AP Biology

## Learning Outcomes

- State where the Krebs cycle occurs in eukaryotic cells and identify its main input from pyruvate oxidation.
- Trace the carbon atoms entering and leaving the cycle as acetyl-CoA, carbon dioxide, and regenerated oxaloacetate.
- Calculate the products of one turn of the Krebs cycle and the products per glucose molecule.
- Explain how NADH and FADH2 connect the Krebs cycle to oxidative phosphorylation.
- Distinguish substrate-level phosphorylation in the Krebs cycle from ATP production by chemiosmosis.
- Predict how low oxygen availability affects the Krebs cycle, the electron transport chain, and fermentation.
- Interpret experimental data involving Krebs cycle activity, such as citrate synthase activity and error bars.

## AP Biology Syllabus

For AP Biology Unit 3, the Krebs cycle is tested as part of cellular energetics: how cells transfer energy from food molecules into ATP and electron carriers. Exam questions often ask you to connect cellular locations, redox reactions, carbon flow, ATP yield, and the dependence of respiration on oxygen through the electron transport chain.

- The Krebs cycle is also called the citric acid cycle.
- In eukaryotic cells, the cycle occurs in the mitochondrial matrix.
- Acetyl-CoA enters the cycle after pyruvate oxidation.
- Each turn releases carbon dioxide and regenerates oxaloacetate.
- Each turn produces reduced electron carriers: NADH and FADH2.
- ATP is made directly in the Krebs cycle by substrate-level phosphorylation.
- The Krebs cycle depends indirectly on oxygen because oxygen allows the electron transport chain to regenerate NAD+ and FAD.
- Data questions may use indicators such as citrate synthase activity to represent Krebs cycle activity.

## Test Your Knowledge

Attempt these questions before reading this article. If you find some difficult or cannot remember the answers, look more closely at that area during your revision.

1. What molecule combines with acetyl-CoA at the start of the Krebs cycle, and what six-carbon molecule is formed?
2. For one turn of the Krebs cycle, how many NADH, FADH2, ATP, and carbon dioxide molecules are produced?
3. Why does the Krebs cycle stop when oxygen is not available, even though oxygen is not a direct reactant in the cycle?
4. Why does each glucose molecule cause the Krebs cycle to turn twice?
5. How could increased citrate synthase activity be used as evidence for increased Krebs cycle activity?

## Introduction

Cellular respiration transfers energy from organic molecules, especially glucose, into ATP. Its overall summary is:

$$
\mathrm{C_6H_{12}O_6}+6\mathrm{O_2}\rightarrow6\mathrm{CO_2}+6\mathrm{H_2O}+\mathrm{ATP}
$$

The Krebs cycle is the stage that completes the oxidation of carbon atoms that originally came from glucose. Glycolysis splits one glucose into two pyruvate molecules. Pyruvate oxidation then converts each pyruvate into acetyl-CoA, which enters the Krebs cycle. The cycle does not make most of the ATP directly. Instead, it produces NADH and FADH2, which carry high-energy electrons to the electron transport chain.

> **Key Term: cellular respiration**
> A set of metabolic processes that release energy from organic molecules and transfer that energy to ATP, electron carriers, and other usable cellular forms.

### From pyruvate to acetyl-CoA: preparing fuel for the cycle

Before the Krebs cycle can begin, pyruvate from glycolysis must be converted into acetyl-CoA. Glycolysis produces two three-carbon pyruvate molecules per glucose. In eukaryotic cells, pyruvate is transported into the mitochondrion, where pyruvate oxidation occurs.

During pyruvate oxidation, one carbon is removed from pyruvate and released as carbon dioxide. The remaining two-carbon acetyl group is attached to coenzyme A, forming acetyl-CoA. At the same time, NAD+ is reduced to NADH. Since each glucose produces two pyruvates, pyruvate oxidation happens twice per glucose.

Per pyruvate, pyruvate oxidation produces:

- 1 acetyl-CoA
- 1 carbon dioxide
- 1 NADH

Per glucose, pyruvate oxidation produces:

- 2 acetyl-CoA
- 2 carbon dioxide
- 2 NADH

> **Key Term: acetyl-CoA**
> A two-carbon acetyl group attached to coenzyme A; it delivers the acetyl group into the Krebs cycle.

This preparation step is often tested with carbon-counting. Glucose has six carbons. Glycolysis does not release carbon dioxide, so the six carbons are still present in two three-carbon pyruvates. Pyruvate oxidation releases two carbons total as carbon dioxide, leaving four carbons in two acetyl groups. Those four remaining carbons are released as carbon dioxide during the Krebs cycle.

> **Test Tip:**
> If a question asks why the Krebs cycle happens twice per glucose, the answer is that glycolysis produces two pyruvates, and pyruvate oxidation produces two acetyl-CoA molecules. Each acetyl-CoA enters the Krebs cycle separately.

### The cyclic pathway: citrate formation and oxaloacetate regeneration

In eukaryotic cells, the Krebs cycle occurs in the mitochondrial matrix, the liquid interior region inside the inner mitochondrial membrane. This location matters because the products of the cycle, especially NADH and FADH2, must pass electrons to the electron transport chain on the inner mitochondrial membrane.

> **Key Term: mitochondrial matrix**
> The inner fluid-filled compartment of a mitochondrion where pyruvate oxidation and the Krebs cycle occur in eukaryotic cells.

The cycle begins when acetyl-CoA donates its two-carbon acetyl group to oxaloacetate, a four-carbon molecule. This produces citrate, also called citric acid, a six-carbon molecule. This first product gives the pathway one of its names: the citric acid cycle.

> **Key Term: Krebs cycle**
> A cyclic pathway in cellular respiration that oxidizes acetyl groups to carbon dioxide while producing NADH, FADH2, and ATP.
>
> **Key Term: oxaloacetate**
> A four-carbon molecule that combines with acetyl-CoA at the start of the Krebs cycle and is regenerated by the end of the cycle.

The cycle then passes citrate through a series of enzyme-controlled reactions. During these reactions, two carbons are released as carbon dioxide. Energy from these reactions is transferred to electron carriers and to ATP. By the end of the cycle, oxaloacetate is regenerated. Because oxaloacetate is restored, it can combine with another acetyl-CoA and allow the cycle to continue.

This regeneration is what makes the pathway a cycle rather than a simple chain. Oxaloacetate is not used up permanently. It is a starting molecule and also an end product. AP questions may compare this feature to the Calvin cycle in photosynthesis: both are cycles because they regenerate a starting molecule.

> **Exam Warning:**
> Do not say that oxygen combines with carbon in the Krebs cycle to make carbon dioxide. Carbon dioxide is released during pyruvate oxidation and the Krebs cycle as carbon atoms are removed from organic intermediates. Oxygen is the final electron acceptor at the end of the electron transport chain, where it forms water.

### Products per turn and products per glucose

For one turn of the Krebs cycle, one acetyl-CoA enters. The main outputs are:

- 2 carbon dioxide
- 3 NADH
- 1 FADH2
- 1 ATP
- regenerated oxaloacetate

A useful summary for one turn is:

$$
\text{1 acetyl-CoA}\rightarrow2\mathrm{CO_2}+3\mathrm{NADH}+1\mathrm{FADH_2}+1\mathrm{ATP}
$$

Because each glucose produces two acetyl-CoA molecules, the Krebs cycle turns twice per glucose. Therefore, per glucose, the Krebs cycle produces:

- 4 carbon dioxide
- 6 NADH
- 2 FADH2
- 2 ATP
- regenerated oxaloacetate for each turn

> **Key Term: NADH**
> A reduced electron carrier that transports high-energy electrons from glycolysis, pyruvate oxidation, and the Krebs cycle to the electron transport chain.
>
> **Key Term: FADH2**
> A reduced electron carrier produced in the Krebs cycle that donates electrons to the electron transport chain.

The ATP made in the Krebs cycle is produced directly by substrate-level phosphorylation. This means a phosphate group is transferred directly to ADP to form ATP, without using the electron transport chain or a proton gradient. This is different from oxidative phosphorylation, which uses the electron transport chain, chemiosmosis, and ATP synthase to make most of the ATP in aerobic respiration.

> **Key Term: substrate-level phosphorylation**
> Direct formation of ATP by transferring a phosphate group to ADP during a metabolic reaction, without using chemiosmosis.

Across the first three stages of cellular respiration, per glucose, the cell has made:

- Glycolysis: 2 net ATP and 2 NADH
- Pyruvate oxidation: 2 NADH
- Krebs cycle: 2 ATP, 6 NADH, and 2 FADH2

So before oxidative phosphorylation, the total is:

- 4 ATP by substrate-level phosphorylation
- 10 NADH
- 2 FADH2
- 6 carbon dioxide released total from pyruvate oxidation and the Krebs cycle

The low direct ATP yield can be misleading. The major energy value of the Krebs cycle is not the 2 ATP per glucose. It is the production of reduced electron carriers that feed oxidative phosphorylation.

> **Test Tip:**
> Memorize the Krebs cycle yield per turn first: 2 carbon dioxide, 3 NADH, 1 FADH2, and 1 ATP. Then double it for one glucose. Many AP errors come from mixing up “per turn” with “per glucose.”

### Linking the Krebs cycle to oxygen, the electron transport chain, and experiments

The Krebs cycle and the electron transport chain are linked by electron carriers. NADH and FADH2 carry electrons from earlier stages of respiration to the electron transport chain. When they donate electrons, NADH is oxidized back to NAD+ and FADH2 is oxidized back to FAD. These oxidized carriers can then return to earlier stages of respiration and accept more electrons.

> **Key Term: electron transport chain**
> A series of membrane proteins that pass electrons from NADH and FADH2, using released energy to help build a proton gradient for ATP production.

Oxygen is the terminal electron acceptor in aerobic cellular respiration. At the end of the electron transport chain, oxygen accepts electrons and protons to form water. If oxygen is unavailable, electrons cannot move through the chain normally. NADH and FADH2 cannot be efficiently oxidized back to NAD+ and FAD. Without enough NAD+ and FAD, pyruvate oxidation and the Krebs cycle slow or stop because they require oxidized electron carriers.

This explains why the Krebs cycle has an indirect dependence on oxygen. Oxygen is not one of the listed inputs of the Krebs cycle, but oxygen is required for the electron transport chain to keep regenerating the electron carriers that the cycle needs.

> **Exam Warning:**
> Avoid writing that the Krebs cycle “uses oxygen” as a direct reactant. A stronger AP answer says that the Krebs cycle depends indirectly on oxygen because oxygen allows oxidative phosphorylation to regenerate NAD+ and FAD.

When oxygen is limited, such as in muscle cells during intense exercise, the electron transport chain cannot keep up with demand. Cellular respiration may slow, and fermentation can take over to regenerate NAD+ so glycolysis can continue. Fermentation does not produce extra ATP beyond glycolysis, but it allows glycolysis to keep making a small ATP supply. In human muscle cells, pyruvate can be converted to lactic acid during fermentation.

> **Key Term: fermentation**
> An anaerobic process that regenerates NAD+ from NADH so glycolysis can continue when oxidative phosphorylation is not available.

Exercise is a useful AP context because ATP demand increases. Muscle cells require more energy, so respiration speeds up when oxygen and substrates are available. Increased activity in the Krebs cycle leads to increased production of NADH and FADH2, which deliver electrons to the electron transport chain. If oxygen becomes too limited, the electron transport chain slows or stops and fermentation becomes more significant.

AP Biology questions may also test Krebs cycle data. One common measure is citrate synthase activity. Citrate synthase is an enzyme involved in the formation of citrate from acetyl-CoA and oxaloacetate. Higher citrate synthase activity can be used as evidence of higher Krebs cycle activity, especially in experimental comparisons before and after exercise.

> **Key Term: citrate synthase**
> An enzyme associated with the first step of the Krebs cycle; its activity is often used as an indicator of Krebs cycle activity.

For example, if a data set shows citrate synthase activity rising after exercise in several participant groups, a valid conclusion is that Krebs cycle activity increased after exercise. If you are asked to graph such data, use the variable being used as the measure of Krebs cycle activity on the y-axis, the groups or treatments on the x-axis, and include error bars if provided. If the question provides standard error or confidence intervals, those values help judge whether apparent differences are likely meaningful.

Suppose one group has a mean value of 72 before exercise with a standard error of 7.9, and 83 after exercise with a standard error of 4.8. The error ranges overlap because 72 + 7.9 = 79.9 and 83 - 4.8 = 78.2. In an AP-style answer, you should not simply say “83 is bigger than 72.” You should refer to the overlap in error bars and conclude that the data do not clearly support an increase for that group.

> **Test Tip:**
> In data questions, earn points by pairing your claim with numbers. A strong answer might say: “The post-exercise mean is higher, but the error bars overlap, so the data do not clearly show an increase.” Claims without data support often lose credit.

A follow-up experimental prediction should match the biological pattern. If citrate synthase activity increases after exercise, another valid measure of Krebs cycle activity would also be expected to increase, assuming it measures the same process. A good justification would connect exercise to increased ATP demand, increased respiration, and increased need for electron carriers feeding the electron transport chain.

## Key Point Checklist

This article has covered the following key knowledge points:

- The Krebs cycle is also called the citric acid cycle.
- In eukaryotic cells, the Krebs cycle occurs in the mitochondrial matrix.
- Pyruvate oxidation converts each pyruvate into acetyl-CoA, carbon dioxide, and NADH.
- Each glucose produces two pyruvates, so pyruvate oxidation and the Krebs cycle each occur twice per glucose.
- Acetyl-CoA combines with oxaloacetate to form citrate, a six-carbon molecule.
- Oxaloacetate is regenerated at the end of the cycle, allowing the pathway to continue.
- One turn of the Krebs cycle produces 2 carbon dioxide, 3 NADH, 1 FADH2, and 1 ATP.
- Per glucose, the Krebs cycle produces 4 carbon dioxide, 6 NADH, 2 FADH2, and 2 ATP.
- ATP made directly in the Krebs cycle is produced by substrate-level phosphorylation.
- NADH and FADH2 carry electrons from the Krebs cycle to the electron transport chain.
- Oxygen is needed indirectly because it allows the electron transport chain to regenerate NAD+ and FAD.
- Citrate synthase activity can be used as an experimental measure of Krebs cycle activity.

## Key Terms and Concepts

- cellular respiration
- Krebs cycle
- citric acid cycle
- mitochondrial matrix
- pyruvate oxidation
- acetyl-CoA
- coenzyme A
- oxaloacetate
- citrate
- carbon dioxide
- NADH
- FADH2
- substrate-level phosphorylation
- electron transport chain
- oxidative phosphorylation
- oxygen
- fermentation
- citrate synthase


Cellular respiration - Krebs (citric acid) cycle