Week 10- Citric Acid Cycle, Electron Transport, Oxidative Phosphorylation

Question 1

Overview (Krebs cycle)

Answer 1

• Overall: metabolic pathway converting C-atoms to CO2 and conserving metabolic free energy that drives ATP synthesis
• Origin of name: citrate is the 1st product of the cycle
• CO2 released: 3 CO2 released, one from pyruvate to acetyl-CoA, two others from cycle
• Position in Metabolism
  1. Citric acid cycle is not a continuation of glucose breakdown
  2. Cycle is central pathway for recovering free energy from metabolic fuels (carbohydrates, fatty acids, and amino acid)
  3. Unlike glycolysis, citric acid cycle always goes back to starting point

Question 2

Citric Acid Cycle in Perspective

Answer 2

• Carbon atoms entering cycle are in the form of acetyl groups derived from carbohydrates, or fatty acids
• Closed cycle allowing oxidation of unlimited number of acetyl groups
• Stage 1 and 2 produce reduced electron carriers (NADH, FADH2)
• In stage 3, electron enter electron transport chain to produce ATP

Question 3

Acetyl-CoA basics

Answer 3

• Thioester bonds
• Its formation conserve delta G of oxidation
• Its cleavage helps to drive metabolic reactions
• Delta G = -31.5 kJ

Question 4

Overview of Citric Acid Cycle (Krebs cycle)

Answer 4

• Eight reactions occurring inside mitochondria
• 1st reaction involves acetyl-CoA (high energy)
• Net reaction: 3 NaD+ + FAD + GDP+ Pi + acetyl-CoA = 3 NaDH + FADH2 + GTP + CoA + 2 CO2
  1. Conserve energy in reduced NADH and FADH2
  2. release one GTP (same energy potential as ATP)
• Energy conservation via electron transport
  1. reduction of 3 NAD+ to 3 NADH take up 3 pair of electron
  2. reduction of 1 FAD to FADH2 takes up 4th pair of electrons
  3. Free energy conserved in these reduced cofactors and GTP

Question 5

Enzyme 8 of Citric Acid Cycle

Answer 5

Malate Dehydrogenase
- oxidation of -OH group in Malate
- formation of Oxaloacetate
- unfavorable reaction (delta G = + 29.7), thus low amount of Oxaloacetate
- Coupling of reaction 8 to reaction 1 (delta G = - 31.5) drives reaction 8

Question 6

Citric Acid Cycle = Energy-generating Catalytic Cycle

Answer 6

• electron carried by NADH and FADH2 channeled to electron transport chain and energy conserved by ATP synthesis
• transfer of electron by each NADH generates 3 ATPs
• transfer of electron by each FADH2 generates 2 ATPs
• Oxidation of 2 acetyl-CoA (one cycle) generates 24 ATPs
• Overall, one glucose breakdown generates 38 ATPs in aerobic condition, but only 2 ATPs in anaerobic condition

Question 7

Regulation of Citric Acid Cycle

Answer 7

• Rate-Limiting Enzymes of Citric Acid Cycle (three irreversible reactions)
  1. citrate synthase (reaction 1): inhibition by citrate
  2. Isocitrate dehydrogenase (reaction 3)
  3. alpha-ketoglutarate dehydrogenase (reaction 4)
• NADH and ATP (high energy state = low metabolic state) inhibit all three enzymes, leading to slowdown of citric acid cycle

Question 8

Electron transport - overview pt 1

Answer 8

• Electron transfer in glycolytic and citric acid cycles
• Breakdown of electron transfer:
  1. Oxidation of glucose atoms: C6H12O6 + 6 H2O = 6 CO2 + 24 H+ + 24 electron (Glycolysis and Citric Acid Cycle)
  2. Reduction of oxygen: 6 O2 + 24H+ + 24 electrons = 12 H2O (Electron transport)

Question 9

Electron transport - overview pt 2

Answer 9

• 12 pairs of electron transfer from glucose oxidation to 10+ NAD+ and 2 FAD
• electron transfer from NADH and FADH2 to other re-dox centres in mitochondria under aerobic conditions, and release delta G
• released delta G used to synthesize ATP
• NADH and FADH2 reoxidized back to NAD+ and FAD for additional electron transfer
• Electrochemical gradient generated by protons expulsion from mitochondria, driving ATP synthesis (Oxidative Phosphorylation)
• Oxidative Phosphorylation represents final stage of metabolism and major source of ATPs

Question 10

Mitochondria - Anatomy

Answer 10

• Outer membrane permeable allowing free diffusion
• Inner membrane not permeable allowing transport of ions
• H+ gradient across inner membrane as result of electron transport
• electron transfer through electron transport chain leads to pumping of H+ from matrix to intermembrane space
• H+ gradient provides one of the basis of the coupling mechanism driving ATP synthesis

Question 11

Mitochondria - transport systems

Answer 11

• Cytosolic Reducing Substances
  1. NADH produced by citric acid cycle contributes to electron transport at inner membrane
  2. NADH produced by glycolysis (reaction 6) unable to cross mitochondria membrane
• Barrier overcome by conversion of oxaloacetate to malate in cytoplasm (reduction), followed by its transport into matrix and conversion to oxaloacetate (oxidation)
• Overall: NADH coupled to electron transport chain

Question 12

Electron transport: organization of complexes

Answer 12

• Complex I: NADH-CoQ oxidoreductase, transfers electron from NADH to CoA
• Complex II: succinate-CoQ oxidoreductase, increase the reduced CoQ
• Complex III: CoQH2-cytochrome c oxidoreductase, transfer electron from CoA to cytochrome c
• Complex IV: cytochrome c oxidase, oxidizes cytochrome c and reduces O2

Question 13

Electron transport - complex I

Answer 13

• transfer electron from NADH to CoQ
• Consists of a chain of redox centres with increasing reduction potentials
• Increasing reduction potentials facilitate electron transfer to CoQ
• Concurrent expulsion of 4 H+ out of mitochondria
• Overall: NaDH + H+ +CoQ = NAD+ + CoQH2

Question 14

Electron Transport- Chemiosmosis and H+ gradient

Answer 14

• Chemiosmosis: H+ translocation derived from electron transport complexes, generating a H+ gradient across inner membrane
• Consequence: imbalance in H+ concentration brings about a force in term of delta G to restore the balance
• Energetics:
  1. delta G required for translocation of one H+ is 20 KJ
  2. 10 H+ translocated for each pair of electron transferred from NADH to CoQ
  3. 10 H+ translocation results in 200 KJ, sufficient to drive ATP synthesis
• Uncoupling Protein (UCP) and Brown Fat Tissue