Chapter 19: Electron Transport Chain

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. 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.

(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.

Give an example of (a) an uncoupler of oxidative phosphorylation, and (b) an inhibitor of respiration. (c) Describe the difference in the effects of such uncouplers and inhibitors on mitochondrial function.

(a) Uncouplers include DNP, valinomycin, and CCCP. (b) Respiration inhibitors include antimycin A, piericidin A, CN-, rotenone, and amytal. (c) Uncouplers stop formation of ATP while allowing electron transfer to continue. Inhibitors of respiration block both electron transfer and phosphorylation

Which of the following statements about the chemiosmotic theory is correct? A) Electron transfer in mitochondria is accompanied by an asymmetric release of protons on one side of the inner mitochondrial membrane. B) It predicts that oxidative phosphorylation can occur even in the absence of an intact inner mitochondrial membrance. C) The effect of uncoupling reagents is a consequence of their ability to carry electrons through membranes. D) The membrane ATP synthase has no significant role in the chemiosmotic theory. E) All of the above are correct.

A. Electron transfer in mitochondria is accompanied by an asymmetric release of protons on one side of the inner mitochondrial membrane

If electron transfer in tightly coupled mitochondria is blocked (with antimycin A) between cytochrome b and cytochrome c1, then: A) all ATP synthesis will stop. B) ATP synthesis will continue, but the P/O ratio will drop to one. C) electron transfer from NADH will cease, but O2 uptake will continue. D) electron transfer from succinate to O2 will continue unabated. E) energy diverted from the cytochromes will be used to make ATP, and the P/O ratio will rise.

A. all synthesis will stop

2,4-Dinitrophenol and oligomycin inhibit mitochondrial oxidative phosphorylation. 2,4-Dinitrophenol is an uncoupling agent; oligomycin blocks the ATP synthesis reaction itself. Therefore, 2,4-dinitrophenol will: A) allow electron transfer in the presence of oligomycin. B) allow oxidative phosphorylation in the presence of oligomycin. C) block electron transfer in the presence of oligomycin. D) diminish O2 consumption in the presence of oligomycin E) do none of the above.

A. allow electron transfer in the presence of oligomycin

Upon the addition of 2,4-dinitrophenol (DNP) to a suspension of mitochondria carrying out oxidative phosphorylation linked to the oxidation of malate, all of the following occur except: A) oxygen consumption decreases. B) oxygen consumption increases. C) the P/O ratio drops from a value of approximately 2.5 to 0. D) the proton gradient dissipates. E) the rate of transport of electrons from NADH to O2 becomes maximal.

A. oxygen consumption decreases

Cyanide, oligomycin, and 2,4-dinitrophenol (DNP) are inhibitors of mitochondrial aerobic phosphorylation. Which of the following statements correctly describes the mode of action of the three inhibitors? A) Cyanide and 2,4-dinitrophenol inhibit the respiratory chain, and oligomycin inhibits the synthesis of ATP. B) Cyanide inhibits the respiratory chain, whereas oligomycin and 2,4-dinitrophenol inhibit the synthesis of ATP. C) Cyanide, oligomycin, and 2,4-dinitrophenol compete with O2 for cytochrome oxidase (Complex IV). D) Oligomycin and cyanide inhibit synthesis of ATP; 2,4-dinitrophenol inhibits the respiratory chain. E) Oligomycin inhibits the respiratory chain, whereas cyanide and 2,4-dinitrophenol prevent the synthesis of ATP.

B. Cyanide inhibits the respiratory chain, whereas oligomycin and 2,4-dinitrophenol inhibit the synthesis of ATP

Antimycin A blocks electron transfer between cytochromes b and c1. If intact mitochondria were incubated with antimycin A, excess NADH, and an adequate supply of O2, which of the following would be found in the oxidized state? A) Coenzyme Q B) Cytochrome a3 C) Cytochrome b D) Cytochrome e E) Cytochrome f

B. cytochrome a3

In the reoxidation of QH2 by purified ubiquinone-cytochrome c reductase (Complex III) from heart muscle, the overall stoichiometry of the reaction requires 2 mol of cytochrome c per mole of QH2 because: A) cytochrome c is a one-electron acceptor, whereas QH2 is a two-electron donor. B) cytochrome c is a two-electron acceptor, whereas QH2 is a one-electron donor. C) cytochrome c is water soluble and operates between the inner and outer mitochondrial membranes D) heart muscle has a high rate of oxidative metabolism, and therefore requires twice as much cytochrome c as QH2 for electron transfer to proceed normally. E) two molecules of cytochrome c must first combine physically before they are catalytically active.

B. cytochrome c is a two-electron acceptor, whereas QH2 is a one-electron donor

Mammals produce heat by using the endogenous uncoupling agent: A) the small molecule 2-4-Dinitrophenol synthesized by the cell. B) the protein thermogenin. C) the protein thioredoxin. D) the protein cytochrome c. E) a modified form of the FoF1 ATPase.

B. the protein thermogenic

Uncoupling of mitochondrial oxidative phosphorylation: A) allows continued mitochondrial ATP formation, but halts O2 consumption. B) halts all mitochondrial metabolism. C) halts mitochondrial ATP formation, but allows continued O2 consumption. D) slows down the citric acid cycle. E) slows the conversion of glucose to pyruvate by glycolysis.

C. halts mitochondrial ATP formation, but allows continued O2 consumption

During oxidative phosphorylation, the proton motive force that is generated by electron transport is used to: A) create a pore in the inner mitochondrial membrane. B) generate the substrates (ADP and Pi) for the ATP synthase. C) induce a conformational change in the ATP synthase. D) oxidize NADH to NAD+. E) reduce O2 to H2O.

C. induce a conformational change in the ATP synthase

The oxidation of a particular hydroxy substrate to a keto product by mitochondria has a P/O ratio of less than 2. The initial oxidation step is very likely directly coupled to the: A) oxidation of a flavoprotein. B) oxidation of a pyridine nucleotide. C) reduction of a flavoprotein. D) reduction of a pyridine nucleotide. E) reduction of cytochrome a3.

C. reduction of a flavoprotein

Which of the following statements about energy conservation in the mitochondrion is false? A) Drug that inhibits the ATP synthase will also inhibit the flow of electrons down the chain of carriers. B) For oxidative phosphorylation to occur, it is essential to have a closed membranous structure with an inside and an outside. C) The yield of ATP per mole of oxidizable substrate depends on the substrate. D) Uncouplers (such as dinitrophenol) have exactly the same effect on electron transfer as inhibitors such as cyanide; both block further electron transfer to oxygen. E) Uncouplers "short circuit" the proton gradient, thereby dissipating the proton motive force as heat.

D. Uncouplers (such as dinitrophenol) have exactly the same effect on electron transfer as inhibitors such as cyanide; both block further electron transfer to oxygen

When the delta G'° of the ATP synthesis reaction is measured on the surface of the ATP synthase enzyme, it is found to be close to zero. This is thought to be due to: A) a very low energy of activation. B) enzyme-induced oxygen exchange. C) stabilization of ADP relative to ATP by enzyme binding. D) stabilization of ATP relative to ADP by enzyme binding. E) none of the above.

D. stabilization of ATP relative to ADP by enzyme binding

The rate of oxidative phosphorylation in mitochondria is controlled primarily by: A) feedback inhibition by CO2. B) the availability of NADH from the TCA cycle. C) the concentration of citrate (or) the glycerol-3-phosphate shuttle. D) the mass-action ratio of the ATD-ADP system. E) the presence of thermogenin.

D. the mass-action ratio of the ATD-ADP system

In normal mitochondria, the rate of NADH consumption (oxidation) will: A) be increased in active muscle, decreased in inactive muscle. B) be very low if the ATP synthase is inhibited, but increase when an uncoupler is added. C) decrease if mitochondrial ADP is depleted. D) decrease when cyanide is used to prevent electron transfer through the cytochrome a + a3 complex. E) All of the above are true.

E. All of the above are true

Which one of the following best describes the role of mitochondria in apoptosis? A) Escape of cytochrome c into the cytoplasm. B) Increased rate of fatty acid B-oxidation. C) Increase in permeability of outer membrane. D) Uncoupling of oxidative phosphorylation. E) Both A and C are correct.

E. Both A and C are correct

Which of the following statements about the chemiosmotic theory is false? A) Electron transfer in mitochondria is accompanied by an asymmetric release of protons on one side of the inner mitochondrial membrane. B) Energy is conserved as a transmembrane pH gradient. C) Oxidative phosphorylation cannot occur in membrane-free preparations. D) The effect of uncoupling reagents is a consequence of their ability to carry protons through membranes. E) The membrane ATPase, which plays an important role in other hypotheses for energy coupling, has no significant role in the chemiosmotic theory.

E. The membrane ATPase, which plays an important role in other hypotheses for energy coupling, has no significant role in the chemiosmotic theory

A new compound isolated from mitochondria is claimed to represent a previously unrecognized carrier in the electron transfer chain. It is given the name coenzyme Z. Which line of evidence do you feel is the least conclusive in assigning this compound a position in the electron transfer chain? A) Alternate oxidation and reduction of the mitochondrion-bound coenzyme Z can be readily demonstrated. B) Removal of coenzyme Z from the mitochondria results in a decreased rate of oxygen consumption. C) The rate of oxidation and reduction of mitochondrion-bound coenzyme is of the same order of magnitude as the overall rate of electron transfer in mitochondria as measured by oxygen consumption. D) The reduction potential of Z is between that of two compounds known to participate in the electron transport chain When added to a mitochondrial suspension, coenzyme Z is taken up very rapidly and specifically by the mitochondria. E) When added to a mitochondrial suspension, coenzyme Z is taken up very quickly rapidly and specifically

E. When added to a mitochondrial suspension, coenzyme Z is taken up very quickly rapidly and specifically

Which of the following is correct concerning the mitochondrial ATP synthase? A) It can synthesize ATP after it is extracted from broken mitochondria. B) It catalyzes the formation of ATP even though the reaction has a large positive delta G'°. C) It consists of F0 and F1 subunits, which are transmembrane (integral) polypeptides. D) It is actually an ATPase and only catalyzes the hydrolysis of ATP. E) When it catalyzes the ATP synthesis reaction, the delta G'° is actually close to zero.

E. When it catalyzes the ATP synthesis reaction, the delta G'° is actually close to zero

The relative concentrations of ATP and ADP control the cellular rates of: A) glycolysis. B) oxidative phosphorylation. C) pyruvate oxidation. D) the citric acid cycle. E) all of the above.

E. all of the above

Almost all of the oxygen (O2) one consumes in breathing is converted to: A) acetyl-CoA. B) carbon dioxide (CO2). C) carbon monoxide and then to carbon dioxide. D) none of the above. E) water.

E. water

Compound X is an inhibitor of mitochondrial ATP synthesis. It was observed that when compound X was added to cells, the NAD+/NADH ratio decreased. Would you expect X to be an uncoupling agent or an inhibitor of respiratory electron transfer? Explain in 30 words or less

It is an inhibitor of electron transfer; its addition lowers the NAD+/NADH ratio because NADH produced by oxidative reactions in mitochondria can no longer be reoxidized by electron flow to O2.

When the F1 portion of the ATP synthetase complex is removed from the mitochondrial membrane and studied in solution, it functions as an ATPase. Why does it not function as an ATP synthetase?

Like all enzymes, the F1 subunit of the ATP synthase catalyzes a reaction in both directions: ADP + Pi ATP + H2O The standard free-energy change (G'°) for ATP hydrolysis is -30.5 kJ/mol. With no proton motive force to drive the reaction toward ATP synthesis, the hydrolysis (ATPase activity) occurs spontaneously.

Describe and explain how each of the following manipulations will affect (1) the rate of NADH consumption and (2) the rate of ATP synthesis in mitochondria. (a) Depletion of ADP (b) Addition of an uncoupler (c) Addition of cyanide (CN-)

NADH consumption & ATP synthesis ————————————————————— deplete ADP- lower & lower add uncoupler - higher, or no change & lower add CN- lower & lower

As you read and answer this question, you are (presumably) consuming oxygen. What single reaction accounts for most of your oxygen consumption?

O2 is converted to H2O by electrons from the respiratory chain. The final step is the one catalyzed by cytochrome oxidase (Complex IV).

Mitochondria carrying out oxidative phosphorylation consume oxygen. Explain what happens to this oxygen, and describe the effect of an uncoupling agent such as 2,4-dinitrophenol on the rate of oxygen consumption. Assume there is a sufficient supply of oxidizable substrate, ADP, and Pi.

O2 is reduced to H2O by electrons passed through the respiratory chain. Addition of 2,4-dinitrophenol, which uncouples phosphorylation (ATP synthesis) from electron flow, actually stimulates O2 consumption slightly by removing the "drag" of ATP synthesis

Show the path of electrons from ubiquinone (Q or coenzyme Q) to oxygen in the mitochondrial respiratory chain. One of the two compounds (Q and O2) has a standard reduction potential (E'°) of 0.82 V, and the other, 0.045 V. Which value belongs to each compound? How did you deduce this?

QH2 > cyt b > cyt c1 > cyt c > cyt (a + a3) > O2 E'° for O2 must be the larger positive value (+0.82) because electron flow occurs spontaneously to the electron acceptor with the more positive E'°.

Although molecular oxygen (O2) does not participate directly in any of the reactions of the citric acid cycle, the cycle operates only when O2 is present. Explain this observation

The citric acid cycle produces NADH, which is normally reoxidized to NAD+ by the passage of electrons through the respiratory chain to O2. With no O2 to accept electrons, NADH accumulates, NAD+ is depleted, and the citric acid cycle slows for lack of NAD+

When the dG'° of the ATP synthesis reaction is measured on the surface of the ATP synthase enzyme, it was found to be close to zero. Describe briefly why this is so

The enzyme binds ATP more tightly than ADP thus stabilizing the former (i.e., the product of the synthesis reaction) relative to the latter (i.e., the reactant in the synthesis reaction)

Explain briefly the current model for how the proton motive force that is generated by electron transport is used to drive the ATP synthesis reaction

The tight binding of ATP by the enzyme stabilizes it and makes the G'° of the synthetic reaction more favorable. Once the reaction has occurred, the ATP product must be released from the enzyme. The proton motive force causes protons to move across the inner mitochondrial membrane through the pore in the Fo complex. This movement leads to conformational changes that decrease the affinity of the F1 portion of the synthase for ATP, resulting in its release from the enzyme

Describe, in simple diagrams and a few words, the chemiosmotic theory for coupling oxidation to phosphorylation in mitochondria.

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).

Using a simple diagram of the chemiosmotic theory, explain why anything that makes the mitochondrial membrane leaky stops ATP synthesis in the mitochondria

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). Anything that makes the membrane leaky to protons (an uncoupler such as 2,4-dinitrophenol, or mechanical breakage of the membrane) prevents formation of a proton gradient. With no proton gradient, there is no energy source for ATP synthesis by FoF1 (ATP synthase)

During electron transfer through the mitochondrial respiratory chain, the overall reaction is: NADH + 1/2 O2 + H+ --> NAD+ + H2O. The difference in reduction potentials for the two half-reactions (dE'°) is +1.14 V. Show how you would calculate the standard free-energy change, dG'°, for the reaction as written above. (The Faraday constant, , is 96.48 kJ/V·mol.)

deltaG'° = -nFdeltaE'° = (-2)(96.48 kJ/V·mol)(1.14V) = -220 kJ/mol