State where each of the four stages in aerobic respiration occurs in eukaryotic cells: glycolysis in the cytoplasm, link reaction in the mitochondrial matrix, Krebs cycle in the mitochondrial matrix, oxidative phosphorylation on the inner membrane of

Respiration – Aerobic Pathway (Cambridge International AS & A Level Biology 9700)

Learning Objectives

• State the cellular location of each of the four stages of aerobic respiration in eukaryotic cells.
• Identify the main products generated at each stage (per molecule of glucose).
• Describe the key biochemical events and the principal enzyme complexes involved.
• Explain the role of ATP, substrate‑level phosphorylation and the respiratory quotient (RQ) in energy metabolism.

12.1 Energy – ATP, RQ and Substrate‑Linked Reactions

ATP – the universal energy carrier

• Contains two high‑energy phosphoanhydride bonds (‑P‑O‑P‑) that release ~‑30 kJ mol⁻¹ on hydrolysis.
• Highly soluble in water; the charged phosphate groups keep it in the cytosol and mitochondrial matrix.
• Hydrolysis to ADP + Pᵢ is rapid and irreversible under cellular conditions, providing a quick energy source.
• Regenerated by oxidative phosphorylation (most ATP) and by substrate‑level phosphorylation (small amounts in glycolysis and the Krebs cycle).

Substrate‑level phosphorylation

• Direct transfer of a phosphate group from a phosphorylated metabolic intermediate to ADP.
• Occurs in:

  • Glycolysis – net gain of 2 ATP per glucose (phosphoglycerate kinase and pyruvate kinase).
  • Krebs cycle – formation of 1 GTP per turn (succinyl‑CoA synthetase), which is readily converted to ATP.

Respiratory Quotient (RQ)

Definition: RQ = (moles of CO₂ produced) ÷ (moles of O₂ consumed) for the oxidation of a substrate.

Worked Example – Glucose

1. Overall oxidation: C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O
2. CO₂ produced = 6 mol; O₂ consumed = 6 mol.
3. RQ = 6 / 6 = 1.0.

12.2 Respiration – Location & Main Products of Each Stage

12.2.1 Glycolysis – Cytoplasm

• Overall reaction: C₆H₁₂O₆ + 2 NAD⁺ + 2 ADP + 2 Pᵢ → 2 pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O
• Key enzyme complexes (regulatory points):

  • Hexokinase (or glucokinase in liver) – phosphorylates glucose; inhibited by its product glucose‑6‑phosphate.
  • Phosphofructokinase‑1 (PFK‑1) – rate‑limiting step; activated by AMP and inhibited by ATP & citrate.
  • Pyruvate kinase – converts phosphoenolpyruvate to pyruvate; activated by fructose‑1,6‑bisphosphate (feed‑forward) and inhibited by ATP.

• Energy yield: substrate‑level phosphorylation gives a net gain of 2 ATP per glucose.
• NADH produced: 2 NADH are generated in the oxidation of glyceraldehyde‑3‑phosphate; they later feed electrons into the mitochondrial ETC (via the malate‑aspartate shuttle in most eukaryotes).

12.2.2 Link Reaction (Pyruvate Oxidation) – Mitochondrial Matrix

• Overall reaction (per pyruvate): Pyruvate + CoA‑SH + NAD⁺ → Acetyl‑CoA + CO₂ + NADH + H⁺
• Enzyme complex: Pyruvate dehydrogenase complex (PDC) – a multi‑enzyme assembly containing three core enzymes (E1: pyruvate dehydrogenase, E2: dihydrolipoamide acetyltransferase, E3: dihydrolipoamide dehydrogenase) and several cofactors (TPP, lipoic acid, FAD, NAD⁺, CoA‑SH).
• Each glucose yields 2 acetyl‑CoA, 2 CO₂ and 2 NADH in this stage.
• Regulation: PDC is activated by ADP, NAD⁺ and Ca²⁺ (muscle) and inhibited by acetyl‑CoA and NADH.

12.2.3 Krebs Cycle – Mitochondrial Matrix

• Overall per acetyl‑CoA: Acetyl‑CoA + 3 NAD⁺ + FAD + ADP + Pᵢ + 2 H₂O → 2 CO₂ + 3 NADH + FADH₂ + GTP + CoA‑SH
• Principal enzyme complexes (one turn of the cycle):

  • Citrate synthase – combines acetyl‑CoA with oxaloacetate to form citrate.
  • Aconitase – isomerises citrate to isocitrate.
  • Isocitrate dehydrogenase – oxidative decarboxylation to α‑ketoglutarate (produces NADH + CO₂).
  • α‑Ketoglutarate dehydrogenase complex – similar to PDC; yields succinyl‑CoA, NADH + CO₂.
  • Succinyl‑CoA synthetase – substrate‑level phosphorylation (GTP/ATP) from succinyl‑CoA to succinate.
  • Succinate dehydrogenase – oxidises succinate to fumarate (produces FADH₂); also part of Complex II of the ETC.
  • Fumarase – hydrates fumarate to malate.
  • Malate dehydrogenase – oxidises malate to oxaloacetate (produces NADH).

• Products per glucose (two turns): 6 NADH, 2 FADH₂, 2 GTP (≈ 2 ATP) and 4 CO₂.
• Regulation: NADH, ATP and succinyl‑CoA inhibit isocitrate dehydrogenase and α‑ketoglutarate dehydrogenase; ADP and Ca²⁺ activate them.

12.2.4 Oxidative Phosphorylation – Inner Mitochondrial Membrane (Cristae)

• Electron‑transport chain (ETC) complexes:

  • Complex I (NADH dehydrogenase): receives electrons from NADH, pumps protons (4 H⁺) from matrix to inter‑membrane space.
  • Complex II (Succinate dehydrogenase): receives electrons from FADH₂; does not pump protons.
  • Complex III (Cytochrome bc₁ complex): transfers electrons to cytochrome c and pumps 4 H⁺.
  • Complex IV (Cytochrome c oxidase): reduces O₂ to H₂O and pumps 2 H⁺.
  • Complex V (ATP synthase): uses the proton‑motive force to synthesise ATP from ADP + Pᵢ (chemiosmosis).

• Proton gradient: ~10 H⁺ are translocated per NADH and ~6 H⁺ per FADH₂; the resulting electrochemical gradient drives ATP synthesis.
• ATP yield (approximate, eukaryotic values):

  • ~2.5 ATP per NADH.
  • ~1.5 ATP per FADH₂.
  • Total from oxidative phosphorylation ≈ 34 ATP per glucose.

• Overall ATP accounting (per glucose):

  • Glycolysis – 2 ATP (substrate‑level) + 2 NADH → ~5 ATP.
  • Link reaction – 2 NADH → ~5 ATP.
  • Krebs cycle – 6 NADH + 2 FADH₂ + 2 GTP → ~20 ATP.
  • Grand total ≈ 34 ATP (plus the 4 ATP from substrate‑level steps) = ≈ 38 ATP in prokaryotes; ≈ 36–38 ATP in eukaryotes (accounting for transport costs).

Overall Equation for Aerobic Respiration of Glucose

C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O + ≈ 38 ATP

In eukaryotic cells the net yield is usually quoted as 36–38 ATP because two NADH produced in glycolysis must be shuttled into the mitochondrion (costing ~1 ATP each).