between requirements and resources reconciled?

Energy harvest. What is the yield of ATP when each of the following substrates is completely oxidized to CO₂ by a mammalian cell homogenate? Assume that glycolysis, the citric acid cycle, and oxidative phosphorylation are fully active.

Potent poisons. What is the effect of each of the following inhibitors on electron transport and ATP formation by the respiratory chain?

A question of coupling. What is the mechanistic basis for the observation that the inhibitors of ATP synthase also lead to an inhibition of the electron-transport chain?

A Brownian ratchet wrench. What causes the c subunits of ATP synthase to rotate? What determines the direction of rotation?

An essential residue. The conduction of protons by the F₀ unit of ATP synthase is blocked by dicyclohexylcarbodiimide, which reacts readily with carboxyl groups. What are the most likely targets of action of this reagent? How might you use site-specific mutagenesis to determine whether this residue is essential for proton conduction?

Alternative routes. The most common metabolic sign of mitochondrial disorders is lactic acidosis. Why?

Connections. How does the inhibition of ATP-ADP translocase affect the citric acid cycle? Glycolysis?

O₂ consumption. Oxidative phosphorylation in mitochondria is often monitored by measuring oxygen consumption. When oxidative phosphorylation is proceeding rapidly, the mitochondria will rapidly consume oxygen. If there is little oxidative phosphorylation, only small amounts of oxygen will be used. You are given a suspension of isolated mitochondria and directed to add the following compounds in the order from a to h. With the addition of each compound, all of the previously added compounds remain present. Predict the effect of each addition on oxygen consumption by the isolated mitochondria.

P : O ratios. The number of molecules of inorganic phosphate incorporated into organic form per atom of oxygen consumed, termed the P : O ratio, was frequently used as an index of oxidative phosphorylation.

Cyanide antidote. The immediate administration of nitrite is a highly effective treatment for cyanide poisoning. What is the basis for the action of this antidote? (Hint: Nitrite oxidizes ferrohemoglobin to ferrihemoglobin.)

Runaway mitochondria 1. Suppose that the mitochondria of a patient oxidize NADH irrespective of whether ADP is present. The P : O ratio for oxidative phosphorylation by these mitochondria is less than normal. Predict the likely symptoms of this disorder.

Recycling device. The cytochrome b component of Q-cytochrome c oxidoreductase enables both electrons of QH₂ to be effectively utilized in generating a proton-motive force. Cite another recycling device in metabolism that brings a potentially dead end reaction product back into the mainstream.

Crossover point. The precise site of action of a respiratory-chain inhibitor can be revealed by the crossover technique. Britton Chance devised elegant spectroscopic methods for determining the proportions of the oxidized and reduced forms of each carrier. This determination is feasible because the forms have distinctive absorption spectra, as illustrated in the adjoining graph for cytochrome c. You are given a new inhibitor and find that its addition to respiring mitochondria causes the carriers between NADH and QH₂ to become more reduced and those between cytochrome c and O₂ to become more oxidized. Where does your inhibitor act?

Runaway mitochondria 2. Years ago, uncouplers were suggested to make wonderful diet drugs. Explain why this idea was proposed and why it was rejected. Why might the producers of antiperspirants be supportive of the idea?

Everything is connected. If actively respiring mitochondria are exposed to an inhibitor of ATP-ADP translocase, the electron-transport chain ceases to operate. Why?

Identifying the inhibition. You are asked to determine whether a chemical is an electron-transport-chain inhibitor or an inhibitor of ATP synthase. Design an experiment to make this determination.

To each according to its needs. It has been noted that the mitochondria of muscle cells often have more cristae than the mitochondria of liver cells. Provide an explanation for this observation.

Opposites attract. An arginine residue (Arg 210) in the a subunit of the E. coli ATP synthase is near the aspartate residue (Asp 61) in the matrix-side proton channel. How might Arg 210 assist proton flow?

Variable c subunits. Recall that the number of c subunits in the c ring appears to range between 8 and 14. This number is significant because it determines the number of protons that must be transported to generate a molecule of ATP. Each 360-degree rotation of the γ subunit leads to the synthesis and release of three molecules of ATP. Thus, if there are 10 c subunits in the ring (as was observed in a crystal structure of yeast mitochondrial ATP synthase), each ATP generated requires the transport of 10/3 = 3.33 protons. How many protons are required to form ATP if the ring has 12 c subunits? 14?

Counterintuitive. Under some conditions, mitochondrial ATP synthase has been observed to actually run in reverse. How would that situation affect the proton-motive force?

Etiology? What does that mean? What does the fact that rotenone appears to increase the susceptibility to Parkinson disease indicate about the etiology of Parkinson disease?

Exaggerating the difference. Why must ATP-ADP translocase (also called adenine nucleotide translocase or ANT) use Mg²⁺-free forms of ATP and ADP?

Respiratory control. The rate of oxygen consumption by mitochondria increases markedly when ADP is added and then returns to its initial value when the added ADP has been converted into ATP (Figure 18.39). Why does the rate decrease?

Same, but different. Why is the electroneutral exchange of H₂PO₄⁻ for OH⁻ indistinguishable from the electroneutral symport of H₂PO₄⁻ and H⁺?

Multiple uses. Give an example of the use of the proton-motive force in ways other than for the synthesis of ATP.

Just obeying the laws. Why do isolated F₁ subunits of ATP synthase catalyze ATP hydrolysis?

The right location. Some cytoplasmic kinases, enzymes that phosphorylate substrates at the expense of ATP, bind to voltage-dependent anion channels. What might the advantage of this binding be?

No exchange. Mice that completely lack ATP-ADP translocase (ANT⁻/ANT⁻) can be made by using the knockout technique. Remarkably, these mice are viable but have the following pathological conditions: (1) high serum levels of lactate, alanine, and succinate; (2) little electron transport; and (3) a six- to eightfold increase in the level of mitochondrial H₂O₂ compared with that in normal mice. Provide a possible biochemical explanation for each of these conditions.

Maybe you shouldn’t take your vitamins. Exercise is known to increase insulin sensitivity and to ameliorate type 2 diabetes (Chapter 27). Recent research suggests that taking antioxidant vitamins might mitigate the beneficial effects of exercise with respect to ROS protection.

Monitoring energy sources. XF technology (Seahorse Bioscience) now allows the measurement of the rate of aerobic respiration and lactic acid fermentation simultaneously in real time in cultured cells. The extent of aerobic respiration is determined by measuring the oxygen consumption rate (OCR, measured in picomoles of oxygen consumed per minute) while the rate of glycolysis correlates with the extracellular acidification rate [ECAR-milli pH per minute (the changes in pH that occur over time)]. The graph below shows the results of an experiment using the new technology.

Dinitrophenol (DNP), the glycolysis inhibitor 2-deoxyglucose (DG), and rotenone were added sequentially to cell cultures.

Mitochondrial disease. A mutation in a mitochondrial gene encoding a component of ATP synthase has been identified. People who have this mutation suffer from muscle weakness, ataxia, and retinitis pigmentosa. A tissue biopsy was performed on each of three patients having this mutation, and submitochondrial particles were isolated that were capable of succinate-sustained ATP synthesis. First, the activity of the ATP synthase was measured on the addition of succinate and the following results were obtained.

Chiral clue. ATPγS, a slowly hydrolyzed analog of ATP, can be used to probe the mechanism of phosphoryl-transfer reactions. Chiral ATPγS has been synthesized containing ¹⁸O in a specific γ position and ordinary ¹⁶O elsewhere in the molecule. The hydrolysis of this chiral molecule by ATP synthase in ¹⁷O-enriched water yields inorganic [¹⁶O,¹⁷O,¹⁸O]thiophosphate having the following absolute configuration. In contrast, the hydrolysis of this chiral ATPγS by a calcium-pumping ATPase from muscle gives thiophosphate of the opposite configuration. What is the simplest interpretation of these data?