explain that, when oxygen is available, pyruvate enters mitochondria to take part in the link reaction

Respiration – The Link Reaction (Aerobic Pathway)

Learning Objective – AO1 (Knowledge & Understanding)

When oxygen is available, the pyruvate produced in glycolysis is transported into the mitochondrial matrix where it undergoes the link reaction. This reaction converts each pyruvate into acetyl‑CoA, releases one molecule of CO₂ and produces NADH, thereby linking glycolysis to the Krebs cycle.

For each glucose molecule the link reaction occurs twice, giving:

  • 2 Acetyl‑CoA (entry substrate for the Krebs cycle)

  • 2 CO₂ (waste)

  • 2 NADH → ≈5 ATP after oxidative phosphorylation (≈2.5 ATP per NADH)

Position of the Link Reaction in the Four‑Stage Model (A‑Level Syllabus – Topic 12)

  • Stage 1 – Glycolysis (cytoplasm)

  • Stage 2 – Link Reaction (mitochondrial matrix) – connects glycolysis to the Krebs cycle

  • Stage 3 – Krebs Cycle (mitochondrial matrix)

  • Stage 4 – Oxidative Phosphorylation (inner mitochondrial membrane)

Overview of the Link Reaction (AO1)

The reaction is catalysed by the pyruvate dehydrogenase complex (PDC). For one molecule of pyruvate:

Pyruvate + CoA‑SH + NAD⁺ → Acetyl‑CoA + CO₂ + NADH + H⁺

Key Steps (AO1)

  1. Transport: Pyruvate crosses the outer and inner mitochondrial membranes via a specific pyruvate carrier.

  2. Decarboxylation & oxidation: The PDC (E1, E2, E3) removes one carbon as CO₂ and transfers the remaining two‑carbon fragment to CoA‑SH.

  3. Formation of acetyl‑CoA: The acetyl group binds to CoA‑SH, yielding acetyl‑CoA.

  4. Reduction of NAD⁺: Electrons released during oxidation reduce NAD⁺ to NADH, which later enters the electron‑transport chain.

Enzyme Complex & Cofactor Functions (AO2)

Component (E)CofactorPrimary Function
E1 – Pyruvate dehydrogenaseThiamine pyrophosphate (TPP)Forms a covalent “active aldehyde” with pyruvate; stabilises the carbanion formed during decarboxylation.
E2 – Dihydrolipoamide acetyl‑transferaseLipoic acid (as a swinging arm)Transfers the acetyl group from the TPP‑bound intermediate to CoA‑SH, producing acetyl‑CoA.
E3 – Dihydrolipoamide dehydrogenaseFlavin adenine dinucleotide (FAD)Re‑oxidises reduced lipoamide, passing the electrons to NAD⁺ (forming NADH) and regenerating the active lipoic acid.

Why Oxygen Is Essential (AO2)

  • Oxygen does not participate directly in the link reaction.

  • It is the final electron acceptor in the electron‑transport chain (ETC).

  • If oxygen is absent, NADH produced by the PDC cannot be oxidised back to NAD⁺ in the ETC.

  • Accumulation of NADH depletes the NAD⁺ pool, halting the PDC and therefore the link reaction.

Link to Overall ATP Yield (AO2)

The 2 NADH generated here each yield ≈2.5 ATP during oxidative phosphorylation. Combined with the ATP produced in the other stages, the complete aerobic breakdown of one glucose molecule gives roughly 30–32 ATP (≈5 ATP from the link reaction, ≈2 ATP from glycolysis, ≈10 ATP from the Krebs cycle and ≈15‑17 ATP from oxidative phosphorylation).

Practical Investigation (AO3 – Suggested Activity)

Investigating NADH Formation from Pyruvate

  1. Prepare a reaction mixture containing purified pyruvate dehydrogenase complex, CoA‑SH, NAD⁺ and a buffer at pH 7.4.

  2. Place the cuvette in a spectrophotometer set at 340 nm (absorbance maximum for NADH).

  3. Record the baseline absorbance (no pyruvate). Add a known amount of pyruvate and monitor the increase in absorbance over time.

  4. Calculate the rate of NADH production using Beer‑Lambert law (A = εcl, ε₃₄₀ ≈ 6.22 × 10³ M⁻¹ cm⁻¹).

  5. Repeat the assay under anaerobic conditions (e.g., nitrogen‑purged cuvette) to demonstrate the effect of lacking oxygen on NAD⁺ regeneration.

Learning points: The rise in absorbance confirms NAD⁺ reduction; the anaerobic trial shows that without downstream oxidation of NADH the reaction slows, illustrating the indirect dependence on oxygen.

Summary Table of Aerobic Respiration Stages (AO1)

StageLocationMain SubstrateMain Products (per glucose)Key Cofactors / Enzymes
GlycolysisCytosolGlucose2 Pyruvate, 2 ATP (net), 2 NADHHexokinase, phosphofructokinase, pyruvate kinase, etc.
Link ReactionMitochondrial matrixPyruvate2 Acetyl‑CoA, 2 CO₂, 2 NADH (≈5 ATP)Pyruvate dehydrogenase complex (E1‑E3), TPP, lipoic acid, FAD, NAD⁺, CoA‑SH
Krebs CycleMitochondrial matrixAcetyl‑CoA6 NADH, 2 FADH₂, 2 GTP (≈2 ATP), 4 CO₂Citrate synthase, isocitrate dehydrogenase, α‑ketoglutarate dehydrogenase, etc.
Oxidative PhosphorylationInner mitochondrial membraneNADH, FADH₂, ADP + Pᵢ≈30–32 ATP, H₂OComplexes I‑IV, ATP synthase, O₂ (final electron acceptor)

Suggested diagram: Flow of pyruvate from the cytosol into the mitochondrion, showing the pyruvate carrier, the pyruvate dehydrogenase complex, production of acetyl‑CoA, CO₂ and NADH, and the connection to the Krebs cycle.