electron transport chain and oxidative phosphorylation

mitochondrion

• double membrane

  • outer membrane is permeable

  • inner membrane involved in electron transport and oxidative phosphorylation

• cristae: invaginations of the inner membrane to increase surface area

• intermembrane space

• matrix: contains pyruvate dehydrogenase complex and citric acid cycle

electron transport chain

• protons are pumped through complexes I, III, and IV as electrons flow through the complexes, generating an electrochemical gradient across the membrane (proton motive force)

• reentry of protons to the matrix through the F₀ channel of ATP synthase (complex V) provides the energy to drive ATP synthesis

protons pumped in electron transport chain

• complex I: 4 H⁺

• complex III: 4 H⁺

• complex IV: 2 H⁺

free energy change of NADH to O₂

• NADH + H⁺ + ½ O₂ → NAD⁺ + H₂O = -220 kJ/mol

• electrons will flow from low to high reduction potential

• energy harnessed and stored in electrochemical gradient

“OXPHOS wars”

• Peter Mitchell: chemi-osmotic theory

• Edward Slater: chemical coupling hypothesis

chemical coupling hypothesis for oxidative phosphorylation

ATP is synthesized from a high energy intermediate of the respiratory chain during oxidation (similar to GAPDH)

evidence for chemi-osmotic coupling

• respiratory chain can function in the absence of phosphate

  • don’t need to make ATP to consume oxygen

• number of moles of ATP generated through NADH oxidation is not an integer

  • not metabolite doing substrate-level phosphorylation

• intact inner mitochondrial membrane needed for OXPHOS

  • ATP cannot be made if a detergent is used to disrupt the membrane (H⁺ gradient can’t be produced)

• key electron transport proteins span the inner mitochondrial membrane

• uncouplers such as 2,4-dinitrophenol (DNP) inhibit ATP synthesis

  • collapse membrane potential

• generating artificial proton gradient permits ATP synthesis without electron transport

reasoning behind large number of redox reactions in oxidative phosphorylation

unlike combustion, where most energy is lost, many reactions allow energy to be harnessed and converted to stored form slowly

P/O ratio

molecules of ATP made per oxygen atom consumed

ATP synthase function

• each 360° rotation produces 3 ATP molecules

• 8 F₀/c subunits in mammals

• 8 H⁺ required per 360° turn + 3 H⁺ from import of P_i

• 11 H⁺ / 3 ATP = 3.7 H⁺/ATP

import of P_i to electron transport chain

since P_i is negatively charged, brought in with proton to remain neutral (no effect on membrane potential)

P/O ratio per NADH

• through complex I → 10 H⁺ pumped

• 10 H⁺/(3.7 H⁺/ATP) = ~2.5 ATP

P/O ratio per FADH₂

• skips complex I, through complex II → 6 H⁺ pumped

• 6 H⁺/(3.7 H⁺/ATP) = ~1.5 ATP

shuttling cytosolic NADH to mitochondria

• NADH doesn’t have a transporter

• must be shuttled into mitochondria using carrier metabolites

NADH shuttles

• dihydroxyacetone phosphate/glycerol-3-phosphate

• malate/aspartate

dihydroxyacetone phosphate/glycerol-3-phosphate NADH shuttle

1. reduction of DHAP by NADH in the cytosol

2. G3P crosses outer mitochondrial membrane

3. reoxidation of G3P and reduction of FAD by G3P dehydrogenase in inner mitochondrial membrane

4. transfer of an electron pair from FADH₂ to coenzyme Q (in complex III of ETC)

5. DHAP returns to cytosol

malate/aspartate NADH shuttle

1. reduction of oxaloacetate to malate by NADH

2. malate crosses both mitochondrial membranes

3. reoxidation of malate to oxaloacetate and reduction of NAD⁺

4. oxidation of NADH by complex I (of ETC)

5. transamination of oxaloacetate by glutamate into aspartate and α-ketoglutarate

6. α-ketoglutarate and aspartate cross both mitochondrial membranes

7. transamination of α-ketoglutarate and aspartate into oxaloacetate and glutamate

8. return of glutamate to mitochondrial matrix