How do your cells extract energy from the food that you eat?

As it turns out, cells have a network of elegant

metabolic pathways dedicated to just this task. Learn more about cellular respiration, fermentation, and other
processes that extract energy from fuel molecules like glucose.
Steps of cellular respiration
Introduction
Cellular respiration is one of the most elegant, majestic, and fascinating metabolic pathways on earth. At the
same time, it’s also one of the most complicated. When I learned about it for the first time, I felt like I had
tripped and fallen into a can of organic-chemistry-flavored alphabet soup!
Luckily, cellular respiration is not so scary once you get to know it. Let's start by looking at cellular
respiration at a high level, walking through the four major stages and tracing how they connect up to one
another.
Steps of cellular respiration

Overview of the steps of cellular respiration.

1. Glycolysis. Six-carbon glucose is converted into two pyruvates (three carbons each). ATP and NADH are made.
These reactions take place in the cytosol.
2. Pyruvate oxidation. Pyruvate travels into the mitochondrial matrix and is converted to a two-carbon molecule
bound to coenzyme A, called acetyl CoA. Carbon dioxide is released and NADH is made.
3. Citric acid cycle. The acetyl CoA combines with a four-carbon molecule and goes through a cycle of reactions,
ultimately regenerating the four-carbon starting molecule. ATP (or, in some cases, GTP), NADH, and FADH_2
are made, and carbon dioxide is released. These reactions take place in the mitochondrial matrix.
4. Oxidative phosphorylation. The NADH and FADH_2 produced in other steps deposit their electrons in the
electron transport chain in the inner mitochondrial membrane. As electrons move down the chain, energy is
released and used to pump protons out of the matrix and into the intermembrane space, forming a gradient.
The protons flow back into the matrix through an enzyme called ATP synthase, making ATP. At the end of the
electron transport chain, oxygen accepts electrons and takes up protons to form water.
During cellular respiration, a glucose molecule is gradually broken down into carbon dioxide and water.
Along the way, some ATP is produced directly in the reactions that transform glucose. Much more ATP,
however, is produced later in a process called oxidative phosphorylation. Oxidative phosphorylation is
powered by the movement of electrons through the electron transport chain, a series of proteins embedded
in the inner membrane of the mitochondrion.
These electrons come originally from glucose and are shuttled to the electron transport chain by electron
carriers \text{NAD}^+NAD+ and \text{FAD}FAD, which
become \text{NADH}NADHand \text{FADH}_2FADH2 when they gain electrons. To be clear, this is what's
happening in the diagram above when it says ++ \text {NADH}NADH or ++ \text{FADH}_2FADH2. The
molecule isn't appearing from scratch, it's just being converted to its electron-carrying form:
\text {NAD}^+NAD+ ++ 2 e^-2e− ++ 2 \text H^+2H+ \rightarrow→ \text {NADH}NADH ++ \text H^+H+
\text {FAD}FAD ++ 2e^-2e− ++ 2 \text H^+2H+ \rightarrow→ \text {FADH}_2FADH2
To see how a glucose molecule is converted into carbon dioxide and how its energy is harvested as ATP
and \text{NADH}NADH//\text{FADH}_2FADH2 in one of your body's cells, let’s walk step by step through the
four stages of cellular respiration.
1. Glycolysis. In glycolysis, glucose—a six-carbon sugar—undergoes a series of chemical transformations. In
the end, it gets converted into two molecules of pyruvate, a three-carbon organic molecule. In these reactions,
ATP is made, and \text{NAD}^+NAD+ is converted to \text{NADH}NADH.
2. Pyruvate oxidation. Each pyruvate from glycolysis goes into the mitochondrial matrix—the innermost
compartment of mitochondria. There, it’s converted into a two-carbon molecule bound to Coenzyme A,
known as acetyl CoA. Carbon dioxide is released and \text{NADH}NADH is generated.
3. Citric acid cycle. The acetyl CoA made in the last step combines with a four-carbon molecule and goes
through a cycle of reactions, ultimately regenerating the four-carbon starting molecule.
ATP, \text{NADH}NADH, and \text{FADH}_2FADH2are produced, and carbon dioxide is released.
4. Oxidative phosphorylation. The \text{NADH}NADH and \text{FADH}_2FADH2 made in other steps deposit
their electrons in the electron transport chain, turning back into their "empty" forms
(\text{NAD}^+NAD+ and \text{FAD}FAD). As electrons move down the chain, energy is released and used to
pump protons out of the matrix, forming a gradient. Protons flow back into the matrix through an enzyme
called ATP synthase, making ATP. At the end of the electron transport chain, oxygen accepts electrons and
takes up protons to form water.
Glycolysis can take place without oxygen in a process called fermentation. The other three stages of cellular
respiration—pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation—require oxygen in
order to occur. Only oxidative phosphorylation uses oxygen directly, but the other two stages can't run
without oxidative phosphorylation.
Each stage of cellular respiration is covered in more detail in other articles and videos on the site. Try
watching the overview video, or jump straight to an article on a particular stage by using the links above.
[References]
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Glycolysis
Glycolysis is the first step in the breakdown of glucose to extract energy for cellular metabolism. Glycolysis
consists of an energy-requiring phase followed by an energy-releasing phase.
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Introduction
Suppose that we gave one molecule of glucose to you and one molecule of glucose to Lactobacillus
acidophilus—the friendly bacterium that turns milk into yogurt. What would you and the bacterium do with
your respective glucose molecules?
Overall, the metabolism of glucose in one of your cells would be pretty different from its metabolism
in Lactobacillus—check out the fermentation article for more details. Yet, the first steps would be the same in
both cases: both you and the bacterium would need to split the glucose molecule in two by putting it through
glycolysis^11.
What is glycolysis?
Glycolysis is a series of reactions that extract energy from glucose by splitting it into two three-carbon
molecules called pyruvates. Glycolysis is an ancient metabolic pathway, meaning that it evolved long ago, and
it is found in the great majority of organisms alive today^{2,3}2,3.
In organisms that perform cellular respiration, glycolysis is the first stage of this process. However, glycolysis
doesn’t require oxygen, and many anaerobic organisms—organisms that do not use oxygen—also have this
pathway.
Highlights of glycolysis
Glycolysis has ten steps, and depending on your interests—and the classes you’re taking—you may want to
know the details of all of them. However, you may also be looking for a greatest hits version of glycolysis,
something that highlights the key steps and principles without tracing the fate of every single atom. Let’s start
with a simplified version of the pathway that does just that.
Glycolysis takes place in the cytosol of a cell, and it can be broken down into two main phases: the energy-
requiring phase, above the dotted line in the image below, and the energy-releasing phase, below the dotted
line.
 Energy-requiring phase. In this phase, the starting molecule of glucose gets rearranged, and two phosphate
groups are attached to it. The phosphate groups make the modified sugar—now called fructose-1,6-
bisphosphate—unstable, allowing it to split in half and form two phosphate-bearing three-carbon sugars.
Because the phosphates used in these steps come from \text{ATP}ATP, two \text{ATP}ATP molecules get
used up.

Simplified diagram of glycolysis.

Energy investment phase. Glucose is first converted to fructose-1,6-bisphosphate in a series of steps that use
up two ATP. Then, unstable fructose-1,6-bisphosphate splits in two, forming two three-carbon molecules
called DHAP and glyceraldehyde-3-phosphae. Glyceraldehyde-3-phosphate can continue with the next steps of
the pathway, and DHAP can be readily converted into glyceraldehyde-3-phosphate.
Energy payoff phase. In a series of steps that produce one NADH and two ATP, a glyceraldehyde-3-phosphate
molecule is converted into a pyruvate molecule. This happens twice for each molecule of glucose since glucose
is split into two three-carbon molecules, both of which will go through the final steps of the pathway.
 The three-carbon sugars formed when the unstable sugar breaks down are different from each other. Only
one—glyceraldehyde-3-phosphate—can enter the following step. However, the unfavorable
sugar, \text{DHAP}DHAP, can be easily converted into the favorable one, so both finish the pathway in the
end
 Energy-releasing phase. In this phase, each three-carbon sugar is converted into another three-carbon
molecule, pyruvate, through a series of reactions. In these reactions, two \text{ATP}ATP molecules and
one \text{NADH}NADHmolecule are made. Because this phase takes place twice, once for each of the two
three-carbon sugars, it makes four \text{ATP}ATP and two \text{NADH}NADH overall.
Each reaction in glycolysis is catalyzed by its own enzyme. The most important enzyme for regulation of
glycolysis is phosphofructokinase, which catalyzes formation of the unstable, two-phosphate sugar
molecule, fructose-1,6-bisphosphate^44. Phosphofructokinase speeds up or slows down glycolysis in
response to the energy needs of the cell.
Overall, glycolysis converts one six-carbon molecule of glucose into two three-carbon molecules of pyruvate.
The net products of this process are two molecules of \text{ATP}ATP (44 \text{ATP}ATP produced -
− 22 \text{ATP}ATP used up) and two molecules of \text{NADH}NADH.
Detailed steps: Energy-requiring phase
We’ve already seen what happens on a broad level during the energy-requiring phase of glycolysis.
Two \text{ATP}ATPs are spent to form an unstable sugar with two phosphate groups, which then splits to
form two three-carbon molecules that are isomers of each other.
Next, we’ll look at the individual steps in greater detail. Each step is catalyzed by its own specific enzyme,
whose name is indicated below the reaction arrow in the diagram below.

Detailed steps of the first half of glycolysis.

1. Glucose is converted to glucose-6-phosphate by hexokinase. This step converts one ATP to ADP.
2. Glucose-6-phosphate is converted into fructose-6-phosphate by phosphoglucose isomerase.
3. Fructose-6-phosphate is converted into fructose-1,6-bisphosphate by phosphofructokinase. This step converts
one ATP to ADP.
4. Fructose-1,6-bisphosphate is split into two three-carbon molecules, glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate (DHAP). The enzyme that catalyzes this step is fructose bisphosphate aldolase.
5. The DHAP is converted into glyceralde-3-phosphate by an enzyme called triose phosphate isomerase. This
reaction can go in either direction, but because glyceraldehyde-3-phosphate is continually being used up in
the rest of the pathway, the equilibrium favors conversion of DHAP to glyceraldehyde-3-phosphate.
Image credit: modified from "Glycolysis: Figure 1" by OpenStax College, Biology, CC BY 3.0
Step 1. A phosphate group is transferred from \text{ATP}ATP to glucose, making glucose-6-phosphate.
Glucose-6-phosphate is more reactive than glucose, and the addition of the phosphate also traps glucose
inside the cell since glucose with a phosphate can’t readily cross the membrane.
Step 2. Glucose-6-phosphate is converted into its isomer, fructose-6-phosphate.
Step 3. A phosphate group is transferred from \text{ATP}ATP to fructose-6-phosphate, producing fructose-
1,6-bisphosphate. This step is catalyzed by the enzyme phosphofructokinase, which can be regulated to speed
up or slow down the glycolysis pathway.
 Step 4. Fructose-1,6-bisphosphate splits to form two three-carbon sugars: dihydroxyacetone phosphate
(\text{DHAP}DHAP) and glyceraldehyde-3-phosphate. They are isomers of each other, but only one—
glyceraldehyde-3-phosphate—can directly continue through the next steps of glycolysis.
Step 5. \text{DHAP}DHAP is converted into glyceraldehyde-3-phosphate. The two molecules exist in
equilibrium, but the equilibrium is “pulled” strongly downward, in the scheme of the diagram above, as
glyceraldehyde-3-phosphate is used up. Thus, all of the \text{DHAP}DHAP is eventually converted.
Detailed steps: Energy-releasing phase
In the second half of glycolysis, the three-carbon sugars formed in the first half of the process go through a
series of additional transformations, ultimately turning into pyruvate. In the process,
four \text{ATP}ATP molecules are produced, along with two molecules of \text{NADH}NADH.
Here, we’ll look in more detail at the reactions that lead to these products. The reactions shown below happen
twice for each glucose molecule since a glucose splits into two three-carbon molecules, both of which will
eventually proceed through the pathway.

Detailed steps of the second half of glycolysis. All of these reactions will happen twice for one molecule of
glucose.
6. Glyceraldehyde-3-phosphate is converted into 1,3-bisphosphoglycerate. This is a redox reaction in which
NAD+ is converted to NADH (with the release of an H+ ion). An inorganic phosphate is also a reactant for this
reaction, which is catalyzed by glyceraldehyde-3-phosphate dehydrogenase.
7. 1,3-bisphosphoglycerate is converted to 3-phosphoglycerate by phosphoglycerate kinase. This step converts
an ADP to an ATP.
8. 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase.
9. 2-phosphoglycerate is converted to phosphoenolpyruvate (PEP) by enolase. This reaction releases a water
molecule.
10. Phosphoenolpyruvate (PEP) is converted to pyruvate by pyruvate kinase. An ADP is converted to an ATP in
this reaction.
Image modified from "Glycolysis: Figure 2," by OpenStax College, Biology (CC BY 3.0).
Step 6. Two half reactions occur simultaneously: 1) Glyceraldehyde-3-phosphate (one of the three-carbon
sugars formed in the initial phase) is oxidized, and 2) \text{NAD}^{+}NAD+ is reduced
to \text{NADH}NADH and \text{H}^+H+. The overall reaction is exergonic, releasing energy that is then used
to phosphorylate the molecule, forming 1,3-bisphosphoglycerate.
Step 7. 1,3-bisphosphoglycerate donates one of its phosphate groups to \text{ADP}ADP, making a molecule
of \text{ATP}ATP and turning into 3-phosphoglycerate in the process.
Step 8. 3-phosphoglycerate is converted into its isomer, 2-phosphoglycerate.
Step 9. 2-phosphoglycerate loses a molecule of water, becoming phosphoenolpyruvate
(\text{PEP}PEP). \text{PEP}PEP is an unstable molecule, poised to lose its phosphate group in the final step
of glycolysis.
Step 10. \text{PEP}PEP readily donates its phosphate group to \text{ADP}ADP, making a second molecule
of \text{ATP}ATP. As it loses its phosphate, \text{PEP}PEP is converted to pyruvate, the end product of
glycolysis.
What happens to pyruvate and \text{NADH}NADH?
At the end of glycolysis, we’re left with two \text{ATP}ATP, two \text{NADH}NADH, and two pyruvate
molecules. If oxygen is available, the pyruvate can be broken down (oxidized) all the way to carbon dioxide in
cellular respiration, making many molecules of \text{ATP}ATP. You can learn how this works in the videos
and articles on pyruvate oxidation, the citric acid cycle, and oxidative phosphorylation.
What happens to the \text{NADH}NADH? It can't just sit around in the cell, piling up. That's because cells
have only a certain number of \text{NAD}^+NAD+ molecules, which cycle back and forth between oxidized
(\text{NAD}^+NAD+) and reduced (\text{NADH}NADH) states:
\text{\blue{NAD}}^+NAD+ ++ 2\text {e}^-2e− ++ 2 \text
{\purple{H}}^+2H+ \rightleftharpoons⇌ \text{\blue{NAD}}NAD\text{\purple{H}}H ++ \text{
\purple{H}}^+ H+
Glycolysis needs \text{NAD}^+NAD+ to accept electrons as part of a specific reaction. If there’s
no \text{NAD}^+NAD+ around (because it's all stuck in its \text{NADH}NADH form), this reaction can’t
happen and glycolysis will come to a halt. So, all cells need a way to turn \text{NADH}NADH back
into \text{NAD}^+NAD+ to keep glycolysis going.
There are two basic ways of accomplishing this. When oxygen is present, \text{NADH}NADH can pass its
electrons into the electron transport chain, regenerating \text{NAD}^+NAD+ for use in glycolysis. (Added
bonus: some \text{ATP}ATP gets made!)
When oxygen is absent, cells may use other, simpler pathways to regenerate \text{NAD}^+NAD+. In these
pathways, \text{NADH}NADH donates its electrons to an acceptor molecule in a reaction that doesn’t
make \text{ATP}ATP but does regenerate \text{NAD}^+NAD+ so glycolysis can continue. This process is
called fermentation, and you can learn more about it in the fermentation videos.
Fermentation is a primary metabolic strategy for lots of bacteria—including our friend from the
introduction, Lactobacillus acidophilus^11. Even some cells in your body, such as red blood cells, rely on
fermentation to make their \text{ATP}ATP.
© 2019 Khan Academy
Terms of use

Pyruvate oxidation
How pyruvate from glycolysis is converted to acetyl CoA so it can enter the citric acid cycle. Pyruvate is
modified by removal of a carboxyl group followed by oxidation, and then attached to Coenzyme A.
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Introduction
Among the four stages of cellular respiration, pyruvate oxidation is kind of the odd one out; it’s relatively
short in comparison to the extensive pathways of glycolysis or the citric acid cycle. But that doesn’t make it
unimportant! On the contrary, pyruvate oxidation is a key connector that links glycolysis to the rest of cellular
respiration.
Overview of pyruvate oxidation
At the end of glycolysis, we have two pyruvate molecules that still contain lots of extractable energy. Pyruvate
oxidation is the next step in capturing the remaining energy in the form of \text{ATP}ATP, although
no \text{ATP}ATP is made directly during pyruvate oxidation.
Simplified diagram of pyruvate oxidation. Pyruvate—three carbons—is converted to acetyl CoA, a two-carbon
molecule attached to coenzyme A. A molecule of coenzyme A is a necessary reactant for this reaction, which
releases a molecule of carbon dioxide and reduces a NAD+ to NADH.
In eukaryotes, this step takes place in the matrix, the innermost compartment of mitochondria. In
prokaryotes, it happens in the cytoplasm. Overall, pyruvate oxidation converts pyruvate—a three-carbon
molecule—into acetyl \text{CoA}CoA—a two-carbon molecule attached to Coenzyme A—producing
an \text{NADH}NADH and releasing one carbon dioxide molecule in the process. Acetyl \text{CoA}CoA acts as
fuel for the citric acid cycle in the next stage of cellular respiration.
Pyruvate oxidation steps
Pyruvate is produced by glycolysis in the cytoplasm, but pyruvate oxidation takes place in the mitochondrial
matrix (in eukaryotes). So, before the chemical reactions can begin, pyruvate must enter the mitochondrion,
crossing its inner membrane and arriving at the matrix.
In the matrix, pyruvate is modified in a series of steps:
 More detailed diagram of the mechanism of pyruvate oxidation.
1. A carboxyl group is removed from pyruvate and released as carbon dioxide.
2. The two-carbon molecule from the first step is oxidized, and NAD+ accepts the electrons to form NADH.
3. The oxidized two-carbon molecule, an acetyl group, is attached to Coenzyme A to form acetyl CoA.
Image credit: "Oxidation of pyruvate and the citric acid cycle: Figure 1" by OpenStax College, Biology, CC BY 3.0
Step 1. A carboxyl group is snipped off of pyruvate and released as a molecule of carbon dioxide, leaving
behind a two-carbon molecule.
Step 2. The two-carbon molecule from step 1 is oxidized, and the electrons lost in the oxidation are picked up
by \text{NAD}^+NAD+ to form \text{NADH}NADH.
Step 3. The oxidized two-carbon molecule—an acetyl group, highlighted in green—is attached to Coenzyme A
(\text{CoA}CoA), an organic molecule derived from vitamin B5, to form acetyl \text{CoA}CoA.
Acetyl \text{CoA}CoA is sometimes called a carrier molecule, and its job here is to carry the acetyl group to
the citric acid cycle.
The steps above are carried out by a large enzyme complex called the pyruvate dehydrogenase complex,
which consists of three interconnected enzymes and includes over 60 subunits. At a couple of stages, the
reaction intermediates actually form covalent bonds to the enzyme complex—or, more specifically, to its
cofactors. The pyruvate dehydrogenase complex is an important target for regulation, as it controls the
amount of acetyl \text{CoA}CoA fed into the citric acid cycle^{1,2,3}1,2,3.
If we consider the two pyruvates that enter from glycolysis (for each glucose molecule), we can summarize
pyruvate oxidation as follows:
 Two molecules of pyruvate are converted into two molecules of acetyl \text{CoA}CoA.
 Two carbons are released as carbon dioxide—out of the six originally present in glucose.
 2 \text{NADH}NADH are generated from \text{NAD}^+NAD+.
Why make acetyl \text{CoA}CoA? Acetyl \text{CoA}CoA serves as fuel for the citric acid cycle in the next stage
of cellular respiration. The addition of \text{CoA}CoA helps activate the acetyl group, preparing it to undergo
the necessary reactions to enter the citric acid cycle.
The citric acid cycle
Overview and steps of the citric acid cycle, also known as the Krebs cycle or tricarboxylic acid (TCA) cycle.
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Introduction
How important is the citric acid cycle? So important that it has not one, not two, but three different names in
common usage today!
The name we'll primarily use here, the citric acid cycle, refers to the first molecule that forms during the
cycle's reactions—citrate, or, in its protonated form, citric acid. However, you may also hear this series of
reactions called the tricarboxylic acid (TCA) cycle, for the three carboxyl groups on its first two intermediates,
or the Krebs cycle, after its discoverer, Hans Krebs.
[See a picture of the first two intermediates]
\text {H}^+

Whatever you prefer to call it, the citric cycle is a central driver of cellular respiration. It takes
acetyl \text{CoA}CoA—produced by the oxidation of pyruvate and originally derived from glucose—as its
starting material and, in a series of redox reactions, harvests much of its bond energy in the form
of \text{NADH}NADH, \text{FADH}_2FADH2, and \text{ATP}ATP molecules. The reduced electron carriers—
\text{NADH}NADH and \text{FADH}_2FADH2—generated in the TCA cycle will pass their electrons into the
electron transport chain and, through oxidative phosphorylation, will generate most of the ATP produced in
cellular respiration.
Below, we’ll look in more detail at how this remarkable cycle works.
Overview of the citric acid cycle
In eukaryotes, the citric acid cycle takes place in the matrix of the mitochondria, just like the conversion of
pyruvate to acetyl \text{CoA}CoA. In prokaryotes, these steps both take place in the cytoplasm. The citric acid
cycle is a closed loop; the last part of the pathway reforms the molecule used in the first step. The cycle
includes eight major steps.
 Simplified diagram of the citric acid cycle. First, acetyl CoA combines with oxaloacetate, a four-carbon
molecule, losing the CoA group and forming the six-carbon molecule citrate. After citrate undergoes a
rearrangement step, it undergoes an oxidation reaction, transferring electrons to NAD+ to form NADH and
releasing a molecule of carbon dioxide. The five-carbon molecule left behind then undergoes a second, similar
reaction, transferring electrons to NAD+ to form NADH and releasing a carbon dioxide molecule. The four-
carbon molecule remaining then undergoes a series of transformations, in the course of which GDP and
inorganic phosphate are converted into GTP—or, in some organisms, ADP and inorganic phosphate are
converted into ATP—an FAD molecule is reduced to FADH2, and another NAD+ is reduced to NADH. At the
end of this series of reactions, the four-carbon starting molecule, oxaloacetate, is regenerated, allowing the
cycle to begin again.
In the first step of the cycle, acetyl \text{CoA}CoA combines with a four-carbon acceptor molecule,
oxaloacetate, to form a six-carbon molecule called citrate. After a quick rearrangement, this six-carbon
molecule releases two of its carbons as carbon dioxide molecules in a pair of similar reactions, producing a
molecule of \text{NADH}NADH each time^11. The enzymes that catalyze these reactions are key regulators of
the citric acid cycle, speeding it up or slowing it down based on the cell’s energy needs^22.
The remaining four-carbon molecule undergoes a series of additional reactions, first making
an \text{ATP}ATP molecule—or, in some cells, a similar molecule called \text{GTP}GTP—then reducing the
electron carrier \text{FAD}FAD to \text{FADH}_2FADH2, and finally generating another \text{NADH}NADH.
This set of reactions regenerates the starting molecule, oxaloacetate, so the cycle can repeat.
Overall, one turn of the citric acid cycle releases two carbon dioxide molecules and produces
three \text{NADH}NADH, one \text{FADH}_2FADH2, and one \text{ATP}ATP or \text{GTP}GTP. The citric
acid cycle goes around twice for each molecule of glucose that enters cellular respiration because there are
two pyruvates—and thus, two acetyl \text{CoA}CoAs—made per glucose.
Steps of the citric acid cycle
You've already gotten a preview of the molecules produced during the citric acid cycle. But how, exactly, are
those molecules made? We’ll walk through the cycle step by step, seeing
how \text{NADH}NADH, \text{FADH}_2FADH2, and \text{ATP}ATP/\text{GTP}GTP are produced and where
carbon dioxide molecules are released.
Step 1. In the first step of the citric acid cycle, acetyl \text{CoA}CoA joins with a four-carbon molecule,
oxaloacetate, releasing the \text{CoA}CoA group and forming a six-carbon molecule called citrate.
Step 2. In the second step, citrate is converted into its isomer, isocitrate. This is actually a two-step process,
involving first the removal and then the addition of a water molecule, which is why the citric acid cycle is
sometimes described as having nine steps—rather than the eight listed here^33.
Step 3. In the third step, isocitrate is oxidized and releases a molecule of carbon dioxide, leaving behind a
five-carbon molecule—α-ketoglutarate. During this step, \text{NAD}^+NAD+ is reduced to
form \text{NADH}NADH. The enzyme catalyzing this step, isocitrate dehydrogenase, is important in
regulating the speed of the citric acid cycle.
Step 4. The fourth step is similar to the third. In this case, it’s α-ketoglutarate that’s oxidized,
reducing \text{NAD}^+NAD+ to \text{NADH}NADH and releasing a molecule of carbon dioxide in the process.
The remaining four-carbon molecule picks up Coenzyme A, forming the unstable compound
succinyl \text{CoA}CoA. The enzyme catalyzing this step, α-ketoglutarate dehydrogenase, is also important
in regulation of the citric acid cycle.
 Detailed diagram of the citric acid cycle, showing the structures of the various cycle intermediates and the
enzymes catalyzing each step.
Step 1. Acetyl CoA combines with oxaloacetate in a reaction catalyzed by citrate synthase. This reaction also
takes a water molecule as a reactant, and it releases a SH-CoA molecule as a product.
Step 2. Citrate is converted into isocitrate in a reaction catalyzed by aconitase.
Step 3. Isocitrate is converted into α-ketoglutarate in a reaction catalyzed by isocitrate dehydrogenase. An
NAD+ molecule is reduced to NADH + H+ in this reaction, and a carbon dioxide molecule is released as a
product.
Step 4. α-ketoglutarate is converted to succinyl CoA in a reaction catalyzed by α-ketoglutarate dehydrogenase.
An NAD+ molecule is reduced to NADH + H+ in this reaction, which also takes a SH-CoA molecule as reactant.
A carbon dioxide molecule is released as a product.
Step 5. Succinyl CoA is converted to succinate in a reaction catalyzed by the enzyme succinyl-CoA synthetase.
This reaction converts inorganic phosphate, Pi, and GDP to GTP and also releases a SH-CoA group.
Step 6. Succinate is converted to fumarate in a reaction catalyzed by succinate dehydrogenase. FAD is reduced
to FADH2 in this reaction.
Step 7. Fumarate is converted to malate in a reaction catalyzed by the enzyme fumarase. This reaction
requires a water molecule as a reactant.
Step 8. Malate is converted to oxaloacetate in a reaction catalyzed by malate dehydrogenase. This reaction
reduces an NAD+ molecule to NADH + H+.
image credit: modified from "Oxidation of pyruvate and citric acid cycle: Figure 2" by OpenStax College,
Biology, CC BY 3.0
Step 5. In step five, the \text{CoA}CoA of succinyl \text{CoA}CoA is replaced by a phosphate group, which is
then transferred to \text{ADP}ADP to make \text{ATP}ATP. In some cells, \text{GDP}GDP—guanosine
diphosphate—is used instead of \text{ADP}ADP, forming \text{GTP}GTP—guanosine triphosphate—as a
product. The four-carbon molecule produced in this step is called succinate.
[Learn more about GTP.]
\text{GTP}\text{ATP}\text{ATP}\text{GTP}
Step 6. In step six, succinate is oxidized, forming another four-carbon molecule called fumarate. In this
reaction, two hydrogen atoms—with their electrons—are transferred to \text{FAD}FAD,
producing \text{FADH}_2FADH2. The enzyme that carries out this step is embedded in the inner membrane
of the mitochondrion, so \text{FADH}_2FADH2 can transfer its electrons directly into the electron transport
chain.
[Why use FAD here?]
\text{FAD}\text{NAD}^+\text{NAD}^+\text{FAD}^{4,5}
Step 7. In step seven, water is added to the four-carbon molecule fumarate, converting it into another four-
carbon molecule called malate.
Step 8. In the last step of the citric acid cycle, oxaloacetate—the starting four-carbon compound—is
regenerated by oxidation of malate. Another molecule of \text{NAD}^+NAD+ is reduced
to \text{NADH}NADH in the process.
Products of the citric acid cycle
Let’s take a step back and do some accounting, tracing the fate of the carbons that enter the citric acid cycle
and counting the reduced electron carriers—\text{NADH}NADH and \text{FADH}_2FADH2—
and \text{ATP}ATP produced.
In a single turn of the cycle,
 two carbons enter from acetyl \text{CoA}CoA, and two molecules of carbon dioxide are released;
 three molecules of \text{NADH}NADH and one molecule of \text{FADH}_2FADH2 are generated; and
 one molecule of \text{ATP}ATP or \text{GTP}GTP is produced.
These figures are for one turn of the cycle, corresponding to one molecule of acetyl \text{CoA}CoA. Each
glucose produces two acetyl \text{CoA}CoA molecules, so we need to multiply these numbers by 22 if we
want the per-glucose yield.
Two carbons—from acetyl \text{CoA}CoA—enter the citric acid cycle in each turn, and two carbon dioxide
molecules are released. However, the carbon dioxide molecules don’t actually contain carbon atoms from the
acetyl \text{CoA}CoA that just entered the cycle. Instead, the carbons from acetyl \text{CoA}CoA are initially
incorporated into the intermediates of the cycle and are released as carbon dioxide only during later turns.
After enough turns, all the carbon atoms from the acetyl group of acetyl \text{CoA}CoA will be released as
carbon dioxide.
Where’s all the \text{ATP}ATP?
You may be thinking that the \text{ATP}ATP output of the citric acid cycle seems pretty unimpressive. All that
work for just one \text{ATP}ATP or \text{GTP}GTP?
It’s true that the citric acid cycle doesn’t produce much \text{ATP}ATP directly. However, it can make a lot
of \text{ATP}ATP indirectly, by way of the \text{NADH}NADH and \text{FADH}_2FADH2 it generates. These
electron carriers will connect with the last portion of cellular respiration, depositing their electrons into the
electron transport chain to drive synthesis of ATP molecules through oxidative phosphorylation.

Oxidative phosphorylation
Overview of oxidative phosphorylation. The electron transport chain forms a proton gradient across the inner
mitochondrial membrane, which drives the synthesis of ATP via chemiosmosis.
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Why do we need oxygen?
You, like many other organisms, need oxygen to live. As you know if you’ve ever tried to hold your breath for
too long, lack of oxygen can make you feel dizzy or even black out, and prolonged lack of oxygen can even
cause death. But have you ever wondered why that’s the case, or what exactly your body does with all that
oxygen?
As it turns out, the reason you need oxygen is so your cells can use this molecule during oxidative
phosphorylation, the final stage of cellular respiration. Oxidative phosphorylation is made up of two closely
connected components: the electron transport chain and chemiosmosis. In the electron transport chain,
electrons are passed from one molecule to another, and energy released in these electron transfers is used to
form an electrochemical gradient. In chemiosmosis, the energy stored in the gradient is used to make ATP.
So, where does oxygen fit into this picture? Oxygen sits at the end of the electron transport chain, where it
accepts electrons and picks up protons to form water. If oxygen isn’t there to accept electrons (for instance,
because a person is not breathing in enough oxygen), the electron transport chain will stop running, and ATP
will no longer be produced by chemiosmosis. Without enough ATP, cells can’t carry out the reactions they
need to function, and, after a long enough period of time, may even die.
In this article, we'll examine oxidative phosphorylation in depth, seeing how it provides most of the ready
chemical energy (ATP) used by the cells in your body.
Overview: oxidative phosphorylation

Simple diagram of the electron transport chain. The electron transport chain is a series of proteins embedded
in the inner mitochondrial membrane.
In the matrix, NADH and FADH2 deposit their electrons in the chain (at the first and second complexes of the
chain, respectively).
The energetically "downhill" movement of electrons through the chain causes pumping of protons into the
intermembrane space by the first, third, and fourth complexes.
Finally, the electrons are passed to oxygen, which accepts them along with protons to form water.
The proton gradient produced by proton pumping during the electron transport chain is used to synthesize
ATP. Protons flow down their concentration gradient into the matrix through the membrane protein ATP
synthase, causing it to spin (like a water wheel) and catalyze conversion of ADP to ATP.
 The electron transport chain is a series of proteins and organic molecules found in the inner membrane of
the mitochondria. Electrons are passed from one member of the transport chain to another in a series of
redox reactions. Energy released in these reactions is captured as a proton gradient, which is then used to
make ATP in a process called chemiosmosis. Together, the electron transport chain and chemiosmosis make
up oxidative phosphorylation. The key steps of this process, shown in simplified form in the diagram above,
include:
 Delivery of electrons by NADH and FADH_22. Reduced electron carriers (NADH and FADH_22) from other
steps of cellular respiration transfer their electrons to molecules near the beginning of the transport chain. In
the process, they turn back into NAD^++ and FAD, which can be reused in other steps of cellular respiration.
 Electron transfer and proton pumping. As electrons are passed down the chain, they move from a higher to
a lower energy level, releasing energy. Some of the energy is used to pump H^++ ions, moving them out of the
matrix and into the intermembrane space. This pumping establishes an electrochemical gradient.
 Splitting of oxygen to form water. At the end of the electron transport chain, electrons are transferred to
molecular oxygen, which splits in half and takes up H^++ to form water.
 Gradient-driven synthesis of ATP. As H^++ ions flow down their gradient and back into the matrix, they
pass through an enzyme called ATP synthase, which harnesses the flow of protons to synthesize ATP.
We'll look more closely at both the electron transport chain and chemiosmosis in the sections below.
The electron transport chain
The electron transport chain is a collection of membrane-embedded proteins and organic molecules, most
of them organized into four large complexes labeled I to IV. In eukaryotes, many copies of these molecules are
found in the inner mitochondrial membrane. In prokaryotes, the electron transport chain components are
found in the plasma membrane.
As the electrons travel through the chain, they go from a higher to a lower energy level, moving from less
electron-hungry to more electron-hungry molecules. Energy is released in these “downhill” electron transfers,
and several of the protein complexes use the released energy to pump protons from the mitochondrial matrix
to the intermembrane space, forming a proton gradient.
[Click to see a free energy diagram]

Image of the electron transport chain. All the components of the chain are embedded in or attached to the
inner mitochondrial membrane. In the matrix, NADH deposits electrons at Complex I, turning into NAD+ and
releasing a proton into the matrix. FADH2 in the matrix deposits electrons at Complex II, turning into FAD and
releasing 2 H+. The electrons from Complexes I and II are passed to the small mobile carrier Q. Q transports
the electrons to Complex III, which then passes them to Cytochrome C. Cytochrome C passes the electrons to
Complex IV, which then passes them to oxygen in the matrix, forming water. It takes two electrons, 1/2 O2,
and 2 H+ to form one water molecule. Complexes I, III, and IV use energy released as electrons move from a
higher to a lower energy level to pump protons out of the matrix and into the intermembrane space,
generating a proton gradient.
Image modified from "Oxidative phosphorylation: Figure 1", by OpenStax College, Biology (CC BY 3.0).
All of the electrons that enter the transport chain come from NADH and FADH_22 molecules produced during
earlier stages of cellular respiration: glycolysis, pyruvate oxidation, and the citric acid cycle.
 NADH is very good at donating electrons in redox reactions (that is, its electrons are at a high energy level),
so it can transfer its electrons directly to complex I, turning back into NAD^++. As electrons move through
complex I in a series of redox reactions, energy is released, and the complex uses this energy to pump protons
from the matrix into the intermembrane space.
 FADH_22 is not as good at donating electrons as NADH (that is, its electrons are at a lower energy level), so it
cannot transfer its electrons to complex I. Instead, it feeds them into the transport chain through complex II,
which does not pump protons across the membrane.
 Because of this "bypass," each FADH_22 molecule causes fewer protons to be pumped (and contributes less to
the proton gradient) than an NADH.
[More about complexes I and II]
_2_2_2
Beyond the first two complexes, electrons from NADH and FADH_22 travel exactly the same route. Both
complex I and complex II pass their electrons to a small, mobile electron carrier called ubiquinone (Q),
which is reduced to form QH_22 and travels through the membrane, delivering the electrons to complex III.
As electrons move through complex III, more H^++ ions are pumped across the membrane, and the electrons
are ultimately delivered to another mobile carrier called cytochrome C (cyt C). Cyt C carries the electrons to
complex IV, where a final batch of H^++ ions is pumped across the membrane. Complex IV passes the
electrons to O_22, which splits into two oxygen atoms and accepts protons from the matrix to form water.
Four electrons are required to reduce each molecule of O_22, and two water molecules are formed in the
process.
[More about complexes III and IV]
^+
_2^1^+
Overall, what does the electron transport chain do for the cell? It has two important functions:
 Regenerates electron carriers. NADH and FADH_22 pass their electrons to the electron transport chain,
turning back into NAD^++ and FAD. This is important because the oxidized forms of these electron carriers
are used in glycolysis and the citric acid cycle and must be available to keep these processes running.
 Makes a proton gradient. The transport chain builds a proton gradient across the inner mitochondrial
membrane, with a higher concentration of H^++ in the intermembrane space and a lower concentration in
the matrix. This gradient represents a stored form of energy, and, as we’ll see, it can be used to make ATP.
Chemiosmosis
Complexes I, III, and IV of the electron transport chain are proton pumps. As electrons move energetically
downhill, the complexes capture the released energy and use it to pump H^++ ions from the matrix to the
intermembrane space. This pumping forms an electrochemical gradient across the inner mitochondrial
membrane. The gradient is sometimes called the proton-motive force, and you can think of it as a form of
stored energy, kind of like a battery.
Like many other ions, protons can't pass directly through the phospholipid bilayer of the membrane because
its core is too hydrophobic. Instead, H^++ ions can move down their concentration gradient only with the
help of channel proteins that form hydrophilic tunnels across the membrane.
In the inner mitochondrial membrane, H^++ ions have just one channel available: a membrane-spanning
protein known as ATP synthase. Conceptually, ATP synthase is a lot like a turbine in a hydroelectric power
plant. Instead of being turned by water, it’s turned by the flow of H^++ ions moving down their
electrochemical gradient. As ATP synthase turns, it catalyzes the addition of a phosphate to ADP, capturing
energy from the proton gradient as ATP.

Overview diagram of oxidative phosphorylation. The electron transport chain and ATP synthase are
embedded in the inner mitochondrial membrane. NADH and FADH2 made in the citric acid cycle (in the
mitochondrial matrix) deposit their electrons into the electron transport chain at complexes I and II,
respectively. This step regenerates NAD+ and FAD (the oxidized carriers) for use in the citric acid cycle. The
electrons flow through the electron transport chain, causing protons to be pumped from the matrix to the
intermembrane space. Eventually, the electrons are passed to oxygen, which combines with protons to form
water. The proton gradient generated by proton pumping during the electron transport chain is a stored form
of energy. When protons flow back down their concentration gradient (from the intermembrane space to the
matrix), their only route is through ATP synthase, an enzyme embedded in the inner mitochondrial
membrane. When protons flow through ATP synthase, they cause it to turn (much as water turns a water
wheel), and its motion catalyzes the conversion of ADP and Pi to ATP.
 Image modified from "Oxidative phosphorylation: Figure 3," by Openstax College, Biology (CC BY 3.0).
This process, in which energy from a proton gradient is used to make ATP, is called chemiosmosis. More
broadly, chemiosmosis can refer to any process in which energy stored in a proton gradient is used to do
work. Although chemiosmosis accounts for over 80% of ATP made during glucose breakdown in cellular
respiration, it’s not unique to cellular respiration. For instance, chemiosmosis is also involved in the light
reactions of photosynthesis.
What would happen to the energy stored in the proton gradient if it weren't used to synthesize ATP or do
other cellular work? It would be released as heat, and interestingly enough, some types of cells deliberately
use the proton gradient for heat generation rather than ATP synthesis. This might seem wasteful, but it's an
important strategy for animals that need to keep warm. For instance, hibernating mammals (such as bears)
have specialized cells known as brown fat cells. In the brown fat cells, uncoupling proteins are produced
and inserted into the inner mitochondrial membrane. These proteins are simply channels that allow protons
to pass from the intermembrane space to the matrix without traveling through ATP synthase. By providing an
alternate route for protons to flow back into the matrix, the uncoupling proteins allow the energy of the
gradient to be dissipated as heat.
ATP yield
How many ATP do we get per glucose in cellular respiration? If you look in different books, or ask different
professors, you'll probably get slightly different answers. However, most current sources estimate that the
maximum ATP yield for a molecule of glucose is around 30-32 ATP^{2,3,4}2,3,4. This range is lower than
previous estimates because it accounts for the necessary transport of ADP into, and ATP out of, the
mitochondrion.
[More details]

 ^{5,6}
Where does the figure of 30-32 ATP come from? Two net ATP are made in glycolysis, and another two ATP
(or energetically equivalent GTP) are made in the citric acid cycle. Beyond those four, the remaining ATP all
come from oxidative phosphorylation. Based on a lot of experimental work, it appears that four H^++ ions
must flow back into the matrix through ATP synthase to power the synthesis of one ATP molecule. When
electrons from NADH move through the transport chain, about 10 H^++ ions are pumped from the matrix to
the intermembrane space, so each NADH yields about 2.5 ATP. Electrons from FADH_22, which enter the
chain at a later stage, drive pumping of only 6 H^++, leading to production of about 1.5 ATP.
With this information, we can do a little inventory for the breakdown of one molecule of glucose:

Stage Direct products (net) Ultimate ATP yield (net)

Glycolysis 2 ATP 2 ATP

2 NADH 3-5 ATP

Pyruvate oxidation 2 NADH 5 ATP

Citric acid cycle 2 ATP/GTP 2 ATP

6 NADH 15 ATP

2 FADH_22 3 ATP

Total 30-32 ATP

 [Click here for a diagram showing ATP production]

One number in this table is still not precise: the ATP yield from NADH made in glycolysis. This is because
glycolysis happens in the cytosol, and NADH can't cross the inner mitochondrial membrane to deliver its
electrons to complex I. Instead, it must hand its electrons off to a molecular “shuttle system” that delivers
them, through a series of steps, to the electron transport chain.
 Some cells of your body have a shuttle system that delivers electrons to the transport chain via FADH_22. In
this case, only 3 ATP are produced for the two NADH of glycolysis.
 Other cells of your body have a shuttle system that delivers the electrons via NADH, resulting in the
production of 5 ATP.
In bacteria, both glycolysis and the citric acid cycle happen in the cytosol, so no shuttle is needed and 5 ATP
are produced.
30-32 ATP from the breakdown of one glucose molecule is a high-end estimate, and the real yield may be
lower. For instance, some intermediates from cellular respiration may be siphoned off by the cell and used in
other biosynthetic pathways, reducing the number of ATP produced. Cellular respiration is a nexus for many
different metabolic pathways in the cell, forming a network that’s larger than the glucose breakdown
pathways alone.

Self-check questions
1. Cyanide acts as a poison because it inhibits complex IV, making it unable to transport electrons. How would
cyanide poisoning affect 1) the electron transport chain and 2) the proton gradient across the inner
mitochondrial membrane?
Choose 1 answer:
Choose 1 answer:

o
(Choice A)
A
The electron transport chain would speed up, and the gradient would become stronger

o
(Choice B)
B
The electron transport chain would stop, and the gradient would decrease

o
(Choice C)
C
Both the electron transport chain and the gradient would stay the same

o
(Choice D)
D
The electron transport chain would be re-routed through complex II, and the gradient would become
weaker
[Hint]
2. Dinitrophenol (DNP) is a chemical that acts as an uncoupling agent, making the inner mitochondrial
membrane leaky to protons. It was used until 1938 as a weight-loss drug. How would DNP affect the
amount of ATP produced in cellular respiration? Why do you think it is now off the market?*
Choose 1 answer:
Choose 1 answer:
o
(Choice A)
A
It would increase ATP production, but could also cause dangerously high body temperature

o
(Choice B)
B
It would decrease ATP production, but could also cause dangerously high body temperature

o
(Choice C)
C
It would decrease ATP production, but could also cause dangerously low body temperature

o
(Choice D)
D
It would increase ATP production, but could also cause dangerously low body temperature