The Krebs Cycle

A2 · 11 min

The Krebs cycle (also called the citric acid cycle) is where the acetyl groups from glucose, fatty acids and amino acids are completely broken down to carbon dioxide. It makes only a little ATP directly. Its real purpose is to strip hydrogen atoms from its intermediates and load them onto NAD and FAD, which carry them to the electron transport chain where most ATP is made. Examiners want you to know the 4C and 6C compounds by name, to explain the decarboxylation and dehydrogenation reactions, and to count products per turn and per glucose.

Overview

The Krebs cycle takes place in the mitochondrial matrix, where its enzymes are dissolved. It is a cycle because the 4C compound that accepts the acetyl group at the start, oxaloacetate, is regenerated at the end. A small amount of oxaloacetate can therefore process an unlimited number of acetyl groups, as long as the cycle keeps turning.

Every turn of the cycle:

accepts one 2C acetyl group from acetyl coenzyme A;
releases two molecules of carbon dioxide (two decarboxylation reactions);
removes eight hydrogen atoms in four dehydrogenation reactions, forming three reduced NAD and one reduced FAD;
makes one ATP by substrate-linked phosphorylation.

Since one glucose produces two pyruvate and therefore two acetyl CoA, the cycle turns twice per glucose.

The steps of the cycle

One turn of the Krebs cycle

Formation of citrate. The acetyl group (2C) from acetyl CoA combines with oxaloacetate (4C) to form citrate (6C). Coenzyme A is released and returns to the link reaction.
Citrate to a 5C compound. Citrate is decarboxylated (one $\ce{CO2}$ removed) and dehydrogenated (hydrogen accepted by NAD, forming reduced NAD), producing a 5C compound.
5C compound to a 4C compound. The 5C compound is decarboxylated (second $\ce{CO2}$ ) and dehydrogenated (second reduced NAD). One ATP is made by substrate-linked phosphorylation.
Regeneration of oxaloacetate. The 4C compound is converted back to oxaloacetate in a series of small steps, including two more dehydrogenations: one reduces FAD (forming reduced FAD) and one reduces NAD (third reduced NAD).
The regenerated oxaloacetate accepts another acetyl group and the cycle repeats.

What is used and made at each step of one turn

Step	Carbon change	$\ce{CO2}$	Reduced NAD	Reduced FAD	ATP
1	acetyl (2C) + oxaloacetate (4C) → citrate (6C)	0	0	0	0
2	citrate (6C) → 5C compound	1	1	0	0
3	5C compound → 4C compound	1	1	0	1
4	4C compound → oxaloacetate (4C)	0	1	1	0
Per turn		2	3	1	1
Per glucose (2 turns)		4	6	2	2

Tip

The full cycle has more intermediates than you need. For reference only (not required by the syllabus): the 5C compound is α-ketoglutarate, and the 4C compounds after it are succinyl CoA, succinate, fumarate and malate. Succinate is dehydrogenated to fumarate by succinate dehydrogenase, which uses FAD; malate is dehydrogenated to oxaloacetate using NAD. In animal cells the substrate-linked step actually makes GTP, which is used to make ATP. Using only "citrate", "oxaloacetate", "5C" and "4C" is enough for full marks.

Decarboxylation and dehydrogenation

The syllabus asks you to explain that the Krebs cycle involves both of these reactions. It is worth being precise.

Definition

Decarboxylation is the removal of carbon dioxide from a molecule, catalysed by a decarboxylase. In the Krebs cycle it converts citrate (6C) to a 5C compound, and the 5C compound to a 4C compound.

Dehydrogenation is the removal of hydrogen atoms from a molecule, catalysed by a dehydrogenase. It is a form of oxidation. The hydrogen is accepted by a coenzyme (NAD or FAD), which is reduced.

These two reactions explain where the products go:

The carbon dioxide diffuses out of the mitochondrion, out of the cell, into the blood, and is excreted at the lungs. All the carbon of the acetyl group is lost this way (although, strictly, the two carbons lost in one turn are not the same two that just entered; they leave on a later turn).
The hydrogen carried by reduced NAD and reduced FAD holds most of the energy that was in glucose. It is delivered to the electron transport chain.

NAD and FAD as hydrogen carriers

Both NAD and FAD (flavin adenine dinucleotide, made from the vitamin riboflavin) are coenzymes that act as hydrogen carriers. Their role, which the syllabus asks you to describe, is to:

accept hydrogen atoms removed from respiratory intermediates by dehydrogenase enzymes, becoming reduced NAD or reduced FAD;
carry the hydrogen to the electron transport chain on the inner mitochondrial membrane;
donate the hydrogen to the first carrier of the chain (reduced FAD hands over its hydrogen at a later point than reduced NAD), so that they are oxidised and regenerated as NAD and FAD;
return to accept more hydrogen, so that the dehydrogenation reactions of glycolysis, the link reaction and the Krebs cycle can continue.

	NAD	FAD
Used in	glycolysis, link reaction, Krebs cycle (and in fermentation)	Krebs cycle only
Reduced form per glucose (aerobic)	10 (2 + 2 + 6)	2
Where it donates hydrogen	at the start of the electron transport chain	further along the chain
ATP produced per molecule (approximate)	about 2.5	about 1.5

Because reduced FAD donates its hydrogen further along the chain, its electrons drive the pumping of fewer protons, so it yields less ATP than reduced NAD.

Why the Krebs cycle needs oxygen

No step of the Krebs cycle uses oxygen directly. Yet the cycle stops almost immediately when oxygen runs out. The reason is the supply of oxidised coenzymes:

Without oxygen, the final electron acceptor of the electron transport chain is missing, so electrons cannot pass along the chain.
Reduced NAD and reduced FAD cannot be oxidised, so they accumulate and the supply of NAD and FAD runs out.
Without NAD and FAD, the dehydrogenation reactions of the link reaction and Krebs cycle cannot occur, so these stages stop.

This is why the Krebs cycle is described as an aerobic process even though oxygen is not a reactant in it.

Totals for glycolysis, link reaction and Krebs cycle

Per glucose	Glycolysis	Link reaction	Krebs cycle	Total
ATP (net, substrate-linked)	2	0	2	4
Reduced NAD	2	2	6	10
Reduced FAD	0	0	2	2
$\ce{CO2}$	0	2	4	6

Only 4 ATP have been made so far. Ten reduced NAD and two reduced FAD carry the rest of the energy to oxidative phosphorylation.

Worked examples

Outline the Krebs cycle

Outline the Krebs cycle. [5]

Solution

Occurs in the mitochondrial matrix. [1]
Acetyl (2C) from acetyl coenzyme A combines with oxaloacetate (4C) to form citrate (6C); coenzyme A is released. [1]
Citrate is converted back to oxaloacetate in a series of small steps: 6C → 5C → 4C. [1]
Decarboxylation releases 2 $\ce{CO2}$ per turn. [1]
Dehydrogenation produces 3 reduced NAD and 1 reduced FAD per turn. [1]
1 ATP is made per turn by substrate-linked phosphorylation. [1]
Oxaloacetate is regenerated, so the cycle continues. [1]

Counting from fatty acids

A fatty acid with 16 carbon atoms is broken down into acetyl groups, which all enter the Krebs cycle. Calculate the number of molecules of carbon dioxide, reduced NAD and reduced FAD produced by the Krebs cycle alone. [3]

Solution

$16 \div 2 = 8$ acetyl groups, so the cycle turns 8 times.

$\ce{CO2} = 8 \times 2 = 16$
Reduced NAD $= 8 \times 3 = 24$
Reduced FAD $= 8 \times 1 = 8$

(The breakdown of the fatty acid into acetyl groups produces additional reduced NAD and reduced FAD, which is part of the reason fatty acids release so much energy.)

A competitive inhibitor

Malonate is a competitive inhibitor of succinate dehydrogenase, the enzyme that removes hydrogen from the 4C compound succinate, using FAD. Mitochondria were supplied with pyruvate and oxygen, and then with malonate.

(a) Explain how malonate inhibits the enzyme. [2]

(b) Predict and explain the effect of malonate on (i) the concentration of succinate and (ii) the rate of oxygen uptake. [4]

Solution

(a) Malonate has a similar shape to succinate, so it binds to the active site of succinate dehydrogenase and prevents succinate binding; it does not react, and its effect can be overcome by raising the succinate concentration. [2]

(b)(i) Succinate accumulates (concentration rises), because it is still being formed from earlier steps of the cycle but cannot be converted to the next intermediate. [2]

(ii) Oxygen uptake falls. The cycle slows or stops beyond succinate, so less oxaloacetate is regenerated and fewer acetyl groups enter; less reduced NAD and reduced FAD are produced, so fewer electrons pass along the electron transport chain to the final electron acceptor, oxygen. [2]

Why a cycle?

Explain the advantage of the Krebs cycle being a cyclic pathway in which oxaloacetate is regenerated. [2]

Solution

Oxaloacetate is regenerated at the end of each turn, so it can accept another acetyl group; it acts like a reusable carrier. [1]
Only a small amount of oxaloacetate is needed to oxidise a large number of acetyl groups, so the cell does not need to keep making new acceptor molecules (saving resources and energy). [1]

Data: labelled acetyl groups

In an experiment, mitochondria were given acetyl CoA in which the acetyl carbons were radioactively labelled. No radioactive carbon dioxide was released during the first turn of the cycle, but it appeared in later turns. Suggest an explanation. [2]

Solution

The two carbon atoms removed as $\ce{CO2}$ in the first turn come from the oxaloacetate part of citrate, not from the acetyl group that has just joined. [1]
The labelled carbons remain in the regenerated oxaloacetate, so they are released as $\ce{CO2}$ when this oxaloacetate passes through the cycle on later turns. [1]

Watch out

Oxaloacetate is 4C and citrate is 6C. Getting these the wrong way round is one of the most frequent errors.
The Krebs cycle produces $\ce{CO2}$ but does not use oxygen. Do not write "oxygen is used in the Krebs cycle".
Per glucose, double the per-turn numbers. If the question says "per turn" or "per acetyl group", do not double.
FAD is reduced only in the Krebs cycle; NAD is reduced in glycolysis, the link reaction and the Krebs cycle.

Exam tip

"Explain the role of NAD in respiration" needs: accepts hydrogen (from dehydrogenation) → reduced NAD → carries hydrogen to the electron transport chain / inner membrane → releases hydrogen (protons and electrons) → NAD regenerated for further dehydrogenation.
When asked to "explain why the Krebs cycle cannot continue without oxygen", you need the chain of reasoning: no final electron acceptor → ETC stops → reduced NAD/FAD not reoxidised → no NAD/FAD for dehydrogenation.
Diagram questions often show the cycle with letters for compounds. Identify them by carbon number: the 6C compound is citrate, the 4C compound that accepts acetyl is oxaloacetate.

Summary

The Krebs cycle takes place in the mitochondrial matrix and turns twice per glucose.
Oxaloacetate (4C) accepts the acetyl (2C) group from acetyl CoA to form citrate (6C), which is converted back to oxaloacetate in small steps (6C → 5C → 4C).
Per turn: 2 $\ce{CO2}$ (decarboxylation), 3 reduced NAD and 1 reduced FAD (dehydrogenation), 1 ATP (substrate-linked phosphorylation).
Decarboxylation removes $\ce{CO2}$ ; dehydrogenation removes hydrogen, which reduces NAD or FAD.
NAD and FAD carry hydrogen to the electron transport chain on the inner membrane, where they are reoxidised.
Oxygen is not used in the cycle, but without it NAD and FAD are not regenerated and the cycle stops.
By the end of the Krebs cycle, glucose has yielded only 4 ATP but 10 reduced NAD and 2 reduced FAD.

Practice

Question

Name the 4C compound that combines with acetyl coenzyme A. [1]
State the number of carbon atoms in citrate. [1]
State the products of one turn of the Krebs cycle. [3]
Define dehydrogenation and state its importance in the Krebs cycle. [2]
Explain why the Krebs cycle is described as part of aerobic respiration even though it does not use oxygen. [3]
How many molecules of reduced NAD are produced from one molecule of glucose in the Krebs cycle only, and how many in glycolysis, the link reaction and the Krebs cycle together? [2]
Explain why the Krebs cycle cannot occur in prokaryotes in the same location as in eukaryotes, and suggest where it takes place in a bacterium. [2]
Some cells use intermediates of the Krebs cycle, such as the 5C compound, to make amino acids. Suggest and explain the effect on the Krebs cycle if a large amount of 5C compound is removed, and how the cell might compensate. [4]
Compare the link reaction and the Krebs cycle. [5]

Answers

Oxaloacetate.
Six.
2 $\ce{CO2}$ , 3 reduced NAD, 1 reduced FAD, 1 ATP (and regenerated oxaloacetate; coenzyme A released).
The removal of hydrogen atoms from a substrate by a dehydrogenase (an oxidation). It produces reduced NAD and reduced FAD, which carry hydrogen to the electron transport chain, where its energy is used to make most of the ATP.
The Krebs cycle depends on a supply of NAD and FAD for its dehydrogenation reactions. These are regenerated only when reduced NAD and reduced FAD are oxidised by the electron transport chain, which requires oxygen as the final electron acceptor. Without oxygen, NAD and FAD run out and the cycle stops.
Krebs cycle only: 6. All three stages: $2 + 2 + 6 = 10$ .
Prokaryotes have no mitochondria (no membrane-bound organelles). The Krebs cycle takes place in the cytoplasm of the bacterium (with the electron transport chain on the cell surface membrane).
Less 5C compound is available, so less 4C compound and less oxaloacetate are regenerated; fewer acetyl groups can enter the cycle, so the cycle slows, producing less $\ce{CO2}$ , reduced NAD, reduced FAD and ATP. The cell could compensate by making oxaloacetate from pyruvate (adding $\ce{CO2}$ ), or by converting certain amino acids into Krebs cycle intermediates, keeping the cycle running.
Similarities: both in the mitochondrial matrix; both involve decarboxylation and dehydrogenation; both produce reduced NAD and $\ce{CO2}$ ; both depend indirectly on oxygen; both involve coenzyme A. Differences: link reaction has no ATP made, Krebs cycle makes 1 ATP per turn; link reaction produces no reduced FAD, Krebs cycle produces 1 per turn; link reaction is a linear pathway, Krebs cycle is cyclic (oxaloacetate regenerated); link reaction attaches acetyl to coenzyme A, Krebs cycle releases coenzyme A; per pyruvate the link reaction releases 1 $\ce{CO2}$ and 1 reduced NAD, the Krebs cycle (per acetyl) 2 $\ce{CO2}$ and 3 reduced NAD.

Overview

The steps of the cycle

Decarboxylation and dehydrogenation

NAD and FAD as hydrogen carriers

Why the Krebs cycle needs oxygen

Totals for glycolysis, link reaction and Krebs cycle

Worked examples

Practice

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