Lecture 19: Oxidative Phosphorylation

Question 1

Mitochondrial Oxidative Phosphorylation

Answer 1

• NADH and FADH2 carry electrons to electron transport chain
• As electrons are passed, a change in redox potential generates free energy
  
  • 1st law of thermodynamics: energy cannot be created or destroyed
• Energy is used to power a conformational change in protein complexes, pumping protons from matrix into intermembrane space
• Proton gradient used by ATP synthase to make ATP in matrix

Question 2

Oxidative Phosphorylation

Answer 2

• Complex 1(-0.32 V): NADH-Q Oxidoreductase, pumps 4 protons out
• Complex 2(+0.03 V): Succinate-Q reductase(Succinate dehydrogenase from TCA cycle), oxidizes FADH2 to FADH, not an electron pump
• Complex 3(+0.04 V): Q-cytochrome c oxidoreductase, pumps 4 protons out
• Complex 4(+0.23 V): Cytochrome c oxidase, pump 2 protons out
• Energy generated by electrons moves protons against conc gradient

Question 3

• Electron Transfer Potential

Answer 3

• Standard Reduction Potential: A molecules tendency to be oxidized and reduced, ΔG = -nFΔE
  
  • F = 96,485J/Vmol
  • n = number of electrons
• positive ΔE, gains e more easily
• negative ΔE, loses e more easily

Question 4

Electron Transport Chain

Answer 4

• Electrons are passed from carrier to carrier
• Electron transfer potential of carriers measured by standard reduction potential, E
  
  • Good reducing agents give up electrons easily and have negative E values
    - Strong oxidizing agents have a greater affinity for electrons and have a positive E value
• Passage of electrons through chain( - to +) results in free energy change that drives confomational changes in the complexes, setting up a proton gradient for ATP synthase

Question 5

Oxidative Phosphorylation Inhibitors

Answer 5

• Rotenone and amytal inhibit electron flow from complex 1 to CoQ
• Antimycin A blocks complex 3
• Cyanide, azide and CO blocks Complex 4(cannot use oxygen as electron acceptor)
• Oligomycin inhibits ATP synthase(complex 5)
• Uncouplers can disrupt proton gradient, affecting ATP synthesis

Question 6

Uncouplers

Answer 6

• Uncouplers are molecules that have hydrophobic groups that allow them to cross the membrane
• Acidic groups can bind H and move them from high to low concentrations, disrupting the proton gradient and ATP synthesis

Question 7

How does ATP synthase make ATP from proton gradient

Answer 7

• ATP synthesis arises due to an electrochemical gradient across the mitochondrial inner membrane
• Proton gradient is produced by e transport using suitable e donors
• Proton-motive force is driving force behind ADP to ATP conversion
• ATP synthase is membrane-bound, reversible, and dependent on proton gradient

Question 8

ATP synthase

Answer 8

• F1 carries out catalytic synthesis of ATP in the matrix
• F0 is the integral membrane protein unit and anchors the complex to the membrane
  
  • Binding of H in the rotor causes rotation in the ring of c subunits of F0
  • Rotation of the ring, rotates the gamma subunit, inducing a conformational change in the beta subunit. H is released into the matrix
  • Conformational change in the F1 beta subunits are responsible for ATP synthesis
• Each beta subunit functions independently and alternates between 3 states
  - Open/empty: ATP leaves
  - Loose: ADP and P bound
  - Tight: ATP bound

Question 9

of H needed for ATP synthesis

Answer 9

• 3 H transported for production of 1 ATP
• 1 extra H needed for ATP export and ADP + P import
  - ATP-ADP translocate and P carrier protein
  - Maintains charge across inner mitochondrial membrane
• 4 H required/ATP made in matrix

Question 10

How much ATP is made

Answer 10

• NADH and FADH2 each donate a pair of e to the ETC, resulting in H being pumped into a pool of protons used by ATP synthase
• P/O ratio tells how many ATP are made per oxygen reduced to water(2 e from donor)
  
  • NADH = 10 H / 4 H per ATP = 2.5 ATP
  • FADH2 = 6 H / 4 H per ATP = 1.5 ATP

Question 11

P/O ratio exceptions

Answer 11

• NADHcyt from glycolysis cannot be imported across inner mitochondrial membrane for use in ETC
  - Glycerophosphate shuttle passes from NADHcyt to FADH2 in mitochondria
  - P/O for NADHcyt = 1.5
  - Malate-aspartate shuttle another possible entry point
• P/O ratio also differs depending on the ATP synthase present(different # of course subunits and # of H needed for a complete rotation to make 1 ATP)

Question 12

Water formation in oxidative phosphorylation

Answer 12

• In electron transport(Complex 4) : 1 H2O formed at last step in electron transfer
• By ATP synthase: 2.5 H2O made when 2.5 ATP made by NADH, 1.5 H2O made when 1.5 ATP made by FADH2
• Water ratio: 3.5 for NADH, 2.5 for FADH2

Question 13

Total ATP made from glycolysis, PDC, TCA, and oxidative phosphorylation

Answer 13

• 2 ATP, 2 NADHcyt(FADH2), 2 H2O made from glycolysis
• 2 NADH made from PDC
• 2 GTP, 6 NADH, 2 FADH2, -4 H2O made from TCA
• 8 NADH, 4 FADH used in ETC, 12 H2O made
• 26 ATP, 26 H2O made from ATP synthase
• Total 28 ATP made, 36 H2O made

Question 14

Overall summary equation for oxidation of glucose

Answer 14

Glucose + ADP + P + O2 -> 6 CO2 + 3 ATP + 36 H2O