explain that during oxidative phosphorylation: hydrogen atoms split into protons and energetic electrons, energetic electrons release energy as they pass through the electron transport chain (details of carriers are not expected), the released energy

Oxidative Phosphorylation (Stage 5 of Aerobic Respiration)

1. Where does it fit in the whole respiration pathway?

2. Quick‑reference box – ATP yield

3. The five key steps

1. Splitting of hydrogen atoms

   • Reduced co‑enzymes (NADH, FADH₂) each carry two hydrogen atoms.
   • When they donate their electrons, each hydrogen atom is separated into a proton (H⁺) and an energetic electron (e⁻).
     

     Example: NADH → NAD⁺ + H⁺ + 2 e⁻

2. Electron transport chain (ETC)

   • Electrons pass “downhill” through a series of membrane‑embedded carriers (Complex I–IV, ubiquinone, cytochrome c).
     

     Note: The Cambridge exam does not require naming each carrier – a generic description is sufficient.

   • As electrons move, they lose free energy because they move to carriers of lower redox potential.

3. Proton pumping & chemiosmotic gradient

   • The energy released in step 2 powers complexes I, III and IV to pump protons from the matrix into the inter‑membrane space.
   • This creates an electrochemical gradient (the proton‑motive force) across the inner membrane – the core of Peter Mitchell’s chemiosmotic hypothesis.

4. ATP synthesis (oxidative phosphorylation)

   • Protons flow back into the matrix through the enzyme ATP synthase (Complex V) by facilitated diffusion.
   • The flow drives conformational changes in ATP synthase that phosphorylate ADP + Pᵢ → ATP.

5. Final electron acceptor – oxygen

   • At Complex IV, electrons combine with molecular oxygen (O₂) and the protons that have been pumped back to form water (H₂O).
   • O₂ prevents a backup of electrons in the chain; without it the ETC would stop and ATP synthesis would cease.

4. Overall simplified reaction

NADH + H⁺ + ½ O₂ → NAD⁺ + H₂O + energy for ATP synthesis

5. Experimental evidence supporting the mechanism (AO3)

• Uncouplers (e.g., dinitrophenol) – carry protons across the membrane, collapsing the gradient. ATP production falls dramatically while heat production rises, confirming that the gradient drives ATP synthesis.
• Inhibitors:

  • Rotenone blocks Complex I → no NADH‑derived electrons → reduced ATP yield.
  • Cyanide or azide block Complex IV → electrons cannot be passed to O₂ → chain backs up, ATP synthesis stops.
  • Oligomycin blocks ATP synthase → protons cannot re‑enter the matrix → gradient builds up, electron flow slows, demonstrating the coupling of the gradient to ATP formation.

6. Practical skill linked to Paper 3 (AO3)

Design a simple investigation to test the effect of a respiratory inhibitor on mitochondrial ATP production:

1. Isolate mitochondria from fresh liver tissue (or use a commercial preparation).
2. Divide the suspension into two cuvettes – one control, one with a known concentration of cyanide.
3. Supply a substrate that generates NADH (e.g., pyruvate + NAD⁺) and measure ATP formed over time using a luciferase‑based luminescence assay.
4. Compare the rate of ATP synthesis between control and inhibited samples; a marked reduction confirms the role of the ETC and oxygen as the final electron acceptor.

7. Exam‑style practice question (AO2)

Question: Explain why molecular oxygen is essential for oxidative phosphorylation.

Answer outline (3‑4 marks):

• O₂ acts as the final electron acceptor at Complex IV.
• It combines with electrons and protons to form H₂O, removing electrons from the chain.
• Removal of electrons allows the ETC to continue flowing “downhill”, keeping the proton pumps active.
• Without O₂ the chain backs up, proton pumping stops, the proton‑motive force collapses, and ATP synthase can no longer produce ATP.

8. Summary table