Chp 21: Oxidative Phosphorylation

Question 1

1. Be able to draw a cartoon demonstrating oxidative phosphorylation. Include NADH, complexes I, II, III and IV, CoQ, cytochrome c, and ATP synthase. Demonstrate the sites for the pumping of protons and the direction of movement. Indicate the excess protons on one side of the membrane. Draw equations showing the synthesis of H2O and ATP (Fig 21.1)

Answer 1

In order from left to right: Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase), CoQ, Complex III (cytochrome b-c1), Cytochrome C, Complex IV (Cytochrome oxidase), ATP synthase

For the position of Complex II, see Fig. 21.5 and remember that electrons from Complex I and II only flow to CoQ. That is, an electron from Complex II cannot flow to Complex I and vice versa

As electrons flow down the ETC, protons must be pumped from the inside of the mitochrondria to the outside of the mitochondria. This means there will always be an excess of protons on the outside of the mitochondrial membrane as compared to the inside.

• Cytochrome oxidase reaction: O2 + 4H+ + 4e-  2H2O
  
  • The O2 comes from hemoglobin in the blood, the 4H+ comes from the solution inside the mitochondria, and the e- come from the ETC. Once an electron enters the ETC, it may only exit by reacting with O2. Without O2, the ETC will become fully reduced and electrons will not flow
• ATP synthase reaction: ADP + Pi + H+(outside)  ATP + H+(inside)
  
  • The energy for the high energy bond of ATP is provided by the proton gradient across the membrane. H+(inside) represents hydrogen ions inside the mitochondria

Other info: The outer mitochondrial membrane has huge pores in it. Compounds with a molecular weight of 700 have no trouble crossing the outer mitochondrial membrane. So when we pump protons across the inner membrane to the intermembrane space, we are really pumping them out of the mitochrondria

Question 2

2. Why does the movement of protons produce ATP?

Answer 2

• ATP synthase links the generation of ATP to proton flow through it from the intermembrane space back into the matrix: ADP + P_i + H⁺(outside) → ATP + H⁺(inside)
• This reaction makes more than 90% of ATP in the cell
• Whenever the concentration of ATP drops, the above reaction will flow to the right to replace the ATP that has been used by other reactions
• The protons on the left will come from the ETC
• If ATP concentrations are high enough, the reaction will reach equilibrium (stop) and no more protons will be pumped out of the mitochondria. Thus, the ETC will stop
• When we say that the pumping of protons creates an electrochemical gradient, the electric gradient comes for the positive sign, i.e., the electrons left behind create a gradient with more negative charge in the mitochondria than on the other side of the membrane. The chemical part of the electrochemical gradient is the gradient of proton, more on the outside than on the inside. Altogether, you have both protons/positive charges at higher concentration outside vs inside. This gradient has negative ∆G that can be used to make ATP from ADP and P_i

Question 3

3. Where is complex II found?

Answer 3

In the inner mitochrondrial membrane but does not span the membrane, and thus does not contain a proton-pumping mechanism. It feeds electrons to CoQ and then to Complex III (Cytochrome b-c1), Cytochrome C, and Complex IV (Cytochrome Oxidase)

Question 4

3. What reaction of the TCA cycle does complex II catalyze?

Answer 4

One component of Complex II is succinate dehydrogenase, which is part of the TCA cycle and catalyzes the oxidation of succinate to form fumarate and the reduction of FAD to form FAD(2H). The FAD(2H) feeds two electrons into the ETC, and two protons and one FAD into the solution

Question 5

3. How much ATP is produced when 2 electrons of succinate are passed through the chain to O2?

Answer 5

If oxygen is present, each FAD(2H) will produce 1.5 ATPs. Other books say two but since we are using this text, 1.5 ATPs

Question 6

3. Which complexes couple proton pumping and succinate oxidation?

Answer 6

Protons are pumped as the electrons pass through Complex III and Complex IV

Question 7

4. What is the function of an electron in the electron transport chain?

Answer 7

The electrons in the ETC have an inherent free energy within them that is slowly released as the electrons are passed down the ETC. The transfer of electrons at Complex I, III, and IV is coupled with the pumping of protons from the mitochondrial matrix into the intermembrane space (which creates an electrochemical gradient along the inner membrane of the mitochondria). The ∆G in the transfer of electrons at Complex I, III, and IV is very negative; the ∆G in the pumping of protons against their electrical and chemical gradients is, comparatively, only somewhat positive. The net result of these coupled reactions is irreversible and the entire ETC is irreversible.

Question 8

5. What is a cytochrome?

Answer 8

• Cytochromes are proteins containing a heme (an Fe atom bound to a porphyrin nucleus similar in structure to heme in hemoglobin). Unlike the heme in hemoglobin, the heme in cytochromes is reduced and oxidized. The oxidation-reduction couple is Fe2+/Fe3+. As they accept electrons, they are reduced to Fe2+. Each cytochrome is reoxidized as the electron passes to the next component of the ETC.
• Each cytochrome contains differences in protein component and small differences in the heme structure. This results in hemes with different free energies. As the electrons progresses down the ETC, each cytochrome has a lower free energy and the reactions are irreversible. Heat is given off.

Question 9

6. Explain how iron deficiency anemia affects oxygen transport in the blood and oxidative phosphorylation in mitochondria.

Answer 9

• Iron deficiency usually affects hemoglobin blood and the cytochromes of the ETC. In either case, iron deficiency may leave you tired and short of breath.
• Anemia is the condition of having a lower-than-normal number of red blood cells or quantity of hemoglobin
• Iron deficiency anemia is a reduction of or below-normal the number of erythrocytes, quantity of hemoglobin, or the volume of packed red blood cells (hematocrit) in the blood. When examined under a microscope, the red blood cells also appear pale or light colored. For this reason, the anemia that occurs with iron deficiency is also called hypochronic microcytic anemia
• Without sufficient heme molecules, less O₂ reaches tissue. Thus less oxidative phosphorylation can be used to make ATP and the body must shift to using other means of ATP production to survive
• Oxidative phosphorylation: Iron deficiency leads to less cytochromes in the mitochrondria. Thus less oxidative phosphorylation can be used to make ATP and the body must shift to using other means of ATP production to survive.

Question 10

7. What reaction is catalyzed by cytochrome oxidase?

Answer 10

O₂ + 4H⁺ + 4e^- → 2H₂O

OR

1/2O₂ + 2H⁺+ 2e^- → H₂O (either reaction is acceptable)

The O₂ comes from hemoglobin in the blood, the 4H⁺ comes from the solution inside the mitochrondria, and the electrons come from the ETC. Once an electrons enters the ETC, it may only exit by reacting with O₂

Question 11

8. Be able to state the chemiosmotic theory.

Answer 11

• Most ATP synthesis in cells is linked to a proton gradient that exists across the mitochrondrial membrane. The oxidation of NADH and FAD(2H) by the ETC and O2 creates the gradient
• Basically, the chemiosmotic theory explains how electrons transferred to oxygen result in a proton gradient and high energy phosphate bonds in ATP
• ATP synthase was discovered as a result of this theory

Question 12

9. How many ATPs are synthesized by ATP synthase for each NADH oxidized by the electron transport chain? How many for FAD(2H)?

Answer 12

2.5 ATP per NADH and 1.5 ATP per FAD(2H)

Other info: It takes four protons to pass through ATP synthase to synthesize one ATP. The two electrons NADH transfers through ETC can pump 10 protons into the intermembrane space: four at Complex I, four at Complex III, two at complex IV. But the two electrons that FADH transfers through the ETC can pump only six protons: four at Complex III, two at Complex IV. Complex II does not pump protons. This difference determines that NADH can produce 2.5 ATP, which FAD(2H) produces 1.5 ATP

Question 13

10. Is oxidative phosphorylation a reversible reaction? Why?

Answer 13

Oxidative phosphorylation is not a reversible reaction because the ∆G for the pathway is so negative. This is a pathway necessary for life. It is designed by nature to go forward to make ATP. The ∆G in the transfer of electrons at Complex I, III, and IV is very negative; the ∆G in the pumping of protons against their electrical and chemical gradients is, comparatively, only somewhat positive. The net result of these coupled reactions is irreversible

Question 14

11. What portion of the ETC is inhibited by CN^-? What is the effect of cyanide inhibition upon proton pumping and ATP synthesis?

Answer 14

Cyanide binds to the iron in the heme of the Cytochrome C Oxidase (Complex IV) and prevents electron transport to oxygen. Once blocked, all the cytochromes of the ETC become fully reduced and protons are no longer pumped out of the mitochrondria. This causes a rapid decline in the proton gradient and ATP synthesis. With enough cyanid, the cell rapidly runs out of energy and dies

Question 15

12. Why does an impairment of the electron transport chain result in lactic acidosis?

Answer 15

The chain of events following cyanide poisoning or lack of oxygen:

• The ETC becomes fully reduced and stops pumping protons
• ATP synthase stops because of lack of a proton gradient
• The ratio of ATP to ADP & AMP decreases
• NADH increases and inhibits the TCA even though the ratio of ATP to ADP & AMP tends to activate
• The ratio of ATP to ADP & AMP activates glycolysis (glucose to pyruvate), producing lots of pyruvate that cannot enter the PDC, so pyruvate concentration builds up
• High concentrations of pyruvate and NADH drive the lactate dehydrogenase reaction, producing lots of lactic acid. The increased lactic acid released into the blood results in lactic acidosis
• Other info: Low ATP and high AMP/ADP causes activation of glycolysis. The increased AMP activates phosphofructokinase-1, which causes increased pyruvate
• Shock is the lack of oxygen at the tissue level (though there are many causes, they all produce this end result). This lack of oxygen, like a defective ETC, will drive anaerobic glycolysis, producing high levels of lactate. This is why trending lactate levels can be useful in treating shock patients

Question 16

13. How does shivering generate heat?

Answer 16

Shivering (involuntary contractions, or twitching, of muscles) increases heat in 2 ways:

• When myosin and actin muscle fibers contract, some of the free energy from the hydrolysis of ATP produces heat
• When electrons move down the ETC, much of the change in free energy is lost as heat

Question 17

14. Be able to go through the series of events whereby increases ATP utilization is coupled to increased O2 utilization.

Answer 17

Increased ATP utilization causes the concentration of ADP to increase. With more ADP available to bind to ATP synthase, more protons will FLOW (rather than being pumped) across the mitochrondrial membrane into the matrix to drive ATP synthase to make the additional needed ATP. This decreases the electrochemical (or H⁺) gradient across the membrane. The ETC then starts to run faster to pump out more protons to restore the gradient; more electrons run through the ETC and through cytochrome oxidase, which increases the amount of O₂ used and reduced. Thus, increased ATP use is coupled to increased O₂ use.

Question 18

15. Be able to go through the series of events whereby pyruvate dehydrogenase is activated by increased ATP utilization.

Answer 18

• When ATP utilization increases, ATP/{ADP&AMP) decreases, ETC increases, NADH/NAD+ decreases, TCA cycle increases, acetyl CoA decreases, and the PDC is activated
• That is, decreased ATP/{ADP&AMP), NADH, and acetyl CoA all activate the PDC complex

Question 19

16. Understand how a chemical uncoupler works. What happens to heat production, proton pumping, ATP synthesis, and NADH utilization following uncoupling?

Answer 19

• A chemical uncoupler (also known as a proton ionophore) is a lipid-soluble compound that allows protons to abnormally reenter the mitochondria. Normally, protons flow back into the mitochondrial matrix through the ATP synthase and the amount of protons flowing back is regulated by the ATP/{ADP&AMP). When an uncoupled is present, the protons flow back without synthesizing any ATP. To the extent that an uncoupler is present, this becomes the preferred path for proton reentry
• Simply speaking, the uncoupler has made a hole in the inner membrane and the normal electrochemical gradient is no longer maintained. As a result, electron flow through the ETC increases and more protons are pumped in an effort to try to maintain the electrochemical gradient. More electrons flowing through the ETC increases the use of NADH, FAD(2H), and O2
• At the same time, as the electrochemical potential is dropping, less and less ATP can be synthesized. If enough chemical uncoupler is present, ATP synthesis will stop and the cell will die
• Other info: Uncouplers generate lots of heat because of all the free energy of reentry of protons is converted to heat. Some examples of chemical uncouplers: dinitrophenol, thyroid hormones, and aspirin

Question 20

17. What is the difference between an inhibitor of electron transport and an uncoupler of electron transport with respect to NADH utilization?

Answer 20

• ETC inhibitor: NADH utilization will decrease because ETC is fully reduced and cannot accept electrons
• Chemical uncoupler: NADH utilization is increased because electrons are flowing faster to try to maintain the proton gradient

Question 21

17. What is the difference between an inhibitor of electron transport and an uncoupler of electron transport with respect to proton pumping?

Answer 21

• ETC inhibitor: Proton pumping will slow or stop because the flow of electrons through the ETC is slowed or stopped
• Chemical uncoupler: Proton pumping will increase as more electrons flow through the ETC

Question 22

17. What is the difference between an inhibitor of electron transport and an uncoupler of electron transport with respect to ATP synthase?

Answer 22

• ETC inhibitor: ATP synthase activity will decrease with the decrease in the proton gradient
• Chemical uncoupler: ATP synthase activity will decrease with the decrease of the proton gradient

Question 23

18. Give an example of both a symport and an antiport that functions in the mitochondrial membrane. (Fig 21.13)

Answer 23

Both symports and antiports are translocases, transmembrane enzymes that function to translocate (transfer) chemicals across the mitochrondrial membrane

Symport (“same direction”): carries two substances across the membrane in the same direction. Examples:

• Phosphate translocase: HPO₄^2-(outside) + H⁺(outside) ⇔HPO₄^2-(inside) + H⁺(inside)
• Pyruvate translocase: CH₃COCOO^-(outside) + H⁺(outside)  CH₃COCOO^-(inside) + H⁺(inside)

Antiport (“opposite direction”): carries substances in opposite directions across a membrane. Examples:

• ATP-ADP translocase: ADP(outside) + ATP(inside) ⇔ ADP(inside) + ATP(outside)

Question 24

19. Concerning Cora Nari: She had a heart attack. Why was nasal oxygen administered?

Answer 24

To try to correct the ischemic state of the heart cells. The limited availability of oxygen to act as an electron acceptor will decrease proton pumping, the generation of the electrochemical gradient across the inner mitochondrial membrane, and the ATP concentration of the ischemic cells.

Question 25

19. Concerning Cora Nari: Why was her blood pressure lowered?

Answer 25

Nitroprusside was administered to decrease ATP demands of the heart. The effect of nitroprusside is vasodilation, which increases the diameter of the vessel and decreases vascular smooth muscle tone, thus decreasing peripheral vascular resistance and ultimately decreasing the workload of the heart (decreasing her blood pressure and using less ATP)

Question 26

19. Concerning Cora Nari: She had a heart attack. What might have been the effect on ATP production if nitroprusside had been continued for several days?

Answer 26

During prolonged infusions of 24-48 hours or more, nitroprusside is converted to cyanide, an inhibitor of the cytochrome oxidase (Complex IV). This would inhibit electron transport to oxygen via the ETC, and mitochrondrial respiration as well as energy production would decrease and eventually cease

Question 27

20. Concerning Cora Nari: What is TPA and how does it dissolve blood clots?

Answer 27

Short story: TPA (tissue plasminogen activator) is a normal substance released from blood vessel walls that activates plasminogen, which hydrolyzes amide bonds in the fibrin proteins of blood clots

Long story: TPA is a protease released from blood vessel walls in response to small unwanted vascular clots. After release, it converts plasminogen to plasmin, a protease that hydrolyzes amide bonds fibrin. Fibrin is a major component of blood clots to digesting the fibrin dissolves the clot. Heart attacks are usually caused by clots that obstruct one or more large vessels in the heart. TPA medication is administered intravenously to break up the thrombi that may be obstructing the coronary arteries in a patient experiencing an acute MI. The TPA converts plasminogen to plasmin by directly cleaving the single chain of plasminogen into two chains which are then linked together by a disulfide bond to form a molecule of plasmin. The plasmin digests the fibrin to dissolve the clot and this allows the blood (i.e., O2) to flow back in and for ATP to be made. TPA is also used to treat patients experiencing an acute massive pulmonary embolism and also patients with acute ischemic stroke

Question 28

21. Concerning Cora Nari: How did the change in the ratio of ATP to ADP and AMP affect anaerobic glycolysis? How did this affect the pH?

Answer 28

• In the heart and other tissues, the control enzyme of glycolysis is affected by the ATP/ADP&AMP ratio. Any time the ATP/ADP&AMP drops, glycolysis will increase. Unlike striated muscle, the healthy heart very seldom uses anaerobic glycolysis.
• With an MI, the heart is deprived of oxygen so the ATP/ADP&AMP drops and the TCA cycle and PDC slow down or stop depending on the degree of ischemia. In the heart deprived of oxygen, decrease in the ATP/ADP&AMP ratio has a major effect upon glycolysis
• Large quantities of pyruvate and NADH are made. The only pathway open to them is the lactate dehydrogenase reaction that produces large amounts of lactic acid. Since lactic acid is acidic, it eventually lowers the blood pH, causing metabolic acidosis
• Note! The healthy heart is well-oxygenated and will remove lactate from the blood to use it for energy

Question 29

22. Concerning X.S.Teefore, Explain how the affect of excess thyroid hormone on oxidative phosphorylation could explain increased appetite and sweating?

Answer 29

• Uncoupling proteins are activated by high concentration of triiodothronine (T₃), allowing protons to leak back across the mitochondrial membrane. Thus more NADH and FAD(2H) will be oxidized in an attempt to maintain the normal level of ATP
• The increased flow of electrons in the ETC helps to explain the excess heat. Also, using more NADH and FAD(2H) requires more energy, and this activates the hunger mechanisms of the body
• In fact, excess T₃ induces the enzymes for many anabolic and catabolic pathways, and they all seem to be more active. All these pathways are using energy (food) and generating heat