Oxidative Phosphorylation

LEARNING OBJECTIVES

By the end of this section, you will be able to do the following:

 Describe how electrons move through the electron transport chain and
explain what happens to their energy levels during this process
 Explain how a proton (H+) gradient is established and maintained by the
electron transport chain
You have just read about two pathways in glucose catabolism—glycolysis and the citric
acid cycle—that generate ATP. Most of the ATP generated during the aerobic
catabolism of glucose, however, is not generated directly from these pathways. Instead,
it is derived from a process that begins by moving electrons through a series of electron
carriers that undergo redox reactions. This process causes hydrogen ions to
accumulate within the matrix space. Therefore, a concentration gradient forms in which
hydrogen ions diffuse out of the matrix space by passing through ATP synthase. The
current of hydrogen ions powers the catalytic action of ATP synthase, which
phosphorylates ADP, producing ATP.

Electron Transport Chain

The electron transport chain ((Figure)) is the last component of aerobic respiration and
is the only part of glucose metabolism that uses atmospheric oxygen. Oxygen
continuously diffuses into plant tissues (typically through stomata), as well as into fungi
and bacteria; however, in animals, oxygen enters the body through a variety of
respiratory systems. Electron transport is a series of redox reactions that resembles a
relay race or bucket brigade in that electrons are passed rapidly from one component to
the next, to the endpoint of the chain where the electrons reduce molecular oxygen and,
along with associated protons, produces water. There are four complexes composed of
proteins, labeled I through IV. The aggregation of these four complexes, together with
associated mobile, accessory electron carriers, is called the electron transport chain.
The electron transport chain is present with multiple copies in the inner mitochondrial
membrane of eukaryotes and within the plasma membrane of prokaryotes.
Figure 1. The electron transport chain is a series of electron transporters embedded in the inner mitochondrial
membrane that shuttles electrons from NADH and FADH2 to molecular oxygen. In the process, protons are
pumped from the mitochondrial matrix to the intermembrane space, and oxygen is reduced to form water.

Complex I

First, two electrons are carried to the first complex via NADH. This complex, labelled I,
is composed of flavin mononucleotide (FMN) and an iron-sulfur (Fe-S)-containing
protein. FMN, which is derived from vitamin B2 (also called riboflavin), is one of several
prosthetic groups or cofactors in the electron transport chain. A prosthetic group is a
non-protein molecule required for the activity of a protein. Prosthetic groups are organic
or inorganic, non-peptide molecules bound to a protein that facilitate its function.
Prosthetic groups include coenzymes, which are the prosthetic groups of enzymes. The
enzyme in complex I is NADH dehydrogenase and is a very large protein, containing 45
amino acid chains. Complex I can pump four hydrogen ions across the membrane from
the matrix into the intermembrane space, and it is in this way that the hydrogen ion
gradient is established and maintained between the two compartments separated by the
inner mitochondrial membrane.
Q and Complex II

Complex II directly receives FADH2—which does not pass through complex I. The
compound connecting the first and second complexes to the third is ubiquinone B. The
Q molecule is lipid soluble and freely moves through the hydrophobic core of the
membrane. Once it is reduced (QH2), ubiquinone delivers its electrons to the next
complex in the electron transport chain. Q receives the electrons derived from NADH
from complex I, and the electrons derived from FADH2 from complex II. This enzyme
and FADH2 form a small complex that delivers electrons directly to the electron transport
chain, bypassing the first complex. Since these electrons bypass and thus do not
energize the proton pump in the first complex, fewer ATP molecules are made from the
FADH2 electrons. The number of ATP molecules ultimately obtained is directly
proportional to the number of protons pumped across the inner mitochondrial
membrane.

Complex III

The third complex is composed of cytochrome b—another Fe-S protein, a Rieske center
(2Fe-2S center), and cytochrome c proteins. This complex is also called cytochrome
oxidoreductase. Cytochrome proteins have a prosthetic group of heme. The heme
molecule is similar to the heme in haemoglobin, but it carries electrons, not oxygen. As
a result, the iron ion at its core is reduced and oxidized as it passes the electrons,
fluctuating between different oxidation states: Fe++ (reduced) and Fe+++ (oxidized). The
heme molecules in the cytochromes have slightly different characteristics due to the
effects of the different proteins binding to them, giving slightly different characteristics to
each complex. Complex III pumps protons through the membrane and passes its
electrons to cytochrome c for transport to the fourth complex of proteins and enzymes.
(Cytochrome c receives electrons from Q; however, whereas Q carries pairs of
electrons, cytochrome c can accept only one at a time.)

Complex IV

The fourth complex is composed of cytochrome proteins c, a, and a3. This complex
contains two heme groups (one in each of the two cytochromes, a, and a3) and three
copper ions (a pair of CuA and one CuB in cytochrome a3). The cytochromes hold an
oxygen molecule very tightly between the iron and copper ions until the oxygen is
completely reduced by the gain of two electrons. The reduced oxygen then picks up two
hydrogen ions from the surrounding medium to make water (H2O). The removal of the
hydrogen ions from the system contributes to the ion gradient that forms the foundation
for the process of chemiosmosis.

Chemiosmosis

In chemiosmosis, the free energy from the series of redox reactions just described is
used to pump hydrogen ions (protons) across the mitochondrial membrane. The uneven
distribution of H+ ions across the membrane establishes both concentration and
electrical gradients (thus, an electrochemical gradient), owing to the hydrogen ions’
positive charge and their aggregation on one side of the membrane.
If the membrane were continuously open to simple diffusion by the hydrogen ions, the
ions would tend to diffuse back across into the matrix, driven by the concentrations
producing their electrochemical gradient. Recall that many ions cannot diffuse through
the nonpolar regions of phospholipid membranes without the aid of ion channels.
Similarly, hydrogen ions in the matrix space can only pass through the inner
mitochondrial membrane by an integral membrane protein called ATP synthase
((Figure)). This complex protein acts as a tiny generator, turned by the force of the
hydrogen ions diffusing through it, down their electrochemical gradient. The turning of
parts of this molecular machine facilitates the addition of a phosphate to ADP, forming
ATP, using the potential energy of the hydrogen ion gradient.
ART CONNECTION
Figure 2. ATP synthase is a complex, molecular machine that uses a proton (H+) gradient to form ATP from ADP and
inorganic phosphate (Pi). (Credit: modification of work by Klaus Hoffmeier)
Dinitrophenol (DNP) is an “uncoupler” that makes the inner mitochondrial membrane “leaky” to
protons. It was used until 1938 as a weight-loss drug. What effect would you expect DNP to
have on the change in pH across the inner mitochondrial membrane? Why do you think this
might be an effective weight-loss drug?
Show Solution
After DNP poisoning, the electron transport chain can no longer form a proton gradient, and
ATP synthase can no longer make ATP. DNP is an effective diet drug because it uncouples
ATP synthesis; in other words, after taking it, a person obtains less energy out of the food he or
she eats. Interestingly, one of the worst side effects of this drug is hyperthermia, or overheating
of the body. Since ATP cannot be formed, the energy from electron transport is lost as heat.
Chemiosmosis ((Figure)) is used to generate 90 percent of the ATP made during
aerobic glucose catabolism; it is also the method used in the light reactions of
photosynthesis to harness the energy of sunlight in the process of
photophosphorylation. Recall that the production of ATP using the process of
chemiosmosis in mitochondria is called oxidative phosphorylation. The overall result of
these reactions is the production of ATP from the energy of the electrons removed from
hydrogen atoms. These atoms were originally part of a glucose molecule. At the end of
the pathway, the electrons are used to reduce an oxygen molecule to oxygen ions. The
extra electrons on the oxygen attract hydrogen ions (protons) from the surrounding
medium, and water is formed. Thus, oxygen is the final electron acceptor in the electron
transport chain.
ART CONNECTION
Figure 3. In oxidative phosphorylation, the pH gradient formed by the electron transport chain is used by ATP
synthase to form ATP.
Cyanide inhibits cytochrome c oxidase, a component of the electron transport chain. If cyanide
poisoning occurs, would you expect the pH of the intermembrane space to increase or
decrease? What effect would cyanide have on ATP synthesis?
Show Solution
After cyanide poisoning, the electron transport chain can no longer pump electrons into the
intermembrane space. The pH of the intermembrane space would increase, the pH gradient
would decrease, and ATP synthesis would stop.

ATP Yield

The number of ATP molecules generated from the catabolism of glucose varies. For
example, the number of hydrogen ions that the electron transport chain complexes can
pump through the membrane varies between species. Another source of variance
stems from the shuttle of electrons across the membranes of the mitochondria. (The
NADH generated from glycolysis cannot easily enter mitochondria.) Thus, electrons are
picked up on the inside of mitochondria by either NAD+ or FAD+. As you have learned
earlier, these FAD+ molecules can transport fewer ions; consequently, fewer ATP
molecules are generated when FAD+ acts as a carrier. NAD+ is used as the electron
transporter in the liver and FAD+ acts in the brain.
Another factor that affects the yield of ATP molecules generated from glucose is the fact
that intermediate compounds in these pathways are also used for other purposes.
Glucose catabolism connects with the pathways that build or break down all other
biochemical compounds in cells, and the result is somewhat messier than the ideal
situations described thus far. For example, sugars other than glucose are fed into the
glycolytic pathway for energy extraction. In addition, the five-carbon sugars that form
nucleic acids are made from intermediates in glycolysis. Certain nonessential amino
acids can be made from intermediates of both glycolysis and the citric acid cycle. Lipids,
such as cholesterol and triglycerides, are also made from intermediates in these
pathways, and both amino acids and triglycerides are broken down for energy through
these pathways. Overall, in living systems, these pathways of glucose catabolism
extract about 34 percent of the energy contained in glucose, with the remainder being
released as heat.
SECTION SUMMARY

The electron transport chain is the portion of aerobic respiration that uses free oxygen as the
final electron acceptor of the electrons removed from the intermediate compounds in glucose
catabolism. The electron transport chain is composed of four large, multiprotein complexes
embedded in the inner mitochondrial membrane and two small diffusible electron carriers
shuttling electrons between them. The electrons are passed through a series of redox reactions,
with a small amount of free energy used at three points to transport hydrogen ions across a
membrane. This process contributes to the gradient used in chemiosmosis. The electrons
passing through the electron transport chain gradually lose energy. High-energy electrons
donated to the chain by either NADH or FADH complete the chain, as low-energy electrons
2
reduce oxygen molecules and form water. The level of free energy of the electrons drops from
about 60 kcal/mol in NADH or 45 kcal/mol in FADH to about 0 kcal/mol in water. The end
2

products of the electron transport chain are water and ATP. A number of intermediate
compounds of the citric acid cycle can be diverted into the anabolism of other biochemical
molecules, such as nonessential amino acids, sugars, and lipids. These same molecules can
serve as energy sources for the glucose pathways.

Art Connections

(Figure) Dinitrophenol (DNP) is an “uncoupler” that makes the inner mitochondrial

membrane “leaky” to protons. It was used until 1938 as a weight-loss drug. What effect
would you expect DNP to have on the change in pH across the inner mitochondrial
membrane? Why do you think this might be an effective weight-loss drug?
Show Solution
(Figure) After DNP poisoning, the electron transport chain can no longer form a proton
gradient, and ATP synthase can no longer make ATP. DNP is an effective diet drug
because it uncouples ATP synthesis; in other words, after taking it, a person obtains
less energy out of the food he or she eats. Interestingly, one of the worst side effects of
this drug is hyperthermia, or overheating of the body. Since ATP cannot be formed, the
energy from electron transport is lost as heat.
(Figure) Cyanide inhibits cytochrome c oxidase, a component of the electron transport
chain. If cyanide poisoning occurs, would you expect the pH of the intermembrane
space to increase or decrease? What effect would cyanide have on ATP synthesis?
Show Solution
(Figure) After cyanide poisoning, the electron transport chain can no longer pump
electrons into the intermembrane space. The pH of the intermembrane space would
increase, the pH gradient would decrease, and ATP synthesis would stop.
REVIEW QUESTIONS

What compound receives electrons from NADH?

1. FMN
2. ubiquinone
3. cytochrome c1
4. oxygen
Show Solution
A
Chemiosmosis involves ________.
1. the movement of electrons across the cell membrane
2. the movement of hydrogen atoms across a mitochondrial membrane
3. the movement of hydrogen ions across a mitochondrial membrane
 4. the movement of glucose through the cell membrane
Show Solution
C

FREE RESPONSE

How do the roles of ubiquinone and cytochrome c differ from the roles of the other components
of the electron transport chain?
Show Solution
Q and cytochrome c are transport molecules. Their function does not result directly in ATP
synthesis in that they are not pumps. Moreover, Q is the only component of the electron
transport chain that is not a protein. Ubiquinone and cytochrome c are small, mobile electron
carriers, whereas the other components of the electron transport chain are large complexes
anchored in the inner mitochondrial membrane.
What accounts for the different number of ATP molecules that are formed through cellular
respiration?
Show Solution
Few tissues except muscle produce the maximum possible amount of ATP from nutrients. The
intermediates are used to produce needed amino acids, fatty acids, cholesterol, and sugars for
nucleic acids. When NADH is transported from the cytoplasm to the mitochondria, an active
transport mechanism is used, which decreases the amount of ATP that can be made. The
electron transport chain differs in composition between species, so different organisms will
make different amounts of ATP using their electron transport chains.

GLOSSARY

ATP synthase

(also F1F0 ATP synthase) membrane-embedded protein complex that adds a phosphate
to ADP with energy from protons diffusing through it

prosthetic group

(also prosthetic cofactor) molecule bound to a protein that facilitates the function of the
protein

ubiquinone

soluble electron transporter in the electron transport chain that connects the first or
second complex to the third