Link Reaction and Krebs Cycle

Spec Mapping — OCR H420 Module 5.2.2 — Respiration, content statements covering the link reaction (pyruvate dehydrogenase complex; pyruvate → acetyl-CoA + CO₂ + NADH) and the Krebs cycle (citric acid cycle / TCA cycle) in the mitochondrial matrix, including the production of reduced NAD, reduced FAD, ATP and CO₂ per turn (refer to the official OCR H420 specification document for exact wording).

If glucose has been converted to pyruvate by glycolysis in the cytoplasm, the next stage is to transport the pyruvate into the mitochondrial matrix and oxidise it fully to CO₂. This happens in two linked pathways: the link reaction (pyruvate → acetyl CoA) and the Krebs cycle (also called the citric acid cycle or TCA cycle). OCR specification module 5.2.2 requires detailed knowledge of both. Together they release most of the CO₂ from aerobic respiration and produce the reduced NAD and reduced FAD that drive the bulk of ATP production in the next stage.

The cycle is named for the British-German biochemist Sir Hans Krebs, who proposed it in 1937 while working at the University of Sheffield. Krebs's paraphrased reasoning was inductive: by adding various organic acids (citrate, succinate, fumarate, malate) to minced pigeon-breast muscle, he observed that each accelerated O₂ consumption and CO₂ evolution, and that the conversions between them happened in a fixed order. Stitching the conversions together gave a cyclic pathway in which oxaloacetate was regenerated at the end and the net oxidation was of an acetyl unit. Krebs received the 1953 Nobel Prize in Physiology or Medicine for this work. The mitochondrial localisation was settled by Eugene Kennedy and Albert Lehninger (1948), who paraphrased: the enzymes of the Krebs cycle and the electron-transport chain co-fractionate with mitochondrial particles, not with the soluble cytoplasm.

Key Definitions:

• Link reaction — the reaction that "links" glycolysis to the Krebs cycle by converting pyruvate to acetyl CoA.
• Acetyl CoA — a 2-carbon acetyl group bound to coenzyme A; the substrate that enters the Krebs cycle.
• Krebs cycle — a cyclic series of reactions in the mitochondrial matrix that completely oxidises the acetyl group from acetyl CoA, producing CO₂, reduced NAD, reduced FAD and ATP.
• Decarboxylation — removal of a carboxyl group as CO₂.
• Dehydrogenation — removal of hydrogen (with electrons) to a coenzyme (NAD or FAD).

Learning objectives — by the end of this lesson you should be able to:

• Describe the link reaction (pyruvate → acetyl-CoA + CO₂ + reduced NAD) and explain the role of coenzyme A as a carrier.
• Describe the Krebs cycle, tracking carbon from acetyl-CoA through to the regeneration of oxaloacetate.
• State the products per turn (2 CO₂, 3 reduced NAD, 1 reduced FAD, 1 ATP) and per glucose (double).
• Explain that Krebs ATP is made by substrate-level phosphorylation, and that the cycle's main purpose is to supply reduced coenzymes to oxidative phosphorylation.
• Explain why the cycle is described as amphibolic and how it is regulated.

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The Krebs Cycle Is Amphibolic — Not Just an Energy Pathway

A frequent misconception is that the Krebs cycle exists solely to oxidise acetyl-CoA for ATP. In reality it is amphibolic — it operates simultaneously as a catabolic (breaking-down) and anabolic (building-up) pathway. Understanding this dual role is one of the clearest routes to AO3 marks on synoptic questions, because it positions the cycle as the true metabolic crossroads of the cell.

Because intermediates are constantly withdrawn for biosynthesis, the cycle would grind to a halt unless they were replenished. Two opposing classes of reaction keep the intermediate pool balanced:

• Cataplerotic reactions drain intermediates out of the cycle for biosynthesis. Citrate can be exported to the cytoplasm to build fatty acids; α-ketoglutarate and oxaloacetate are transaminated to the amino acids glutamate and aspartate; succinyl-CoA is diverted to make haem for haemoglobin and the cytochromes.
• Anaplerotic reactions ("filling-up" reactions) replenish intermediates. The most important is catalysed by pyruvate carboxylase, which fixes CO₂ onto pyruvate to make oxaloacetate:

$\text{pyruvate} + \text{CO}_2 + \text{ATP} \xrightarrow{\text{pyruvate carboxylase}} \text{oxaloacetate} + \text{ADP} + \text{P}_i$pyruvate+CO2​+ATPpyruvate carboxylase​oxaloacetate+ADP+Pi​

This reaction explains a subtle exam point: if oxaloacetate is withdrawn faster than it is regenerated, the cycle stalls, and even fat cannot be respired efficiently (because acetyl-CoA needs oxaloacetate to enter the cycle). This is why the classic aphorism "fats burn in the flame of carbohydrates" is true — carbohydrate metabolism supplies the oxaloacetate that keeps the cycle turning. In prolonged starvation, when oxaloacetate is diverted to gluconeogenesis, excess acetyl-CoA cannot enter the cycle and is instead converted to ketone bodies (ketosis).

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Regulation of the Cycle in Detail — Three Control Points

The brief "high ATP slows the cycle" statement can be sharpened into a genuine A-Level-depth account. The Krebs cycle is regulated at its three most thermodynamically irreversible, exergonic steps — the same logic used to regulate glycolysis at PFK:

• Citrate synthase (acetyl-CoA + OAA → citrate) is inhibited by ATP and by its own product citrate. When energy is plentiful, entry into the cycle slows.
• Isocitrate dehydrogenase (the rate-limiting step) is activated by ADP and inhibited by ATP and reduced NAD. A high reduced-NAD:NAD ratio signals that the electron transport chain cannot keep up, so the cycle slows to avoid over-reducing the coenzyme pool.
• α-Ketoglutarate dehydrogenase is inhibited by its products succinyl-CoA and reduced NAD.

The unifying principle is respiratory control: the cycle runs fast only when ADP is high and reduced coenzymes are being re-oxidised (i.e. when ATP is being used and oxygen is available). This couples the rate of the whole of aerobic respiration to cellular energy demand — the same feedback logic that governs PFK in glycolysis and, ultimately, oxidative phosphorylation itself.

A further layer of control operates in muscle: calcium ions (Ca²⁺), released to trigger muscle contraction, simultaneously activate isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. This is an elegant piece of physiological integration — the very signal that commands the muscle to contract (and therefore to consume ATP) also accelerates the Krebs cycle that will replenish it. It is a favourite synoptic link between the respiration module and muscle physiology, and demonstrates why regulation of metabolism cannot be understood in isolation from the demands of the whole organism.

Where amino acids feed in

Because the cycle is amphibolic, deaminated amino acids enter it at several defined points — a detail that turns a good answer into an excellent one on substrate-metabolism questions:

• Alanine → pyruvate (then to acetyl-CoA via the link reaction)
• Glutamate → α-ketoglutarate (a single transamination step)
• Aspartate → oxaloacetate (a single transamination step)
• Valine, isoleucine, methionine → succinyl-CoA

The reverse routes (cycle intermediate → amino acid) are the cataplerotic reactions described above. The same carbon skeletons therefore flow into the cycle when amino acids are respired and out of it when amino acids are synthesised — the essence of the amphibolic character.

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The Link Reaction

After glycolysis, pyruvate is actively transported into the mitochondrial matrix. Here, each pyruvate (3C) undergoes three steps:

flowchart LR
    PYR[Pyruvate - 3C] -->|Decarboxylation| C2[2C fragment + CO2]
    C2 -->|Dehydrogenation| NAD[Reduced NAD]
    C2 -->|CoA added| ACo[Acetyl CoA - 2C]

Step-by-step

1. Decarboxylation: pyruvate loses a CO₂ molecule. This leaves a 2-carbon acetyl group.
2. Dehydrogenation: the acetyl group is oxidised — a pair of hydrogens (with electrons) is removed and transferred to NAD, forming reduced NAD.
3. Addition of coenzyme A: the acetyl group is attached to coenzyme A, forming acetyl CoA.

Products Per Pyruvate

• 1 CO₂ (released as waste)
• 1 reduced NAD
• 1 acetyl CoA

Per Glucose (since each glucose produces 2 pyruvates)

• 2 CO₂
• 2 reduced NAD
• 2 acetyl CoA

No ATP is made directly in the link reaction — it is purely a preparation step.

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The Krebs Cycle

The Krebs cycle (named after Hans Krebs, who worked it out in 1937) takes the acetyl group from acetyl CoA and oxidises it completely to CO₂. It is called a "cycle" because the starting molecule is regenerated at the end, allowing it to accept another acetyl group.

flowchart TB
    ACo[Acetyl CoA - 2C] -->|+ Oxaloacetate 4C| CIT[Citrate - 6C]
    CIT -->|Decarboxylation + NAD reduced| C5[5C compound]
    C5 -->|Decarboxylation + NAD reduced + ATP made| C4a[4C compound]
    C4a -->|FAD reduced| C4b[4C intermediate]
    C4b -->|NAD reduced| OAA[Oxaloacetate - 4C]
    OAA --> CIT
    CIT -. 2 CO2 out .-> OUT1[CO2]
    C5 -. CO2 out .-> OUT1

Step-by-step (Per Turn = Per Acetyl CoA)

1. Condensation: acetyl CoA (2C) combines with oxaloacetate (4C) to form citrate (6C). Coenzyme A is released and recycled.
2. First decarboxylation and dehydrogenation: citrate is converted to a 5-carbon compound, releasing 1 CO₂ and reducing 1 NAD → 1 reduced NAD.
3. Second decarboxylation and dehydrogenation: the 5C compound is converted to a 4-carbon compound, releasing 1 more CO₂ and reducing another NAD → 1 reduced NAD. At the same time, 1 ATP is made by substrate-level phosphorylation.
4. Third dehydrogenation: a FAD molecule is reduced → 1 reduced FAD.
5. Fourth dehydrogenation: another NAD is reduced → 1 reduced NAD, regenerating oxaloacetate ready to accept another acetyl CoA.

Products Per Turn of the Krebs Cycle

Product	Amount
CO₂	2
Reduced NAD	3
Reduced FAD	1
ATP	1

Per Glucose (2 turns, because 2 acetyl CoAs from one glucose)

Product	Amount
CO₂	4
Reduced NAD	6
Reduced FAD	2
ATP	2

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Cumulative Tally So Far (Per Glucose)

Stage	ATP	Reduced NAD	Reduced FAD	CO₂
Glycolysis	2 (net)	2	0	0
Link reaction	0	2	0	2
Krebs cycle	2	6	2	4
Total	4	10	2	6

Six CO₂ is the total from one glucose — exactly what the summary equation predicts. The bulk of the energy, however, is now locked up in reduced NAD and reduced FAD, not in ATP directly. These reduced coenzymes will release their stored energy in the next stage: oxidative phosphorylation.

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Where Do The Carbons Go?

This is a favourite OCR question. Let's track all six carbons of the original glucose molecule:

• Glucose (6C) → 2 pyruvates (3C each) in glycolysis. No CO₂ released.
• 2 Pyruvates → 2 acetyl CoAs (2C each) + 2 CO₂ in link reaction.
• 2 Acetyl CoAs through Krebs → 4 CO₂ (2 per turn × 2 turns).

Total: 6 CO₂ per glucose, matching the summary equation exactly. Each carbon of glucose has been oxidised to CO₂.

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Where Does The Hydrogen Go?

The hydrogens from glucose are not lost as H₂O at this stage. They are transferred (together with their electrons) to NAD and FAD to become reduced NAD and reduced FAD. These reduced coenzymes will take the hydrogens (and their electrons) to the inner mitochondrial membrane, where they will enter the electron transport chain. Only at the very end, when the electrons are passed to oxygen, will the hydrogens combine with oxygen to form water.

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Why Is It A Cycle?

The Krebs cycle starts and ends with oxaloacetate. This means:

• Only a small amount of oxaloacetate is needed — it is continuously recycled.
• Each acetyl CoA that enters triggers one complete turn.
• The cycle can handle a continuous supply of acetyl CoA indefinitely, as long as oxaloacetate is regenerated each time.
• If oxaloacetate runs out (e.g. in starvation, when glucose is scarce), the cycle stops — which is why even fat metabolism (which produces acetyl CoA) requires some background carbohydrate metabolism.

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Regulation

The Krebs cycle is regulated by the cellular ATP:ADP ratio and the reduced NAD:NAD ratio:

• High ATP or high reduced NAD → cycle slows (plenty of energy already).
• Low ATP or low reduced NAD → cycle speeds up (more energy needed).

Regulation ensures that energy production is matched to demand and that substrates are not wasted.

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The Krebs Cycle as a Metabolic Hub

Krebs cycle intermediates are not just energy-producers — they are also starting points for the biosynthesis of many other molecules:

Intermediate	Used to make
α-ketoglutarate (5C)	Some amino acids (glutamate, glutamine)
Succinyl CoA	Haem (in haemoglobin)
Oxaloacetate	Aspartate and asparagine
Citrate	Fatty acids (can be exported as cytosolic acetyl CoA)

This makes the Krebs cycle a metabolic crossroads rather than just a "respiration pathway". OCR only occasionally mentions this, but it is an elegant point that can earn bonus marks in synoptic questions.

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Exam Tip

OCR loves to ask "Describe the link reaction and explain the role of coenzyme A." A model answer: "Pyruvate enters the mitochondrial matrix and is decarboxylated (losing CO₂) and dehydrogenated (reducing NAD to reduced NAD). The remaining 2-carbon acetyl group is attached to coenzyme A to form acetyl CoA. Coenzyme A acts as a carrier, transferring the acetyl group into the Krebs cycle where it is released and combined with oxaloacetate." Missing the role of CoA as a carrier is the most common lost mark.

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Common Exam Mistakes