Krebs Cycle (Citric Acid Cycle): 8 Steps, Enzymes and Yield (IB C1.2, NEET)

1 Introduction to the Krebs cycle

Syllabus link: IB Biology C1.2 Cell respiration (first assessment 2025; the link reaction and Krebs cycle are C1.2.11 and C1.2.12, Higher Level); NCERT Class XI Respiration in Plants (tricarboxylic acid cycle)

NEET = point NCERT and NEET stress   IB HL = detail the IB guide expects at Higher Level

Aerobic respiration releases energy from glucose in four stages: glycolysis in the cytoplasm, the link reaction, the Krebs cycle and the electron transport chain with oxidative phosphorylation. Glycolysis and the link reaction leave most of the energy of glucose still locked in the acetyl group of acetyl CoA. The Krebs cycle finishes the job of oxidation: it strips hydrogen (electrons) from the acetyl group, loads it onto the carriers NAD⁺ and FAD, and releases the carbon as CO₂.

The cycle is named after Hans Krebs, who worked it out in 1937 and shared the 1953 Nobel Prize in Physiology or Medicine. It is also called the citric acid cycle, because citric acid (citrate) is the first product, and the tricarboxylic acid (TCA) cycle, because citrate and isocitrate each carry three carboxyl groups. All three names describe the same pathway.

This chapter covers where the cycle runs, how acetyl CoA is made, each of the eight steps with its enzyme and products, the yield per acetyl CoA and per glucose, the amphibolic role, regulation, a comparison with glycolysis, the NCERT points that NEET tests and the traps examiners set. For the bigger picture, start from the photosynthesis and cell respiration hub.

2 Where does the Krebs cycle happen?

In eukaryotes the Krebs cycle takes place in the mitochondrial matrix, the fluid-filled space inside the inner mitochondrial membrane (see mitochondria among the cell organelles). The matrix holds the soluble Krebs enzymes, the coenzymes NAD⁺ and FAD, and a pool of CoA.

• Exception inside the mitochondrion: succinate dehydrogenase (step 6) is the only Krebs enzyme bound to the inner mitochondrial membrane. It is also complex II of the electron transport chain, so its FADH₂ passes electrons straight into the chain.
• Prokaryotes have no mitochondria. In aerobic bacteria the Krebs cycle runs in the cytoplasm and the electron transport chain sits in the plasma membrane, which fits the endosymbiotic origin of mitochondria from aerobic bacteria.
• Cells without mitochondria, such as mature mammalian red blood cells, cannot run the Krebs cycle at all and rely on glycolysis (see blood cells and circulation).
• The large internal volume of the matrix and the folded cristae of the inner membrane are adaptations of the mitochondrion for the Krebs cycle and the electron transport chain.

3 From pyruvate to acetyl CoA: the link reaction

Pyruvate (3C) made in glycolysis is carried into the matrix by membrane transport proteins. There, the link reaction (oxidative decarboxylation of pyruvate) is catalysed by the pyruvate dehydrogenase complex:

Pyruvate + CoA + NAD+ →(pyruvate dehydrogenase, Mg2+)→ Acetyl CoA + CO2 + NADH + H+

• One CO₂ is removed (decarboxylation) and hydrogen is removed and passed to NAD⁺ (oxidation).
• The remaining 2C acetyl group is attached to coenzyme A, giving acetyl CoA, which feeds the Krebs cycle.
• Per glucose the link reaction runs twice: 2 CO₂ and 2 NADH. These are not counted as Krebs cycle products.

IB HL The IB guide stresses that both carbohydrates and lipids are broken down to acetyl groups (2C), which coenzyme A transfers to the Krebs cycle. What happens to pyruvate when oxygen is absent, and more detail on the link reaction, is covered in fate of pyruvate after glycolysis.

4 The 8 steps of the Krebs cycle

The cycle has eight steps, each catalysed by a specific enzyme. Follow the carbon count: 2C + 4C → 6C → 6C → 5C → 4C, and the 4C compounds are then reshaped back into oxaloacetate.

NEET NEET often asks you to match enzymes with steps, or to pick the steps that release CO₂, reduce NAD⁺ or FAD, or make GTP. IB HL IB HL asks you to name only citrate (6C) and oxaloacetate (4C), and to know that the cycle includes two decarboxylations and four oxidations, and that the oxidations are dehydrogenation reactions.

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Step 1: formation of citrate (condensation)

The 2C acetyl group of acetyl CoA joins the 4C acceptor oxaloacetate to form the 6C compound citrate (citric acid). Water is used and coenzyme A is released, free to collect another acetyl group in the link reaction. The enzyme is citrate synthase. This is the step that gives the cycle its name.

Acetyl CoA + Oxaloacetate + H2O →(citrate synthase)→ Citrate + CoA

Step 2: isomerisation of citrate

Citrate is rearranged into its isomer isocitrate, moving the hydroxyl group so that it can be oxidised in the next step. The enzyme aconitase does this by removing and then adding back water, through the short-lived intermediate cis-aconitate. Carbon number stays at 6 and no carrier is reduced.

Citrate ↔(aconitase)↔ Isocitrate

Step 3: first oxidative decarboxylation

Isocitrate is oxidised and decarboxylated to the 5C compound α-ketoglutarate (α-ketoglutaric acid). Hydrogen is passed to NAD⁺, forming NADH, and the first CO₂ of the cycle is released. The enzyme is isocitrate dehydrogenase, an important control point. Older texts show oxalosuccinate as a bound intermediate in this step.

Isocitrate + NAD+ →(isocitrate dehydrogenase)→ α-Ketoglutarate + CO2 + NADH + H+

Step 4: second oxidative decarboxylation

α-Ketoglutarate is oxidised and decarboxylated by the α-ketoglutarate dehydrogenase complex, which works like the pyruvate dehydrogenase complex. The second CO₂ is released, a second NADH is formed and the 4C remainder is attached to CoA as succinyl CoA. After this step the cycle has released two carbons, as many as entered in acetyl CoA.

α-Ketoglutarate + CoA + NAD+ →(α-ketoglutarate dehydrogenase)→ Succinyl CoA + CO2 + NADH + H+

Step 5: substrate-level phosphorylation

The high-energy thioester bond of succinyl CoA is broken by succinyl CoA synthetase (succinate thiokinase), forming succinate (succinic acid) and releasing CoA. The energy is used to make GTP from GDP and Pi in animal cells; the GTP then transfers its phosphate to ADP to give ATP. Plant mitochondria make ATP directly. This is the only step of the cycle that makes ATP directly, by substrate-level phosphorylation.

Succinyl CoA + GDP + Pi →(succinyl CoA synthetase)→ Succinate + GTP + CoA; GTP + ADP → GDP + ATP

Step 6: oxidation of succinate

Succinate is oxidised to fumarate (fumaric acid) by removal of two hydrogen atoms. The hydrogen acceptor here is FAD, not NAD⁺, because the energy change is too small to reduce NAD⁺; FAD is reduced to FADH₂. The enzyme, succinate dehydrogenase, is bound to the inner mitochondrial membrane. Malonate, which resembles succinate, is the classic competitive inhibitor of this enzyme.

Succinate + FAD →(succinate dehydrogenase)→ Fumarate + FADH2

Step 7: hydration of fumarate

Water is added across the double bond of fumarate to form malate (malic acid). The enzyme is fumarase (fumarate hydratase). This is the second step of the cycle that uses water.

Fumarate + H2O ↔(fumarase)↔ Malate

Step 8: regeneration of oxaloacetate

Malate is oxidised to oxaloacetate (oxaloacetic acid, OAA), reducing the third NAD⁺ of the turn to NADH. The enzyme is malate dehydrogenase. Oxaloacetate is now ready to accept another acetyl group, so the cycle can turn again. Because oxaloacetate is regenerated, a small amount can process a large number of acetyl groups.

Malate + NAD+ ↔(malate dehydrogenase)↔ Oxaloacetate + NADH + H+

5 Krebs cycle yield per acetyl CoA and per glucose

One glucose gives two pyruvate, so two acetyl CoA, so the Krebs cycle turns twice per glucose. Double every per-turn figure.

Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → 2 CO2 + 3 NADH + FADH2 + GTP + 2 H+ + CoA

Per pyruvate (link reaction plus one turn), NCERT gives: pyruvic acid + 4 NAD⁺ + FAD + 2 H₂O + ADP + Pi → 3 CO₂ + 4 NADH + 4 H⁺ + FADH₂ + ATP. All six carbons of glucose leave as CO₂ by the end of the cycle: 2 in the link reaction and 4 in the Krebs cycle. The CO₂ diffuses out of the cell and is carried in the blood to the lungs (see how carbon dioxide is transported).

How much ATP does that make?

The cycle makes only 1 ATP per turn directly. Most of its value lies in the NADH and FADH₂, which are reoxidised in the electron transport chain to make ATP by oxidative phosphorylation.

For the glycolysis side of the balance sheet see the net ATP yield of glycolysis.

6 Amphibolic nature of the Krebs cycle

A pathway that is both catabolic (breaking down) and anabolic (building up) is called amphibolic. The Krebs cycle is the hub of metabolism: many substrates are broken down into its intermediates, and its intermediates are drawn off to make new molecules.

Catabolic role: feeding the cycle

• Carbohydrates: glucose → pyruvate → acetyl CoA.
• Fats: fatty acids are broken down to acetyl CoA (by β-oxidation); glycerol enters glycolysis as PGAL (glyceraldehyde 3-phosphate).
• Proteins: amino acids are deaminated and their carbon skeletons enter as pyruvate, acetyl CoA or Krebs intermediates such as α-ketoglutarate and oxaloacetate. The removed amino groups become urea (see nitrogenous excretory products).

These substrates come from the absorption of digested food or from stored reserves.

Anabolic role: drawing intermediates off

• α-Ketoglutarate → glutamate and other amino acids.
• Oxaloacetate → aspartate, and → glucose by gluconeogenesis in the liver (stimulated by glucagon when blood glucose falls).
• Succinyl CoA → porphyrins, the ring of haem and of chlorophyll.
• Citrate is exported to the cytoplasm, where it gives acetyl CoA for fatty acid and sterol synthesis.

Withdrawing intermediates would stop the cycle, so they are topped up by anaplerotic (filling-up) reactions, mainly pyruvate carboxylase (pyruvate + CO₂ → oxaloacetate) in animals and PEP carboxylase in plants. NEET NCERT concludes that the respiratory pathway is better described as amphibolic than catabolic, because fatty acids are broken down to acetyl CoA for respiration but acetyl CoA is also withdrawn to make fatty acids.

7 Regulation of the Krebs cycle

The cycle runs only as fast as the cell needs ATP. Control works through the ratio of energy molecules and through a few irreversible steps.

• Supply of NAD⁺ and FAD: the dehydrogenases need oxidised carriers, which are regenerated only when the electron transport chain passes electrons to oxygen. Without oxygen the carriers stay reduced and the cycle stops. This is why the Krebs cycle is aerobic even though no step uses O₂.
• Citrate synthase is inhibited by ATP, NADH, succinyl CoA and its own product citrate.
• Isocitrate dehydrogenase is activated by ADP and Ca²⁺ and inhibited by ATP and NADH. It is usually described as the main rate-limiting step.
• α-Ketoglutarate dehydrogenase is inhibited by its products succinyl CoA and NADH and activated by Ca²⁺.
• Pyruvate dehydrogenase (link reaction) controls entry of carbohydrate carbon: it is inhibited by acetyl CoA, NADH and ATP.

For a deeper, university-level account of each enzyme and how it is controlled, see the StatPearls review of citric acid cycle biochemistry on NCBI Bookshelf.

When ATP is plentiful, citrate builds up, leaves the mitochondrion and inhibits phosphofructokinase, slowing glycolysis as well (see regulation of glycolysis by phosphofructokinase). These are examples of end-product (feedback) inhibition, which you meet in enzymes (IB C1.1). Ca²⁺ released during muscle contraction speeds the cycle up when demand rises.

8 Krebs cycle compared with glycolysis

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Glucose can also be oxidised by a side route, the pentose phosphate pathway, which makes NADPH and 5C sugars rather than ATP. For a step-by-step comparison with all four stages, see the comparison of respiration stages in the glycolysis chapter.

9 NEET favourite points (NCERT)

Also revise the mitochondrion itself from Cell: The Unit of Life, because NEET likes to ask that the matrix is the site of the TCA cycle and the inner membrane the site of the electron transport chain.

10 Common exam traps

11 Key terms

12 Common doubts about the Krebs cycle

These are the questions students ask us most often about the Krebs cycle.

Where does the Krebs cycle take place?

In eukaryotes the Krebs cycle takes place in the mitochondrial matrix; only succinate dehydrogenase is bound to the inner mitochondrial membrane. In aerobic prokaryotes, which have no mitochondria, it takes place in the cytoplasm.

Does the Krebs cycle need oxygen?

No step of the cycle uses oxygen directly, but it is part of aerobic respiration. Oxygen is the final electron acceptor of the electron transport chain, which regenerates the NAD⁺ and FAD the cycle needs. Without oxygen the carriers stay reduced and the cycle stops.

How many ATP are produced in the Krebs cycle?

Directly, 1 ATP (via GTP) per turn and 2 per glucose, by substrate level phosphorylation. Counting the NADH and FADH₂ at the NCERT values of 3 and 2 ATP, the cycle is worth 12 ATP per turn and 24 per glucose. Modern values give about 10 per turn.

How many CO2, NADH and FADH2 are produced per glucose?

The Krebs cycle turns twice per glucose and gives 4 CO₂, 6 NADH and 2 FADH₂. The link reaction adds a further 2 CO₂ and 2 NADH, which are not part of the cycle.

Why is it called the citric acid cycle or TCA cycle?

Citric acid is the first product formed when the acetyl group joins oxaloacetic acid. Citric acid and isocitric acid have three carboxyl groups, so the pathway is also called the tricarboxylic acid or TCA cycle. Krebs cycle honours Hans Krebs, who discovered it.

Why is the Krebs cycle called a cycle?

Oxaloacetate, which accepts the acetyl group in step 1, is regenerated in step 8, so the same sequence of reactions can repeat. The carbon atoms that enter as acetyl groups leave as CO₂.

Why is the Krebs cycle described as amphibolic?

It breaks down acetyl groups from carbohydrates, fats and proteins, which is catabolism, and its intermediates are also used to make amino acids, glucose, haem and fatty acids, which is anabolism. A pathway with both roles is amphibolic.

Which step of the Krebs cycle makes FADH2?

Step 6, the oxidation of succinate to fumarate by succinate dehydrogenase. It is the only step that uses FAD instead of NAD⁺ as the hydrogen acceptor.

13 Practice questions

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