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Following glycolysis, pyruvate enters the mitochondria where it is further oxidised in the link reaction and the Krebs cycle. These stages generate reduced coenzymes (reduced NAD and reduced FAD) and a small amount of ATP, while releasing CO₂ as a waste product. This lesson covers both stages in detail for the Edexcel A-Level Biology (9BI0) specification.
Before examining the biochemistry, it is essential to understand the mitochondrial structure, as this directly relates to function.
| Structure | Description | Function |
|---|---|---|
| Outer membrane | Smooth, contains porins | Permeable to small molecules; defines the organelle |
| Inner membrane | Highly folded into cristae | Site of the electron transport chain and ATP synthase |
| Intermembrane space | Narrow space between membranes | Accumulates H⁺ ions for chemiosmosis |
| Matrix | Gel-like interior | Contains enzymes for the link reaction and Krebs cycle, mitochondrial DNA, ribosomes |
| Cristae | Folds of the inner membrane | Increase surface area for oxidative phosphorylation |
Exam Tip: The matrix is the site of the link reaction and Krebs cycle. The inner membrane (cristae) is the site of oxidative phosphorylation. Always specify the correct location in your answers.
The link reaction connects glycolysis (in the cytoplasm) to the Krebs cycle (in the mitochondrial matrix). It occurs in the mitochondrial matrix.
For each molecule of pyruvate:
Pyruvate (3C) + NAD⁺ + CoA → Acetyl CoA (2C) + CO₂ + Reduced NAD
The enzyme that catalyses this reaction is the pyruvate dehydrogenase complex, a large multi-enzyme complex.
Since glycolysis produces 2 pyruvate per glucose, the link reaction occurs twice per glucose:
| Product | Per pyruvate | Per glucose |
|---|---|---|
| Acetyl CoA | 1 | 2 |
| CO₂ | 1 | 2 |
| Reduced NAD | 1 | 2 |
Exam Tip: Remember that all quantities from the link reaction onwards must be doubled per glucose molecule, because glycolysis produces two pyruvate molecules.
The Krebs cycle was described by Sir Hans Krebs in 1937. It is a cyclic series of enzyme-controlled reactions that takes place in the mitochondrial matrix. The primary function is to oxidise the acetyl group from acetyl CoA, generating reduced coenzymes for oxidative phosphorylation.
| Step | Reaction | Key molecules |
|---|---|---|
| 1 | Acetyl CoA (2C) + Oxaloacetate (4C) → Citrate (6C) | CoA is released and recycled. Catalysed by citrate synthase. |
| 2 | Citrate (6C) → 5C compound | Decarboxylation releases 1 CO₂. Oxidation produces 1 reduced NAD. |
| 3 | 5C compound → 4C compound | Decarboxylation releases 1 CO₂. Oxidation produces 1 reduced NAD. |
| 4 | 4C compound → another 4C compound | Substrate-level phosphorylation produces 1 ATP (via GTP in some organisms). |
| 5 | 4C compound → another 4C compound | Oxidation produces 1 reduced FAD. |
| 6 | 4C compound → Oxaloacetate (4C) | Oxidation produces 1 reduced NAD. The cycle is ready to begin again. |
| Product | Amount per turn |
|---|---|
| CO₂ | 2 |
| Reduced NAD | 3 |
| Reduced FAD | 1 |
| ATP (via substrate-level phosphorylation) | 1 |
| Product | Per glucose |
|---|---|
| CO₂ | 4 |
| Reduced NAD | 6 |
| Reduced FAD | 2 |
| ATP | 2 |
The following diagram summarises the main steps of one turn of the Krebs cycle:
graph TD
A["Acetyl CoA<br/>(2C)"] -->|"Combines with<br/>Oxaloacetate (4C)"| B["Citrate<br/>(6C)"]
B -->|"Decarboxylation<br/>+ Dehydrogenation"| C["5C compound"]
C -->|"CO₂ released<br/>NAD → NADH"| D["4C compound"]
D -->|"FAD → FADH₂<br/>ATP produced"| E["Oxaloacetate<br/>(4C)"]
E --> A
These are hydrogen carriers (coenzymes) that are essential for transferring electrons and protons to the electron transport chain.
| Coenzyme | Reduced form | Where it donates H⁺/e⁻ |
|---|---|---|
| NAD⁺ | Reduced NAD (NADH + H⁺) | Complex I of the ETC |
| FAD | Reduced FAD (FADH₂) | Complex II of the ETC |
The oxidation of substrates in the Krebs cycle is coupled with the reduction of these coenzymes. When reduced NAD and reduced FAD donate their hydrogen atoms to the ETC, they are re-oxidised, regenerating NAD⁺ and FAD for reuse.
Exam Tip: Coenzymes are not consumed in the reaction — they are recycled. Their role is to act as carriers. Reduced NAD is the most important product of the Krebs cycle because it feeds the electron transport chain, which produces the majority of ATP.
Two key types of reaction occur repeatedly in the link reaction and Krebs cycle:
| Reaction type | What happens | Why it matters |
|---|---|---|
| Decarboxylation | A carboxyl group is removed as CO₂ | Accounts for the CO₂ exhaled in respiration |
| Oxidation | Hydrogen atoms (H⁺ + e⁻) are removed and transferred to NAD⁺ or FAD | Produces reduced coenzymes for the ETC |
In total, from one glucose molecule, 6 CO₂ molecules are produced:
This accounts for all 6 carbons in the original glucose molecule.
| Stage | ATP | Reduced NAD | Reduced FAD | CO₂ |
|---|---|---|---|---|
| Glycolysis | 2 (net) | 2 | 0 | 0 |
| Link reaction (×2) | 0 | 2 | 0 | 2 |
| Krebs cycle (×2) | 2 | 6 | 2 | 4 |
| Total (before ETC) | 4 | 10 | 2 | 6 |
The 10 reduced NAD and 2 reduced FAD molecules carry their hydrogen atoms to oxidative phosphorylation, where the bulk of ATP is produced.
The Krebs cycle is regulated at several points to match the cell's energy demands:
| Regulatory point | Effector | Effect |
|---|---|---|
| Citrate synthase | ATP, citrate (inhibitors) | Slows the cycle when energy is abundant |
| Isocitrate dehydrogenase | ADP (activator), ATP (inhibitor) | Key control point; responds to ATP/ADP ratio |
| α-ketoglutarate dehydrogenase | Reduced NAD (inhibitor) | Slows when reduced NAD accumulates |
Exam Tip: The Krebs cycle does not directly require oxygen. However, it stops in anaerobic conditions because NAD⁺ and FAD are not regenerated (the ETC requires O₂ as the final electron acceptor). Without NAD⁺ and FAD, the oxidation reactions in the cycle cannot proceed.
| Term | Definition |
|---|---|
| Link reaction | The conversion of pyruvate to acetyl CoA with the release of CO₂ and production of reduced NAD |
| Acetyl CoA | A 2-carbon compound attached to coenzyme A; enters the Krebs cycle |
| Krebs cycle | A cyclic series of reactions in the mitochondrial matrix that oxidises acetyl CoA, producing CO₂, reduced NAD, reduced FAD and ATP |
| Oxaloacetate | The 4-carbon molecule that accepts the acetyl group to form citrate |
| Decarboxylation | The removal of a carbon atom as CO₂ from a molecule |
| Oxidation | The removal of hydrogen atoms (or electrons) from a molecule |
This material sits in Edexcel 9BI0 Topic 5 (On the Wild Side — Photosynthesis, Energy and Ecosystems) and concerns the mitochondrial entry pathway of glucose-derived carbon: the link reaction (oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex) and the Krebs cycle, which together strip carbon as CO2 and load reducing power onto NAD+ and FAD. Synoptic links run backwards to lesson 4 (glycolysis) — supplier of pyruvate, with PFK reinforced by Krebs-exported citrate — and forwards to lesson 6 (oxidative phosphorylation) — re-oxidising the reduced NAD and reduced FAD produced here. Links also reach Topic 1 (biological molecules) for acetyl-CoA, citrate, oxaloacetate, the cofactor lineage of pantothenic acid → CoA and thiamine pyrophosphate as a PDH cofactor; and Topic 8 (mitochondrial genetics and disease) for inherited disorders of PDH, succinate dehydrogenase and other Krebs enzymes. Refer to the official Pearson Edexcel 9BI0 specification document for exact wording.
Question (8 marks):
A respiring liver cell is consuming glucose at steady state with adequate oxygen. Trace the carbon, ATP, reduced-NAD and reduced-FAD balance through the link reaction and the Krebs cycle for one glucose molecule.
(a) State precisely how many times the link reaction occurs per glucose, and account for the CO2, reduced NAD and acetyl-CoA produced. (2)
(b) State precisely how many times the Krebs cycle turns per glucose, and account for the CO2, reduced NAD, reduced FAD and ATP produced. (3)
(c) Combine glycolysis, link and Krebs to give the total ATP made by substrate-level phosphorylation per glucose, and the total CO2 released, accounting for the carbons in the original glucose. (3)
Solution with mark scheme:
(a) M1 (AO1) — Glycolysis produces 2 pyruvate per glucose, so the link reaction runs twice. PDH decarboxylates each pyruvate: 1 C leaves as CO2, H is donated to NAD+ (reduced NAD), and the 2C acetyl group is loaded onto CoA as acetyl-CoA.
A1 (AO2) — Per glucose: 2 acetyl-CoA, 2 reduced NAD, 2 CO2. No ATP directly.
(b) M1 (AO1) — Each acetyl-CoA drives one turn, so the cycle runs twice per glucose. Per turn: acetyl-CoA + oxaloacetate (4C) → citrate (6C); two oxidative decarboxylations release 2 CO2 and 2 reduced NAD; substrate-level phosphorylation at succinyl-CoA → succinate yields 1 ATP; SDH produces 1 reduced FAD; malate dehydrogenase regenerates oxaloacetate and produces a third reduced NAD.
M1 (AO1) — Per turn: 2 CO2, 3 reduced NAD, 1 reduced FAD, 1 ATP.
A1 (AO2) — Per glucose (2 turns): 4 CO2, 6 reduced NAD, 2 reduced FAD, 2 ATP.
(c) M1 (AO2) — Substrate-level ATP per glucose: 2 (glycolysis net) + 0 (link) + 2 (Krebs) = 4 ATP. The bulk (~26+ ATP) arrives downstream at oxidative phosphorylation.
M1 (AO2) — CO2 per glucose: 0 (glycolysis) + 2 (link) + 4 (Krebs) = 6 CO2, matching the 6 C of glucose. Carbon is conserved.
A1 (AO3) — All 6 glucose C leave as CO2 before the ETC; oxidative phosphorylation captures only the electrons. Krebs is the carbon-stripping stage; oxidative phosphorylation is the energy-capture stage. (Total: 8 marks; M5 A3.)
Question (6 marks): A patient with a partial deficiency of the pyruvate dehydrogenase (PDH) complex presents with elevated blood lactate, neurological symptoms and exercise intolerance. Glycolysis runs normally; oxygen supply and the electron transport chain are unaffected.
Use your knowledge of the link reaction and the Krebs cycle to explain why PDH deficiency raises blood lactate and limits ATP yield, and identify which downstream stages of respiration are starved of substrate.
Mark scheme decomposition by AO:
| Mark | AO | Earned by |
|---|---|---|
| 1 | AO1.1 | Stating that PDH catalyses the link reaction — the oxidative decarboxylation of pyruvate to acetyl-CoA in the mitochondrial matrix |
| 2 | AO2.1 | Recognising that PDH deficiency causes pyruvate to accumulate in the cytoplasm because mitochondrial entry of carbon is blocked |
| 3 | AO2.2 | Explaining that accumulated pyruvate is reduced to lactate by lactate dehydrogenase (regenerating NAD+ for glycolysis), raising blood lactate |
| 4 | AO3.1 | Identifying that without acetyl-CoA, the Krebs cycle is starved of substrate — citrate synthase has no acetyl input — so cycle flux falls |
| 5 | AO3.2 | Recognising that the 2 ATP, 6 reduced NAD and 2 reduced FAD per glucose normally produced by Krebs are lost, and oxidative phosphorylation downstream is consequently starved of reduced coenzyme input |
| 6 | AO3.3 | Concluding that ATP yield collapses from ~30+ to 2 per glucose (glycolysis only) — explaining the exercise intolerance — while the brain (highly aerobic, mitochondria-dependent) shows the most severe symptoms |
Total: 6 marks (UMS-band-anchored at A; AO1 = 1, AO2 = 2, AO3 = 3). This question structure mirrors Edexcel's preference for applying a core pathway to a clinical or whole-organism context (mitochondrial disease, exercise intolerance, anaerobic shift) and for tracking how a single enzymatic block propagates through downstream stages.
Lesson 4 (glycolysis) — pyruvate is the input. The link reaction depends on glycolysis for pyruvate; mitochondrial entry uses the mitochondrial pyruvate carrier (MPC), an inner-membrane symporter co-importing H+. Without pyruvate, no acetyl-CoA is produced and Krebs flux collapses.
Lesson 6 (oxidative phosphorylation) — reduced NAD and reduced FAD are the outputs. Per glucose, link + Krebs produce 8 reduced NAD (2 link + 6 Krebs) and 2 reduced FAD (Krebs only). These feed Complex I (~2.5 ATP each) and Complex II (~1.5 ATP each) respectively. Succinate dehydrogenase is itself Complex II of the ETC — uniquely both a Krebs enzyme and an ETC complex, embedded in the inner membrane while all other Krebs enzymes are matrix-soluble.
Topic 1 (biological molecules) — cofactor structures. Coenzyme A is built from pantothenic acid (vitamin B5) plus a thiol (-SH) group that forms the high-energy thioester bond with the acetyl group — what makes acetyl-CoA energy-rich enough to drive citrate formation. Thiamine pyrophosphate (TPP) (vitamin B1) is the essential cofactor for the PDH E1 subunit, which is why thiamine deficiency (beriberi, Wernicke–Korsakoff syndrome) causes lactic acidosis: PDH stalls, pyruvate accumulates, lactate rises.
Amphibolic role — Krebs intermediates feed biosynthesis. The cycle is amphibolic: α-ketoglutarate is the carbon skeleton for glutamate (and via transamination, other amino acids); oxaloacetate is the precursor of aspartate and (via PEP carboxykinase) of gluconeogenesis; cytoplasmic citrate is cleaved to acetyl-CoA + oxaloacetate to fuel fatty acid synthesis. Removed intermediates must be replenished by anaplerotic reactions — chiefly pyruvate carboxylase (pyruvate + CO2 + ATP → oxaloacetate).
Allosteric regulation — the PDH checkpoint. PDH is the committed step for glucose-derived carbon entering oxidation. It is allosterically inhibited by high acetyl-CoA, NADH and ATP (end-product inhibition), and covalently regulated by reversible phosphorylation: PDH kinase inactivates (when ATP is high); PDH phosphatase reactivates (when Ca2+ rises, e.g. in contracting muscle). PDH thus integrates fast (allosteric) and slow (covalent) energy signals.
Topic 8 (mitochondrial disease) — Krebs enzyme mutations. SDH-subunit mutations cause familial paraganglioma/phaeochromocytoma: succinate accumulates, inhibits prolyl hydroxylases, and stabilises HIF-1α, driving a pseudohypoxic transcriptional response and tumourigenesis. PDH-complex mutations more commonly cause congenital lactic acidosis with neurological involvement.
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