The Krebs Cycle

In 1937, the German-born biochemist Hans Krebs, working in Sheffield, England, pieced together a remarkable pathway: a circle of reactions in which a two-carbon fragment from food is oxidised completely to carbon dioxide, while a four-carbon molecule is regenerated at the end of every turn to accept the next fragment. The Krebs cycle, also called the citric acid cycle or tricarboxylic acid (TCA) cycle, is the central hub of metabolism. It earned Krebs a share of the 1953 Nobel Prize in Physiology or Medicine.

Where it happens and what goes in

The cycle runs in the matrix of mitochondria (in bacteria, in the cytoplasm). Its fuel is acetyl-CoA: a two-carbon acetyl group attached to coenzyme A by a high-energy thioester bond (see cofactors and coenzymes).

Acetyl-CoA comes from:

• carbohydrates, via glycolysis and the link reaction;
• fatty acids, via β-oxidation;
• some amino acids.

The link reaction (before the cycle)

Pyruvate from glycolysis is converted to acetyl-CoA by the pyruvate dehydrogenase complex:

pyruvate (3C) + CoA + NAD⁺ → acetyl-CoA (2C) + CO₂ + NADH

This is an oxidative decarboxylation: one carbon is removed as CO₂, and the remaining two-carbon unit is oxidised, passing electrons to NAD⁺.

The cycle, step by step

Carbon counts are shown in brackets.

1. Acetyl-CoA (2C) + oxaloacetate (4C) → citrate (6C)

• Enzyme: citrate synthase
• The acetyl group joins oxaloacetate in an aldol-type condensation, and CoA is released. Hydrolysis of the thioester drives the reaction forward.

2. Citrate (6C) → isocitrate (6C)

• Enzyme: aconitase
• The –OH group moves to a neighbouring carbon (via removal and re-addition of water), setting up the molecule for oxidation.

3. Isocitrate (6C) → α-ketoglutarate (5C) + CO₂

• Enzyme: isocitrate dehydrogenase
• Oxidative decarboxylation: the –OH is oxidised to a ketone, and a carboxyl group leaves as CO₂.
• NAD⁺ → NADH
• This is a key control point of the cycle.

4. α-Ketoglutarate (5C) → succinyl-CoA (4C) + CO₂

• Enzyme: α-ketoglutarate dehydrogenase complex (chemically very similar to pyruvate dehydrogenase)
• A second oxidative decarboxylation: another CO₂ leaves, and the product is attached to CoA through a high-energy thioester.
• NAD⁺ → NADH

At this point, two carbons have left as CO₂, matching the two carbons that entered as acetyl. (Isotope-labelling experiments show that the specific carbon atoms lost in the first turn aren’t the ones that just came in, but the carbon count balances.)

5. Succinyl-CoA (4C) → succinate (4C)

• Enzyme: succinyl-CoA synthetase
• The energy of the thioester bond is used to make GTP (or ATP, depending on the tissue) from GDP and phosphate: substrate-level phosphorylation.

6. Succinate (4C) → fumarate (4C)

• Enzyme: succinate dehydrogenase
• A C–C single bond is oxidised to a C=C double bond. The electrons go to FAD, forming FADH₂.
• This enzyme is unusual: it’s embedded in the inner mitochondrial membrane and is also Complex II of the electron transport chain. It’s competitively inhibited by malonate (see enzyme inhibition).
• FAD is used instead of NAD⁺ because oxidising a C–C bond releases less energy than oxidising a C–O bond.

7. Fumarate (4C) → malate (4C)

• Enzyme: fumarase
• Hydration: water is added across the double bond.

8. Malate (4C) → oxaloacetate (4C)

• Enzyme: malate dehydrogenase
• The –OH group is oxidised to a ketone. NAD⁺ → NADH
• Oxaloacetate is regenerated, ready to accept another acetyl group.

Products of one turn

Product	Per turn	Per glucose (2 turns)
CO₂	2	4
NADH	3	6
FADH₂	1	2
GTP/ATP	1	2

Adding the link reaction (2 CO₂ and 2 NADH per glucose) and glycolysis, all six carbons of glucose have now left as CO₂. Most of glucose’s energy isn’t in ATP yet, though: it’s held in the high-energy electrons of NADH and FADH₂, which go on to the electron transport chain. (See cellular respiration for the full tally.)

Why is it a cycle?

A cycle is an efficient way to oxidise a small, awkward two-carbon unit completely. Directly oxidising acetate to two CO₂ would be chemically difficult. Instead, the acetyl group is attached to a four-carbon carrier, and the resulting six-carbon molecule has functional groups (hydroxyl, ketone and carboxyl groups) arranged so that decarboxylations and oxidations are easy. The carrier is regenerated at the end, so a small amount of oxaloacetate can process a huge amount of acetyl-CoA, rather like a catalyst.

Where oxygen comes in

Oxygen doesn’t appear in any step of the Krebs cycle. Yet the cycle stops without oxygen. Why? It needs a steady supply of NAD⁺ and FAD, and these are only regenerated when the electron transport chain passes their electrons on to oxygen. No oxygen → NADH builds up → no NAD⁺ → the dehydrogenases stop.

The oxygen atoms in the CO₂ produced come from the molecules of the cycle and from water added in steps like 2 and 7, not from the O₂ you breathe (which ends up as water).

Regulation

The cycle’s speed is matched to the cell’s energy needs:

• High NADH, ATP or succinyl-CoA (energy plentiful) inhibit citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.
• High ADP and Ca²⁺ (for example in working muscle) activate them.
• Pyruvate dehydrogenase is switched on and off by adding and removing phosphate groups.

The hub of metabolism

The Krebs cycle isn’t only for breaking things down. Its intermediates are also building blocks:

• α-ketoglutarate and oxaloacetate are used to make amino acids (glutamate and aspartate);
• citrate is exported to the cytoplasm to supply acetyl-CoA for making fatty acids and cholesterol;
• succinyl-CoA is used to make haem;
• oxaloacetate can be used to make glucose in the liver.

Because intermediates are drawn off, cells also run “topping-up” reactions (called anaplerotic reactions) to replace them, such as converting pyruvate directly into oxaloacetate.

Remembering the cycle

Students often find the eight intermediates hard to remember. A popular mnemonic lists them in order starting from citrate: “Citrate Is Krebs’ Starting Substrate For Making Oxaloacetate”, standing for citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate and oxaloacetate. It also helps to notice the pattern of reactions: two oxidative decarboxylations (steps 3 and 4) remove the two carbons; one substrate-level phosphorylation (step 5) makes GTP; and then a repeating trio of oxidation (step 6), hydration (step 7) and oxidation (step 8) turns succinate back into oxaloacetate. The same oxidation–hydration–oxidation trio appears in the breakdown of fatty acids, which makes both pathways easier to learn together.

It’s also worth tracking the numbers: every turn needs one acetyl-CoA, three NAD⁺ and one FAD, and gives back two CO₂, three NADH, one FADH₂ and one GTP. If you can reproduce that balance sheet and name the two steps that release CO₂, you have the essentials.

Key takeaways

• The Krebs cycle runs in the mitochondrial matrix and oxidises the acetyl group of acetyl-CoA to 2 CO₂ per turn.
• Acetyl (2C) + oxaloacetate (4C) → citrate (6C) → … → oxaloacetate (4C), regenerated each turn.
• Per turn: 3 NADH, 1 FADH₂, 1 GTP/ATP, 2 CO₂ (double per glucose).
• It needs oxygen indirectly, to regenerate NAD⁺ and FAD through the electron transport chain.
• It’s the hub of metabolism, linking carbohydrate, fat and protein breakdown and supplying building blocks for biosynthesis.