Pyruvate oxidation & the Krebs cycle

Into the mitochondrion: pyruvate oxidation

Glycolysis leaves each glucose as two pyruvates in the cytosol. When O₂ is available, a carrier protein moves each pyruvate across the inner mitochondrial membrane into the matrix, the space inside the inner membrane. There, a huge enzyme complex, pyruvate dehydrogenase (PDH), prepares it for the cycle:

1. 1One carbon is removed from pyruvate and released as CO₂. This is the first CO₂ to come from glucose.
2. 2The remaining two-carbon piece is oxidised, and its electrons go to NAD⁺, making NADH.
3. 3The two-carbon acetyl group is attached to a carrier called coenzyme A (CoA), forming acetyl-CoA.

Per glucose this happens twice, giving 2 CO₂, 2 NADH and 2 acetyl-CoA. Acetyl-CoA is the fuel that enters the cycle. Fatty acids and some amino acids are broken down to acetyl-CoA too, which is why the cycle can burn fat and protein as well as sugar.

Once around the cycle

The Krebs cycle, also called the citric acid cycle or TCA cycle, is a loop of eight enzyme-catalysed steps in the matrix. The number in brackets is how many carbons each molecule has.

1. 1Acetyl-CoA (2) joins oxaloacetate (4) to make citrate (6). CoA is released to pick up another acetyl group.
2. 2Citrate is rearranged into its isomer, isocitrate (6).
3. 3Isocitrate is oxidised: a CO₂ is released and NADH is made, leaving α-ketoglutarate (5).
4. 4α-Ketoglutarate is oxidised in the same way: another CO₂ and another NADH, leaving succinyl-CoA (4).
5. 5The energy of succinyl-CoA's bond is used to make GTP (or ATP in some tissues) by substrate-level phosphorylation, leaving succinate (4). GTP can pass its phosphate to ADP, so it counts as one ATP.
6. 6Succinate is oxidised to fumarate (4), and the electrons go to FAD, making FADH₂.
7. 7Water is added to fumarate, making malate (4).
8. 8Malate is oxidised back to oxaloacetate (4), making one more NADH. The cycle is ready for the next acetyl-CoA.

Keeping count

Each turn of the cycle takes in one acetyl group (2 carbons) and releases 2 CO₂, so carbon in equals carbon out. Each turn also makes 3 NADH, 1 FADH₂ and 1 GTP (≈ ATP). Because one glucose gives two acetyl-CoA, the cycle turns twice per glucose.

• Pyruvate oxidation (×2): 2 CO₂, 2 NADH.
• Krebs cycle (×2): 4 CO₂, 6 NADH, 2 FADH₂, 2 ATP (as GTP).
• Together: 6 CO₂, 8 NADH, 2 FADH₂, 2 ATP. Add glycolysis's 2 NADH and 2 ATP, and one glucose has given 10 NADH, 2 FADH₂ and 4 ATP by substrate-level phosphorylation.

All six carbons of glucose have now left as CO₂: two at pyruvate oxidation, four in the cycle. That's the CO₂ you breathe out. The O₂ you breathe in doesn't become CO₂; it's used at the end of the electron transport chain and becomes water.

Not the main ATP factory

The Krebs cycle sits in the middle of every respiration diagram, but it makes only 2 ATP per glucose directly. Its real product is loaded electron carriers. The cycle strips electrons from the carbon atoms of fuel, loads them onto NAD⁺ and FAD, and releases the carbon skeleton as CO₂.

Those carriers are then cashed in by oxidative phosphorylation. At about 2.5 ATP per NADH and 1.5 per FADH₂, the 10 NADH and 2 FADH₂ from one glucose give about 26–28 ATP, depending on how glycolysis's NADH enters the mitochondrion. Add the 4 made directly and you get the familiar total of about 30–32.

No oxygen, no cycle, even though it never touches O₂

Look back at the eight steps: none of them uses oxygen. Yet the cycle stops within moments if O₂ runs out. Why?

Four of the steps (three in the cycle plus PDH) need NAD⁺ to accept electrons, and one needs FAD. The matrix holds only a limited pool of these carriers. The only thing that turns NADH back into NAD⁺ in the matrix is the electron transport chain, which passes the electrons to O₂. No O₂, and the chain backs up. NADH piles up, NAD⁺ runs out, and the dehydrogenases stop for lack of somewhere to put their electrons. Blocking the chain with a poison such as cyanide has exactly the same effect, even with plenty of O₂ around.

A hub, not just a furnace

The cycle is also where the cell gets many raw materials. Citrate can be moved out of the mitochondrion to make fatty acids; α-ketoglutarate is used to make the amino acid glutamate; oxaloacetate is used to make aspartate. Taking intermediates out drains the cycle, so cells top it up with anaplerotic ("filling-up") reactions. The main one uses the enzyme pyruvate carboxylase to make oxaloacetate directly from pyruvate.

The cycle is also tightly regulated. High NADH and ATP, signs that the cell has plenty of energy, slow down PDH, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. PDH has an extra switch: a kinase turns it off by adding a phosphate, and a phosphatase turns it back on by removing it. In working muscle, Ca²⁺ activates that phosphatase, so the cycle speeds up just when energy is needed.

Key terms

Pyruvate dehydrogenase (PDH)

The matrix enzyme complex that turns pyruvate into acetyl-CoA, releasing CO₂ and making NADH.

Acetyl-CoA

A two-carbon acetyl group attached to coenzyme A; the fuel that enters the Krebs cycle.

Krebs (citric acid) cycle

An eight-step loop in the matrix that oxidises acetyl groups to CO₂, making 3 NADH, 1 FADH₂ and 1 GTP per turn.

Oxaloacetate

The four-carbon molecule that combines with acetyl-CoA to form citrate and is regenerated at the end of each turn.

Decarboxylation

Removal of a carbon as CO₂, as at PDH, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase.

Succinate dehydrogenase

The cycle enzyme that makes FADH₂; it is embedded in the inner membrane and is also Complex II.

Anaplerotic reaction

A reaction that refills Krebs cycle intermediates, such as pyruvate carboxylase making oxaloacetate.

Check yourself

Try answering in your head before you open each answer.

• 1.Cells are given plenty of O₂ and glucose, but a drug blocks Complex I of the electron transport chain. Predict what happens to the ratio of NADH to NAD⁺ in the matrix and to the rate of the Krebs cycle.Show answerHide

  NADH builds up and NAD⁺ falls, because NADH can no longer hand its electrons to the chain. With little NAD⁺, PDH and the cycle's three NAD⁺-using dehydrogenases slow to a crawl, so the cycle nearly stops, even though O₂ is available.

• 2.In an experiment, both carbons of the acetyl group of acetyl-CoA are labelled with ¹⁴C. Will labelled CO₂ be released during the first turn of the cycle?Show answerHide

  No. The two CO₂ released in the first turn come from carbons that were already in oxaloacetate. The labelled acetyl carbons end up in the regenerated oxaloacetate and are only released as CO₂ in later turns.

• 3.A liver cell pulls a lot of α-ketoglutarate out of the cycle to make amino acids. What would happen to the cycle if nothing replaced it, and how does the cell prevent this?Show answerHide

  Every intermediate after α-ketoglutarate would run low, including oxaloacetate. Without oxaloacetate, acetyl-CoA can't enter the cycle, so the cycle would slow. The cell tops it up with anaplerotic reactions, mainly pyruvate carboxylase, which makes oxaloacetate from pyruvate.