Bioenergetics and Oxidative Phosphorylation

1. What is cellular respiration?

→ The processes by which cells consume O₂ and produce CO₂ while oxidizing organic fuel molecules such as glucose, fatty acids, and amino acids. The energy released is converted into bioenergy and stored as ATP.

2. What are the major fuel sources that feed cellular metabolism?

→ Carbohydrates, fatty acids, and amino acids.

3. What is ATP's primary role in the cell?

→ ATP is the cell's direct energy carrier and energy source for most cellular processes and reactions.

4. How is cellular ATP consumption approximately distributed?

→ 30% Na⁺/K⁺ and Ca²⁺ pumps
→ 30% protein synthesis
→ 20% gluconeogenesis, ureagenesis, and carbohydrate/lipid turnover
→ 20% other processes.

5. What is the approximate standard free-energy change associated with hydrolysis of each ATP phosphate bond?

→ ΔG° ≈ −7.3 kcal/mol.

6. What are reducing equivalents?

→ Molecules that carry high-energy electrons generated during oxidation of nutrients, primarily NADH and FADH₂.

7. What is the overall role of mitochondria in cellular respiration?

→ Mitochondria are the major site of oxidative metabolism, the electron transport chain, and oxidative phosphorylation, allowing energy from nutrients to be converted into ATP.

8. Where does glycolysis occur, and what reducing equivalent does it produce?

→ Glycolysis occurs in the cytosol and converts:
1 glucose → 2 pyruvate + 2 NADH.

9. What happens to pyruvate after glycolysis?

→ Pyruvate enters the mitochondria and is converted to acetyl-CoA by pyruvate dehydrogenase, producing NADH.

10. What are the major mitochondrial sources of NADH and FADH₂?

→ Pyruvate dehydrogenase, β-oxidation, and the TCA cycle.

11. What does the TCA cycle produce from acetyl-CoA?

→ Acetyl-CoA is oxidized to CO₂, producing 3 NADH and 1 FADH₂ per acetyl-CoA.

12. Why can mitochondrial NADH directly enter the ETC but cytosolic NADH cannot?

→ NADH produced in the cytosol cannot directly cross the inner mitochondrial membrane, so its electrons must be transferred into mitochondria through electron shuttles.

13. What are the two electron shuttles that transfer cytosolic NADH electrons into mitochondria?

→ Glycerol phosphate shuttle and malate-aspartate shuttle.

14. How does the glycerol phosphate shuttle transfer cytosolic NADH electrons?

→ Cytosolic NADH reduces dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Mitochondrial glycerol phosphate dehydrogenase reoxidizes it and transfers the electrons to FAD, producing FADH₂.

15. What is the ATP yield of the glycerol phosphate shuttle?

→ 1 cytosolic NADH → 1 FADH₂ equivalent → ~1.5 ATP.
→ It therefore loses approximately 1 ATP compared with transferring electrons at the NADH level.

16. Where is the glycerol phosphate shuttle particularly active?

→ Brain and skeletal muscle.

17. How does the malate-aspartate shuttle differ from the glycerol phosphate shuttle?

→ It transfers cytosolic NADH electrons into mitochondria at the NADH level, preserving the full energetic yield.

18. What is the ATP yield of the malate-aspartate shuttle, and where is it especially active?

→ 1 cytosolic NADH → ~2.5 ATP.
→ Particularly active in liver, kidney, and heart.

19. What is the overall purpose of the electron transport chain (ETC)?

→ Electrons from NADH and FADH₂ flow through the ETC to O₂, and the released energy pumps H⁺ across the inner mitochondrial membrane to create a proton gradient.

20. What is the final electron acceptor of the ETC?

→ Oxygen (O₂), which is ultimately reduced to H₂O at Complex IV.

21. Which ETC complexes pump protons from the matrix into the intermembrane space?

→ Complexes I, III, and IV.
→ Complex II does not pump protons.

22. How do electrons enter the ETC from NADH versus FADH₂?

→ NADH → Complex I
→ FADH₂ → Complex II/ubiquinone level

23. What are the major mobile electron carriers of the ETC?

→ Coenzyme Q (ubiquinone) carries electrons between membrane complexes, while cytochrome c carries electrons from Complex III to Complex IV.

24. What is Complex I and what does it do?

→ NADH dehydrogenase/NADH:ubiquinone.
→ Transfers electrons from NADH → FMN → CoQ, reducing CoQ to QH₂.
→ Pumps 4 H⁺.

25. What is the role of flavin mononucleotide (FMN) in Complex I?

→ FMN is a tightly bound prosthetic group that accepts electrons from NADH, becoming FMNH₂, and then transfers them onward toward CoQ.

26. What is Complex II and what does it do?

→ Succinate dehydrogenase/succinate oxidase.
→ It is also a TCA cycle enzyme and transfers electrons from succinate → FAD/FADH₂ → CoQ.

27. Why does FADH₂ generate less ATP than NADH?

→ FADH₂ enters the ETC at Complex II, bypassing Complex I, so it misses the opportunity for Complex I to pump 4 H⁺.

28. What is unique about Complex II compared with Complexes I, III, and IV?

→ Complex II is not a transmembrane proton-pumping complex and does not pump H⁺.

29. What is Complex III and what does it do?

→ Cytochrome b-c₁ complex/ubiquinone:cytochrome c oxidoreductase.
→ Transfers electrons from QH₂ → cytochrome c through iron-sulfur proteins and cytochromes.
→ Pumps 4 H⁺.

30. What is Complex IV and what does it do?

→ Cytochrome oxidase.
→ Transfers electrons from cytochrome c → O₂, producing H₂O.
→ Pumps 2 H⁺.

31. What additional role does Complex IV play in establishing the proton gradient?

→ It consumes matrix H⁺ during the reduction of O₂ to H₂O, further contributing to the proton gradient across the inner mitochondrial membrane.

32. What are the major components of Complex III?

→ Cytochrome b, cytochrome c₁, cytochrome c, and two iron-sulfur proteins.

33. What are the major cytochromes associated with Complex IV?

→ Cytochromes a and a₃.

34. What is Complex V?

→ ATP synthase, also called F₁F₀ ATPase.

35. What are the two major portions of ATP synthase?

→ F₁: mushroom-shaped catalytic portion projecting into the mitochondrial matrix.
→ F₀: membrane-integrated proton channel through which H⁺ flows into the matrix.

36. How does the proton gradient drive ATP synthesis?

→ H⁺ flows from the intermembrane space → matrix through F₀, and the energy from this proton movement drives F₁ to synthesize ATP from ADP + Pi.

37. Approximately how many protons are required to generate one ATP through ATP synthase?

→ Approximately 4 H⁺ per ATP.

38. What are the approximate ATP yields from NADH and FADH₂?

→ NADH → 10 H⁺ → ~2.5 ATP
→ FADH₂ → 6 H⁺ → ~1.5 ATP

39. How many protons are pumped per pair of electrons entering at Complex I versus Complex II?

→ NADH/Complex I: ~10 H⁺
→ FADH₂/Complex II: ~6 H⁺
→ Contributions: Complex I = 4, III = 4, IV = 2.

40. What is oxidative phosphorylation (OXPHOS)?

→ Production of ATP from ADP + Pi using energy released when reduced substrates such as NADH and FADH₂ are oxidized and their electrons ultimately transferred to O₂.

41. Why is an intact inner mitochondrial membrane essential for oxidative phosphorylation?

→ It creates a closed compartment that allows H⁺ to accumulate in the intermembrane space and establish the proton gradient required for ATP synthesis.

42. How does ATP leave the mitochondria after it is synthesized?

→ ATP/ADP translocase exports ATP from the matrix in exchange for ADP from the cytosol, allowing continued ATP synthesis.

43. What happens when Complexes I–IV are inhibited?

→ Proton gradient ↓
→ Cellular respiration ↓
→ ATP synthesis ↓
→ Mitochondrial ATP concentration ↓

44. What is an uncoupler?

→ A substance that collapses the proton gradient across the inner mitochondrial membrane, allowing respiration to continue without efficiently producing ATP.

45. What happens to respiration and ATP production in the presence of an uncoupler?

→ Proton gradient ↓
→ Respiration ↑ as the cell attempts to restore the gradient
→ ATP synthesis ↓.

46. What are the major ETC inhibitors and their targets?

→ Rotenone, amytal → Complex I
→ Antimycin A → Complex III
→ Cyanide, azide → Complex IV
→ Oligomycin → Complex V/ATP synthase
→ Atractyloside and bongkrekic acid → ATP/ADP translocase

47. What happens when Complex V/ATP synthase is inhibited by oligomycin?

→ H⁺ cannot efficiently return to the matrix → proton gradient increases → respiration decreases due to the high proton gradient → ATP synthesis decreases.

48. What are valinomycin and 2,4-dinitrophenol (DNP), and what do they do?

→ Both disrupt mitochondrial energy coupling.
→ Valinomycin is an ionophore that transports K⁺ across membranes, disrupting the electrical component of the proton-motive force.
→ DNP acts as a proton carrier that dissipates the proton gradient.
→ Both decrease efficient ATP production.

49. What happens when ATP/ADP translocase is inhibited, and what are the clinical consequences of respiratory inhibition?

→ Atractyloside and bongkrekic acid inhibit ATP/ADP exchange, decreasing mitochondrial ADP availability → OXPHOS and respiration decrease.
→ When cellular respiration is inhibited, NADH and FADH₂ cannot be efficiently reoxidized, slowing the TCA cycle and contributing to lactate/ketoacid accumulation and acidosis.

50. What are the major clinical correlations involving ETC inhibition and uncoupling?

→ Cyanide poisoning: cyanide inhibits Complex IV (cytochrome oxidase), blocking cellular respiration. Treatment can involve nitrite to generate methemoglobin and thiosulfate to facilitate conversion of cyanide to thiocyanate, which can then be excreted.
→ Brown adipose tissue: UCP1/thermogenin provides a proton-conductive pathway that uncouples the proton gradient from ATP synthesis, releasing energy as heat (thermogenesis).
→ DNP and other uncouplers: dissipate the proton gradient and reduce ATP production.