C. Metabolic Processes · Metabolic ProcessesPyruvate Oxidation and the Krebs Cycle
C3.1 C2.1 C3.4 A2.2About 70 minutes
Learning goals- I can describe how pyruvate is converted to acetyl-CoA, and name the products.
- I can outline the Krebs cycle, including the entry of acetyl-CoA, the regeneration of oxaloacetate, and the steps that release CO₂, NADH, FADH₂ and ATP.
- I can account for all six carbons of glucose and total the products of pyruvate oxidation and the Krebs cycle per glucose.
- I can explain why the Krebs cycle is a cycle and why it depends indirectly on oxygen.
Words to know
pyruvate oxidation
The conversion of pyruvate (3C) to acetyl-CoA (2C) in the mitochondrial matrix, releasing CO₂ and making NADH. Also called the link reaction or transition reaction.
coenzyme A (CoA)
A carrier molecule, made from vitamin B5, that carries acetyl groups into the Krebs cycle.
acetyl-CoA
A two-carbon acetyl group attached to coenzyme A; the fuel that enters the Krebs cycle.
Krebs cycle
An eight-step cycle in the mitochondrial matrix that oxidizes acetyl groups to CO₂, producing NADH, FADH₂ and ATP (also called the citric acid cycle or tricarboxylic acid (TCA) cycle).
oxaloacetate
The four-carbon molecule that combines with acetyl-CoA to start the Krebs cycle and is regenerated at the end of each turn.
citrate
The six-carbon molecule (citric acid) formed in step 1 of the Krebs cycle.
decarboxylation
A reaction that removes a carbon atom as CO₂.
Where does the carbon you breathe out come from?
Every breath you exhale carries carbon dioxide. Some of the carbon atoms in it were in your breakfast a few hours ago. They left your food in two stages that happen inside your mitochondria: pyruvate oxidation and the Krebs cycle. By the end of this lesson, all six carbons of glucose will have become CO₂, and nearly all the energy that was in glucose will be held by electron carriers, ready for the final stage.
Remember from Lesson 4
In Lesson 4 you saw that glycolysis turns 1 glucose into 2 pyruvate, with a net gain of 2 ATP and 2 NADH, and that the next stages happen in the mitochondrial matrix. Remember to double everything: each glucose sends two pyruvates into the mitochondrion.
Stage 2: pyruvate oxidation (the link reaction)
If oxygen is available, pyruvate is moved from the cytosol into the mitochondrial matrix by a carrier protein. There, a large enzyme complex, pyruvate dehydrogenase, does three things to each pyruvate:
- Decarboxylation: one carbon is removed as CO₂. (Pyruvate's 3 carbons become a 2-carbon acetyl group.)
- Oxidation: the acetyl group is oxidized and NAD⁺ is reduced to NADH.
- Attachment: the acetyl group is attached to coenzyme A, making acetyl-CoA.
pyruvate + NAD⁺ + CoA → acetyl-CoA + NADH + CO₂
Per glucose (×2): 2 acetyl-CoA, 2 NADH, 2 CO₂. No ATP is made in this step. Acetyl-CoA is an important crossroads: fatty acids and some amino acids are also broken down into acetyl-CoA, so many fuels enter the Krebs cycle here.
Stage 3: the Krebs cycle
The British-German biochemist Hans Krebs worked out this cycle in 1937 using minced pigeon breast muscle. He shared the 1953 Nobel Prize in Physiology or Medicine with Fritz Lipmann, who discovered coenzyme A. The cycle has eight steps, all in the matrix (one enzyme, succinate dehydrogenase, sits in the inner membrane).
- Entry: acetyl-CoA (2C) joins oxaloacetate (4C) to form citrate (6C). CoA is released to be reused.
- Citrate is rearranged into isocitrate (6C).
- Isocitrate is oxidized and decarboxylated: α-ketoglutarate (5C) + CO₂ + NADH.
- α-ketoglutarate is oxidized and decarboxylated, and CoA is added: succinyl-CoA (4C) + CO₂ + NADH.
- Substrate-level phosphorylation: succinate (4C) and one ATP (in some cells GTP, which transfers its phosphate to ADP).
- Succinate is oxidized to fumarate (4C), and this time the electron carrier is FAD, making FADH₂.
- Water is added: malate (4C).
- Malate is oxidized to oxaloacetate (4C) + NADH. Oxaloacetate is ready to accept the next acetyl-CoA.
It is a cycle because the molecule that starts it (oxaloacetate) is regenerated at the end, so a small amount of oxaloacetate can process many acetyl groups.
Per turn: 2 CO₂, 3 NADH, 1 FADH₂, 1 ATP. Per glucose (2 turns): 4 CO₂, 6 NADH, 2 FADH₂, 2 ATP.
Where did glucose's carbon and energy go?
After glycolysis, pyruvate oxidation and two turns of the Krebs cycle:
- Carbon: 2 CO₂ (pyruvate oxidation) + 4 CO₂ (Krebs) = 6 CO₂. All six carbons of glucose are gone. (The two carbons released in a turn are not the same two that just entered; those leave in later turns. The totals still balance.)
- ATP: only 4 so far (2 from glycolysis + 2 from Krebs).
- Electron carriers: 2 + 2 + 6 = 10 NADH and 2 FADH₂. These hold most of the free energy that was in glucose.
The Krebs cycle uses no oxygen directly. Yet it stops quickly without oxygen. Why? It needs a supply of NAD⁺ and FAD. These are regenerated only when NADH and FADH₂ give their electrons to the electron transport chain, which needs O₂ as the final acceptor. No O₂ → no NAD⁺ → no Krebs cycle.
| Stage | CO₂ | ATP | NADH | FADH₂ |
|---|
| Glycolysis | 0 | 2 (net) | 2 | 0 |
| Pyruvate oxidation (×2) | 2 | 0 | 2 | 0 |
| Krebs cycle (×2 turns) | 4 | 2 | 6 | 2 |
| Total per glucose | 6 | 4 | 10 | 2 |
Worked exampleLet's count the products of a chosen number of turns
A liver cell breaks down 3 glucose molecules completely through the Krebs cycle. How many turns of the Krebs cycle occur, and how many CO₂, NADH, FADH₂ and ATP are produced in pyruvate oxidation plus the Krebs cycle combined?
- Each glucose gives 2 pyruvate → 2 acetyl-CoA → 2 turns. For 3 glucose: 6 turns.
- Pyruvate oxidation for 6 pyruvate: 6 CO₂, 6 NADH.
- Krebs cycle, 6 turns: 12 CO₂, 18 NADH, 6 FADH₂, 6 ATP.
- Add: CO₂ = 6 + 12 = 18; NADH = 6 + 18 = 24; FADH₂ = 6; ATP = 6.
- Check carbon: 3 glucose × 6 C = 18 C = 18 CO₂ ✔
6 turns; 18 CO₂, 24 NADH, 6 FADH₂, 6 ATP.
Worked exampleLet's interpret a classic experiment
In the 1930s, researchers including Albert Szent-Györgyi and Hans Krebs added small amounts of citrate or succinate to minced pigeon muscle and measured oxygen use. Adding a little succinate increased oxygen consumption by much more than the amount needed to oxidize just the added succinate. Malonate (which blocks succinate dehydrogenase) caused succinate to build up, even when citrate was the substance added. Explain what these results suggest.
- If succinate were used once and gone, extra O₂ use would match the succinate added. Instead, it was much larger.
- That suggests the added molecule is regenerated and used again and again: a catalytic, cyclic role.
- Blocking one enzyme (succinate → fumarate) made succinate accumulate even when citrate was added, showing that citrate is converted to succinate: they are linked in one pathway, with citrate upstream.
- Together, these support a cycle in which intermediates are regenerated and acetyl groups are fully oxidized.
The intermediates act like a reusable cycle (they catalyse the oxidation of more fuel than their own amount), and blocking one step causes earlier intermediates to pile up, which revealed the order of steps.
Just for funNeed to remember the eight Krebs intermediates in order? Try: Citrate Is Krebs' Starting Substrate For Making Oxaloacetate (citrate, isocitrate, α-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate). Every turn, the cycle ends exactly where it started, like a hockey Zamboni at intermission.
Hands-on activityDetect the CO₂ you breathe out
You need: red cabbage (a few leaves), hot tap water, a sieve, 2 clear glasses, 2 drinking straws, a timer.
Safety note: tear the cabbage by hand or chop it on a cutting board, and use hot tap water, not boiling water. Breathe out only through the straw; do not suck up the liquid.
Make an indicator: tear or chop the cabbage leaves, soak them in hot tap water for 10 minutes, then strain. The purple-blue liquid is a pH indicator: it turns more pink-red in acid.
- Pour equal amounts of indicator into two glasses. Glass A is the control.
- Through a straw, gently blow bubbles into glass B for 1 minute at rest. Compare colours against a white background.
- Now do 30 jumping jacks (or climb stairs for a minute), then blow into a fresh glass of indicator for the same time.
- Record colour changes in a table.
Questions:
- CO₂ dissolves in water to form carbonic acid. What does the colour change tell you?
- Which stages of respiration produced the CO₂? How many CO₂ come from one glucose?
- Why might the change be faster after exercise?
What to look for: glass B shifts from purple-blue toward violet or pinkish-purple, showing that exhaled air contains CO₂ that makes the solution more acidic. CO₂ is produced in pyruvate oxidation (2 per glucose) and the Krebs cycle (4 per glucose). After exercise, cells respire faster, so you exhale more CO₂ per breath and the colour may change faster. Colour changes can be subtle: compare side by side with the control.
Your turn
1Write a word equation for pyruvate oxidation.
pyruvate + NAD⁺ + CoA → acetyl-CoA + NADH + CO₂.
2What molecule combines with acetyl-CoA at the start of the Krebs cycle, and what is formed?
Oxaloacetate (4C) combines with acetyl-CoA (2C) to form citrate (6C).
3Why is the Krebs cycle described as a cycle?
Because oxaloacetate, which starts the cycle, is regenerated at the end of each turn and can combine with another acetyl-CoA.
4List the products of one turn of the Krebs cycle.
2 CO₂, 3 NADH, 1 FADH₂, 1 ATP (or GTP).
5In which steps of the Krebs cycle is CO₂ released?
In the conversion of isocitrate → α-ketoglutarate and α-ketoglutarate → succinyl-CoA (the two decarboxylation steps).
6Explain why the Krebs cycle stops when a cell runs out of oxygen, even though it does not use oxygen directly.
Without O₂, the electron transport chain stops, so NADH and FADH₂ can't be oxidized back to NAD⁺ and FAD. The Krebs cycle needs NAD⁺ and FAD as electron acceptors, so it soon stops.
7How many ATP per glucose have been made by the end of the Krebs cycle? By what process?
4 ATP (2 in glycolysis, 2 in the Krebs cycle), all by substrate-level phosphorylation.
More practice: warm-up
1Where does the Krebs cycle take place?
In the mitochondrial matrix.
2How many carbons are in acetyl-CoA's acetyl group?
3How many turns of the Krebs cycle happen per glucose?
4Which electron carrier is reduced in the succinate → fumarate step?
5Name the scientist the Krebs cycle is named after.
More practice: on your own
1Account for all six carbons of one glucose by the end of the Krebs cycle.
Glycolysis releases none (6 C are in 2 pyruvate). Pyruvate oxidation releases 2 CO₂ (1 per pyruvate). The Krebs cycle releases 4 CO₂ (2 per turn × 2 turns). Total 6 CO₂.
2A cell is given a drug that blocks the enzyme that converts succinate to fumarate. Predict which intermediate builds up and what happens to NADH production.
Succinate (and intermediates before it) builds up. Fumarate, malate and oxaloacetate run out, so the cycle stops, and NADH production by the Krebs cycle falls (no new citrate can form without oxaloacetate).
3Why is acetyl-CoA called a metabolic crossroads?
Carbohydrates (via pyruvate), fatty acids and many amino acids can all be converted to acetyl-CoA, so they all enter respiration through the Krebs cycle. Acetyl-CoA can also be used to build fatty acids.
4Compare the amount of ATP and NADH made in glycolysis with the amount made in pyruvate oxidation plus the Krebs cycle, per glucose.
Glycolysis: 2 ATP, 2 NADH. Pyruvate oxidation + Krebs: 2 ATP, 8 NADH and 2 FADH₂. The mitochondrial stages capture much more energy in electron carriers.
5Vitamin B5 (pantothenic acid) is used to make coenzyme A. Predict the effect of a severe B5 deficiency on cellular respiration.
Without enough CoA, pyruvate can't be converted to acetyl-CoA (and α-ketoglutarate to succinyl-CoA), so the Krebs cycle slows, less NADH/FADH₂ is produced, and less ATP is made by the ETC. The person may feel tired and weak.
Challenge
1In an experiment, the carbon in an acetyl group is labelled with radioactive ¹⁴C. After one turn of the Krebs cycle, the labelled carbons are found in oxaloacetate, not in the CO₂ released. Explain how this is possible and what happens on later turns.
The two carbons released as CO₂ in a turn come from the oxaloacetate part of citrate, not the newly added acetyl. The labelled acetyl carbons stay in the 4C intermediates and become part of the regenerated oxaloacetate. In later turns, that oxaloacetate joins new acetyl-CoA, and the labelled carbons are eventually released as CO₂. Over time, every carbon is oxidized; the totals still balance (2 CO₂ out for each 2 C in).
2Some cells remove Krebs intermediates (for example, α-ketoglutarate) to build amino acids. Predict the effect on the cycle and suggest how the cell might compensate.
Removing intermediates reduces the amount of oxaloacetate regenerated, so fewer acetyl-CoA can enter and the cycle slows. The cell must replenish intermediates, for example by making oxaloacetate directly from pyruvate (by adding CO₂), so the cycle keeps running.
3A muscle cell has plenty of glucose and O₂, but a mutation makes its NAD⁺ supply very small. Explain which stages of respiration are affected and why the effect is so large.
NAD⁺ is needed in glycolysis (G3P oxidation), pyruvate oxidation and three Krebs steps. With little NAD⁺, all of these slow, so fewer NADH reach the ETC and far less ATP is made. The effect is large because NAD⁺ is a recycled carrier: a small pool must be reused rapidly, and almost every oxidation step depends on it.
Quiz
1. Pyruvate oxidation produces, per pyruvate:
2. The Krebs cycle begins when acetyl-CoA combines with:
3. Per glucose, the Krebs cycle produces:
4. Where in the cell does the Krebs cycle take place?
5. Why does the Krebs cycle stop without oxygen?
6. By the end of the Krebs cycle, how many ATP per glucose have been made in total?
Big idea: Pyruvate oxidation and the Krebs cycle finish oxidizing glucose's carbon to CO₂ and load the energy into NADH and FADH₂ (C3.1, C2.1). Students should be able to account for all six carbons and total the products per glucose.
How to help: A quick oral quiz works well: "Per glucose, how many CO₂ from pyruvate oxidation? From Krebs? How many NADH in total?" Remind your teen to double everything after glycolysis. The cabbage indicator activity is safe (the leaves can be torn by hand), and the student should only blow out through the straw.
Common difficulties:
- Forgetting to double for two pyruvates per glucose.
- Thinking the Krebs cycle uses oxygen directly.
- Mixing up oxaloacetate (start/end, 4C) and citrate (first product, 6C).
- Expecting a lot of ATP from the Krebs cycle; its main job is to make NADH and FADH₂.