Bioenergetics and Metabolism - Mitochondria, Chloroplasts, and Peroxisomes
In addition to being involved in protein sorting and transport, cytoplasmic organelles provide specialized
compartments in which a variety of metabolic activities take place. The generation of metabolic energy is a
major activity of all cells, and two cytoplasmic organelles are specifically devoted to energy metabolism and the
production of ATP. Mitochondria are responsible for generating most of the useful energy derived from the
breakdown of lipids and carbohydrates, and chloroplasts use energy captured from sunlight to generate both
ATP and the reducing power needed to synthesize carbohydrates from CO2 and H2O. The third organelle
discussed in this chapter, the peroxisome, contains enzymes involved in a variety of different metabolic
pathways, including the breakdown of fatty acids and the metabolism of a by-product of photosynthesis.
Mitochondria, chloroplasts, and peroxisomes differ from the organelles discussed in the preceding chapter not
only in their functions, but also in their mechanism of assembly. Rather than being synthesized on membranebound ribosomes and translocated into the endoplasmic reticulum, proteins destined for peroxisomes,
mitochondria, and chloroplasts are synthesized on free ribosomes in the cytosol and imported into their target
organelles as completed polypeptide chains. Mitochondria and chloroplasts also contain their own genomes,
which include some genes that are transcribed and translated within the organelle. Protein sorting to the
cytoplasmic organelles discussed in this chapter is thus distinct from the pathways of vesicular transport that
connect the endoplasmic reticulum, Golgi apparatus, lysosomes, and plasma membrane.
Mitochondria
Mitochondria play a critical role in the generation of metabolic energy in eukaryotic cells. They are responsible
for most of the useful energy derived from the breakdown of carbohydrates and fatty acids, which is converted
to ATP by the process of oxidative phosphorylation. Most mitochondrial proteins are translated on free
cytosolic ribosomes and imported into the organelle by specific targeting signals. In addition, mitochondria are
unique among the cytoplasmic organelles already discussed in that they contain their own DNA, which encodes
tRNAs, rRNAs, and some mitochondrial proteins. The assembly of mitochondria thus involves proteins
encoded by their own genomes and translated within the organelle, as well as proteins encoded by the nuclear
genome and imported from the cytosol.
Organization and Function of Mitochondria
Figure 10.1. Structure of a mitochondrion Mitochondria are bounded by a
double-membrane system, consisting of inner and outer membranes. Folds of the
inner membrane (cristae) extend into the matrix.
Mitochondria are surrounded by a double-membrane system,
consisting of inner and outer mitochondrial membranes separated
by an intermembrane space (Figure 10.1). The inner membrane
forms numerous folds (cristae), which extend into the interior (or
matrix) of the organelle. Each of these components plays distinct functional roles, with the matrix and inner
membrane representing the major working compartments of mitochondria.
The matrix contains the mitochondrial genetic system as
well as the enzymes responsible for the central reactions of
oxidative metabolism (Figure 10.2).
Figure 10.2. Metabolism in the matrix of mitochondria Pyruvate and
fatty acids are imported from the cytosol and converted to acetyl CoA in
the mitochondrial matrix. Acetyl CoA is then oxidized to CO2 via the
citric acid cycle, the central pathway of oxidative metabolism.
The oxidative breakdown of glucose and fatty acids is the principal source of metabolic energy in animal cells.
The initial stages of glucose metabolism (glycolysis) occur in the cytosol, where glucose is converted to
pyruvate. Pyruvate is then transported into mitochondria, where its complete oxidation to CO2 yields the bulk of
usable energy (ATP) obtained from glucose metabolism. This involves the initial oxidation of pyruvate to acetyl
CoA, which is then broken down to CO2 via the citric acid cycle. The oxidation of fatty acids also yields acetyl
CoA, which is similarly metabolized by the citric acid cycle in mitochondria. The enzymes of the citric acid
cycle (located in the matrix of mitochondria) thus are central players in the oxidative breakdown of both
carbohydrates and fatty acids.
The oxidation of acetyl CoA to CO2 is coupled to the reduction of NAD+ and FAD to NADH and FADH2,
respectively. Most of the energy derived from oxidative metabolism is then produced by the process of
oxidative phosphorylation (discussed in detail in the next section), which takes place in the inner mitochondrial
membrane. The high-energy electrons from NADH and FADH2 are transferred through a series of carriers in the
membrane to molecular oxygen. The energy derived from these electron transfer reactions is converted to
potential energy stored in a proton gradient across the membrane, which is then used to drive ATP synthesis.
The inner mitochondrial membrane thus represents the principal site of ATP generation, and this critical role is
reflected in its structure. First, its surface area is substantially increased by its folding into cristae. In addition,
the inner mitochondrial membrane contains an unusually high percentage (greater than 70%) of proteins, which
are involved in oxidative phosphorylation as well as in the transport of metabolites (e.g., pyruvate and fatty
acids) between the cytosol and mitochondria. Otherwise, the inner membrane is impermeable to most ions and
small molecules a property critical to maintaining the proton gradient that drives oxidative phosphorylation.
In contrast to the inner membrane, the outer mitochondrial membrane is freely permeable to small molecules.
This is because it contains proteins called porins, which form channels that allow the free diffusion of
molecules smaller than about 6000 daltons. The composition of the intermembrane space is therefore similar to
the cytosol with respect to ions and small molecules. Consequently, the inner mitochondrial membrane is the
functional barrier to the passage of small molecules between the cytosol and the matrix and maintains the
proton gradient that drives oxidative phosphorylation.
The Genetic System of Mitochondria
Mitochondria contain their own genetic system, which is separate and distinct from the nuclear genome of the
cell. Mitochondria are thought to have evolved from bacteria that developed a symbiotic relationship in which
they lived within larger cells (endosymbiosis). This hypothesis has recently been substantiated by the results of
DNA sequence analysis, which revealed striking similarities between the genomes of mitochondria and of the
bacterium Rickettsia prowazekii. Rickettsia are intracellular parasites which, like mitochondria, are only able to
reproduce within eukaryotic cells. Consistent with their similar symbiotic lifestyles, the genomic DNA
sequences of Rickettsia and mitochondria suggest that they share a common ancestor, from which the genetic
system of present-day mitochondria evolved.
Mitochondrial genomes are usually circular DNA molecules, like those of bacteria, which are present in
multiple copies per organelle. They vary considerably in size between different species. The genomes of human
and most other animal mitochondria are only about 16 kb, but substantially larger mitochondrial genomes are
found in yeasts (approximately 80 kb) and plants (more than 200 kb). However, these larger mitochondrial
genomes are composed predominantly of noncoding sequences and do not appear to contain significantly more
genetic information. For example, the largest sequenced mitochondrial genome is that of the plant Arabidopsis
thaliana. Although Arabidopsis mitochondrial DNA is approximately 367 kb, it encodes only 32 proteins: just
more than twice the number encoded by human mitochondrial DNA. The largest number of mitochondrial
genes has been found in mitochondrial DNA of the protozoan Reclinomonas americana, which is 69 kb and
contains 97 genes. The mitochondrial genome of Reclinomonas appears to more closely resemble the bacterial
genome from which mitochondria evolved than most present-day mitochondrial genomes, which encode only a
small number of proteins that are essential components of the oxidative phosphorylation system. In addition,
mitochondrial genomes encode all of the ribosomal RNAs and most of the transfer RNAs needed for translation
of these protein-coding sequences within mitochondria. Other mitochondrial proteins are encoded by nuclear
genes, which are thought to have been transferred to the nucleus from the ancestral
mitochondrial genome.The human mitochondrial genome encodes 13 proteins
involved in electron transport and oxidative phosphorylation (Figure 10.3).
Figure 10.3. The human mitochondrial genome The genome contains 13 proteincoding sequences,
which are designated as components of respiratory complexes I, III, IV, or V. In addition, the genome
contains genes for 12S and 16S rRNAs and for 22 tRNAs, which are designated by the one-letter code
for the corresponding amino acid. The region of the genome designated "D loop" contains an origin of
DNA replication and transcriptional promoter sequences.
In addition, human mitochondrial DNA encodes 16S and 12S rRNAs and 22 tRNAs,
which are required for translation of the proteins encoded by the organelle genome. The two rRNAs are the only
RNA components of animal and yeast mitochondrial ribosomes, in contrast to the three rRNAs of bacterial
ribosomes (23S, 16S, and 5S). Plant mitochondrial DNAs, however, also encode a third rRNA of 5S. The
mitochondria of plants and protozoans also differ in importing and utilizing tRNAs encoded by the nuclear as
well as the mitochondrial genome, whereas in animal mitochondria, all the tRNAs are encoded by the organelle.
The small number of tRNAs encoded by the mitochondrial genome highlights an important feature of the
mitochondrial genetic system the use of a slightly different genetic code, which is distinct from the
"universal" genetic code used by both prokaryotic and eukaryotic cells.There are 64 possible triplet codons, of
which 61 encode the 20 different amino acids incorporated into proteins. Many tRNAs in both prokaryotic and
eukaryotic cells are able to recognize more than a single codon in mRNA because of "wobble," which allows
some mispairing between the tRNA anticodon and the third position of certain complementary codons.
However, at least 30 different tRNAs are required to translate the universal code according to the wobble rules.
Yet human mitochondrial DNA encodes only 22 tRNA species, and these are the only tRNAs used for
translation of mitochondrial mRNAs. This is accomplished by an extreme form of wobble in which U in the
anticodon of the tRNA can pair with any of the four bases in the third codon position of mRNA, allowing four
codons to be recognized by a single tRNA. In addition, some codons specify different amino acids in
mitochondria than in the universal code.
Like the DNA of nuclear genomes, mitochondrial DNA can be altered by mutations, which are frequently
deleterious to the organelle. Since almost all the mitochondria of fertilized eggs are contributed by the oocyte
rather than by the sperm, germ-line mutations in mitochondrial DNA are transmitted to the next generation by
the mother. Such mutations have been associated with a number of diseases. For example, Leber's hereditary
optic neuropathy, a disease that leads to blindness, can be caused by mutations in mitochondrial genes that
encode components of the electron transport chain. In addition, the progressive accumulation of mutations in
mitochondrial DNA during the lifetime of individuals has been suggested to contribute to the process of aging.
Protein Import and Mitochondrial Assembly
In contrast to the RNA components of the mitochondrial translation apparatus (rRNAs and tRNAs), most
mitochondrial genomes do not encode the proteins required for DNA replication, transcription, or translation.
Instead, the genes that encode proteins required for the replication and expression of mitochondrial DNA are
contained in the nucleus. In addition, the nucleus contains the genes that encode most of the mitochondrial
proteins required for oxidative phosphorylation and all of the enzymes involved in mitochondrial metabolism
(e.g., enzymes of the citric acid cycle). The proteins encoded by these genes (more than 95% of mitochondrial
proteins) are synthesized on free cytosolic ribosomes and imported
into mitochondria as completed polypeptide chains. Because of the
double-membrane structure of mitochondria, the import of proteins
is considerably more complicated than the transfer of a
polypeptide across a single phospholipid bilayer. Proteins targeted
to the matrix have to cross both the inner and outer mitochondrial
membranes, while other proteins need to be sorted to distinct
compartments within the organelle (e.g., the intermembrane space).
The import of proteins to the matrix is the best-understood aspect
of mitochondrial protein sorting (Figure 10.4).
Figure 10.4. Import of proteins into mitochondria Proteins are targeted for
mitochondria by an amino-terminal presequence containing positively charged
amino acids. Proteins are maintained in a partially unfolded state by association
with a cytosolic Hsp70 and are recognized by a receptor on the surface of
mitochondria. The unfolded polypeptide chains are then translocated through the
Tom complex in the outer membrane and transferred to the Tim complex in the
inner membrane. The voltage component of the electrochemical gradient is
required for translocation across the inner membrane. The presequence is cleaved
by a matrix protease, and a mitochondrial Hsp70 binds the polypeptide chain as it
crosses the inner membrane, driving further protein translocation. A
mitochondrial Hsp60 then facilitates folding of the imported polypeptide within
the matrix
Most proteins are targeted to mitochondria by amino-terminal sequences of 20 to 35 amino acids (called
presequences) that are removed by proteolytic cleavage following their import into the organelle. The
presequences of mitochondrial proteins, first characterized by Gottfried Schatz, contain multiple positively
charged amino acid residues, usually in an amphipathic  helix. The first step in protein import is the binding of
these presequences to receptors on the surface of mitochondria. The polypeptide chains are then inserted into a
protein complex that directs translocation across the outer membrane (the translocase of the outer membrane or
Tom complex). The proteins are then transferred to a second protein complex in the inner membrane (the
translocase of the inner membrane or Tim complex). Continuing protein translocation requires the
electrochemical potential established across the inner mitochondrial membrane during electron transport. As
discussed in the next section of this chapter, the transfer of high-energy electrons from NADH and FADH2 to
molecular oxygen is coupled to the transfer of protons from the mitochondrial matrix to the intermembrane
space. Since protons are charged particles, this transfer establishes an electric potential across the inner
membrane, with the matrix being negative. During protein import, this electric potential drives translocation of
the positively charged presequence.
To be translocated across the mitochondrial membrane, proteins must be at least partially unfolded.
Consequently, protein import into mitochondria requires molecular chaperones in addition to the membrane
proteins involved in translocation (see Figure 10.4). On the cytosolic side, members of the Hsp70 family of
chaperones maintain proteins in a partially unfolded state so that they can be inserted into the mitochondrial
membrane. As they cross the inner membrane, the unfolded polypeptide chains bind to another member of the
Hsp70 family, which is associated with the Tim complex and acts as a motor that drives protein import. The
polypeptide is then transferred to a chaperone of the Hsp60 family (a chaperonin), within which protein folding
takes place. Since these interactions of polypeptide chains with molecular chaperones depend on ATP, protein
import requires ATP both outside and inside the mitochondria, in addition to the electric potential across the
inner membrane.
As noted above, some mitochondrial proteins are targeted to the outer membrane, inner membrane, or
intermembrane space rather than to the matrix, so additional mechanisms are needed to direct these proteins to
the correct submitochondrial compartment. These proteins are targeted to their destinations by a second sorting
signal following the positively charged presequence that directs mitochondrial import. The targeting of proteins
to the mitochondrial membranes appears to be mediated by hydrophobic stop-transfer sequences that halt
translocation of the polypeptide chains
through the Tim or Tom complexes,
leading to their insertion into the inner
or outer mitochondrial membranes,
respectively (Figure 10.5).
Figure 10.5. Insertion of mitochondrial
membrane proteins Proteins targeted for the
mitochondrial membranes contain
hydrophobic stop-transfer sequences that halt
their translocation through the Tom or Tim
complexes and lead to their incorporation into
the outer or inner membranes, respectively.
Proteins may be targeted to the intermembrane space by several different mechanisms (Figure 10.6).
Figure 10.6. Sorting
proteins to the
intermembrane space
Proteins can be targeted
to the intermembrane
space by several
mechanisms. Some
proteins (I) are
translocated through the
Tom complex and
released into the
intermembrane space. Other proteins (II) are transferred from the Tom complex to the Tim complex, but they contain hydrophobic stoptransfer sequences that halt translocation through the Tim complex. These stop-transfer sequences are then cleaved to release the proteins
into the intermembrane space. Still other proteins (III) are imported to the matrix, as depicted in Figure 10.4. Removal of the
presequence within the matrix then exposes a hydrophobic signal sequence, which targets the protein back across the inner membrane to
the intermembrane space.
Some proteins are transferred across the outer membrane through the Tom complex but are then released within
the intermembrane space instead of being transferred to the Tim complex. Other proteins are transferred to the
Tim complex but are then released into the intermembrane space as a result of cleavage of hydrophobic stoptransfer sequences. Still other proteins may be completely imported into the mitochondrial matrix and then
exported back across the inner membrane to the intermembrane space.
Not only the proteins, but also the phospholipids of mitochondrial membranes are imported from the cytosol. In
animal cells, phosphatidylcholine and phosphatidylethanolamine are synthesized in the ER and carried to
mitochondria by phospholipid transfer proteins, which extract single phospholipid molecules from the
membrane of the ER. The lipid can then be transported through the aqueous environment of the cytosol, buried
in a hydrophobic binding site of the protein, and released when the complex reaches a new membrane, such as
that of mitochondria. The mitochondria then synthesize
phosphatidylserine from phosphatidylethanolamine, in addition
to catalyzing the synthesis of the unusual phospholipid
cardiolipin, which contains four fatty acid chains (Figure 10.7).
Figure 10.7. Structure of cardiolipin Cardiolipin is an unusual "double"
phospholipid, containing four fatty acid chains, that is found primarily in the
inner mitochondrial membrane.
The Mechanism of Oxidative Phosphorylation
Most of the usable energy obtained from the breakdown of carbohydrates or fats is derived by oxidative
phosphorylation, which takes place within mitochondria. For example, the breakdown of glucose by glycolysis
and the citric acid cycle yields a total of four molecules of ATP, ten molecules of NADH, and two molecules of
FADH2. Electrons from NADH and FADH2 are then transferred to molecular oxygen, coupled to the formation
of an additional 32 to 34 ATP molecules by oxidative phosphorylation. Electron transport and oxidative
phosphorylation are critical activities of protein complexes in the inner mitochondrial membrane, which
ultimately serve as the major source of cellular energy.
The Electron Transport Chain
During oxidative phosphorylation, electrons derived from NADH and FADH2 combine with O2, and the energy
released from these oxidation/
reduction reactions is used to drive the
synthesis of ATP from ADP. The
transfer of electrons from NADH to
O2 is a very energy-yielding reaction,
with G°´ = -52.5 kcal/mol for each
pair of electrons transferred. To be
harvested in usable form, this energy
must be produced gradually, by the
passage of electrons through a series
of carriers, which constitute the
electron transport chain. These
carriers are organized into four
complexes in the inner mitochondrial
membrane. A fifth protein complex
then serves to couple the energyyielding reactions of electron transport
to ATP synthesis.
Electrons from NADH enter the
electron transport chain in complex I,
which consists of nearly 40
polypeptide chains (Figure 10.8).
Figure 10.8. Transport of electrons from NADH
These electrons are initially transferred from NADH to flavin mononucleotide and then, through an iron-sulfur
carrier, to coenzyme Q an energy-yielding process with G°´ = -16.6 kcal/mol. Coenzyme Q (also called
ubiquinone) is a small, lipid-soluble molecule that carries electrons from complex I through the membrane to
complex III, which consists of about ten polypeptides. In complex III, electrons are transferred from
cytochrome b to cytochrome c an energy-yielding reaction with G°´ = -10.1 kcal/mol. Cytochromec, a
peripheral membrane protein bound to the outer face of the inner membrane, then carries electrons to complex
IV (cytochrome oxidase), where they are finally transferred to O2 (G°´ = -25.8 kcal/mol).
A distinct protein complex
(complex II), which consists of four
polypeptides, receives electrons
from the citric acid cycle
intermediate, succinate (Figure
10.9).
Figure 10.9. Transport of electrons from
FADH2 Electrons from succinate enter the
electron transport chain via FADH2 in
complex II. They are then transferred to
coenzyme Q and carried through the rest of
the electron transport chain as described in
Figure 10.8. The transfer of electrons from
FADH2 to coenzyme Q is not associated
with a significant decrease in free energy, so
protons are not pumped across the
membrane at complex II.
These electrons are transferred to
FADH2, rather than to NADH, and
then to coenzyme Q. From coenzyme Q, electrons are transferred to complex III and then to complex IV as
already described. In contrast to the transfer of electrons from NADH to coenzyme Q at complex I, the transfer
of electrons from FADH2 to coenzyme Q is not associated with a significant decrease in free energy and,
therefore, is not coupled to ATP synthesis. Consequently, the passage of electrons derived from FADH2 through
the electron transport chain yields free energy only at complexes III and IV.
The free energy derived from the passage of electrons through complexes I, III, and IV is harvested by being
coupled to the synthesis of ATP. Importantly, the mechanism by which the energy derived from these electron
transport reactions is coupled to ATP synthesis is fundamentally different from the synthesis of ATP during
glycolysis or the citric acid cycle. In the latter cases, a high-energy phosphate is transferred directly to ADP
from the other substrate of an energy-yielding reaction. For example, in the final reaction of glycolysis, the
high-energy phosphate of phosphoenolpyruvate is transferred to ADP, yielding pyruvate plus ATP. Such direct
transfer of high-energy phosphate groups does not occur during electron transport. Instead, the energy derived
from electron transport is coupled to the generation of a proton gradient across the inner mitochondrial
membrane. The potential energy stored in this gradient is then harvested by a fifth protein complex, which
couples the energetically favorable flow of protons back across the membrane to the synthesis of ATP.
Chemiosmotic Coupling
The mechanism of coupling electron transport to ATP generation, chemiosmotic coupling, is a striking example
of the relationship between structure and function in cell biology. The hypothesis of chemiosmotic coupling was
first proposed in 1961 by Peter Mitchell, who suggested that ATP is generated by the use of energy stored in the
form of proton gradients across biological membranes, rather than by direct chemical transfer of high-energy
groups. Biochemists were initially highly skeptical of this proposal, and the chemiosmotic hypothesis took more
than a decade to win general acceptance in the scientific community. Overwhelming evidence eventually
accumulated in its favor, however, and chemiosmotic coupling is now recognized as a general mechanism of
ATP generation, operating not only in mitochondria but also in chloroplasts and in bacteria, where ATP is
generated via a proton gradient across the plasma membrane.
Electron transport through complexes I, III, and IV is coupled to the transport of protons out of the interior of
the mitochondrion (see Figure 10.8). Thus, the energy-yielding reactions of electron transport are coupled to the
transfer of protons from the matrix to the intermembrane space, which establishes a proton gradient across the
inner membrane. Complexes I and IV appear to act as proton pumps that transfer protons across the membrane
as a result of conformational changes induced by electron transport. In complex III, protons are carried across
the membrane by coenzyme Q, which accepts protons from the matrix at complexes I or II and releases them
into the intermembrane space at complex III. Complexes I and III each transfer four protons across the
membrane per pair of electrons. In complex IV, two protons per pair of electrons are pumped across the
membrane and another two protons per pair of electrons are combined with O2 to form H2O within the matrix.
Thus, the equivalent of four protons per pair of electrons are transported out of the mitochondrial matrix at each
of these three complexes. This transfer of protons from the matrix to the intermembrane space plays the critical
role of converting the energy derived from the oxidation/reduction reactions of electron transport to the
potential energy stored in a proton gradient.
Because protons are electrically charged particles, the potential energy stored in the proton gradient is electric
as well as chemical in nature. The electric component corresponds to the voltage difference across the inner
mitochondrial membrane, with the matrix of the mitochondrion negative and the intermembrane space positive.
The corresponding free energy is given by the equation
where F is the Faraday constant and V is the membrane potential. The additional free energy corresponding to
the difference in proton concentration across the membrane is given by the equation
where [H+]i and [H+]o refer, respectively, to the proton concentrations inside and outside the mitochondria.
In metabolically active cells, protons are typically pumped
out of the matrix such that the proton gradient across the
inner membrane corresponds to about one pH unit, or a
tenfold lower concentration of protons within
mitochondria (Figure 10.10).
Figure 10.10. The electrochemical nature of the proton gradient
Since protons are positively charged, the proton gradient established
across the inner mitochondrial membrane has both chemical and
electric components. The chemical component is the proton
concentration, or pH, gradient, which corresponds to about a tenfold
higher concentration of protons on the cytosolic side of the inner
mitochondrial membrane (a difference of one pH unit). In addition,
there is an electric potential across the membrane, resulting from the net
increase in positive charge on the cytosolic side.
The pH of the mitochondrial matrix is therefore about 8, compared to the neutral pH
(approximately 7) of the cytosol and intermembrane space. This gradient also generates
an electric potential of approximately 0.14 V across the membrane, with the matrix
negative. Both the pH gradient and the electric potential drive protons back into the
matrix from the cytosol, so they combine to form an electrochemical gradient across the
inner mitochondrial membrane, corresponding to a G of approximately -5 kcal/mol per
proton.
Because the phospholipid bilayer is impermeable to ions, protons are able to cross the
membrane only through a protein channel. This restriction allows the energy in the
electrochemical gradient to be harnessed and converted to ATP as a result of the action of
the fifth complex involved in oxidative phosphorylation, complex V, or ATP synthase
(see Figure 10.8). ATP synthase is organized into two structurally distinct components, F0
and F1, which are linked by a slender stalk (Figure 10.11).
Figure 10.11. Structure of ATP synthase The mitochondrial ATP synthase (complex V) consists of two multisubunit components, F 0
and F1, which are linked by a slender stalk. F0 spans the lipid bilayer, forming a channel through which protons can cross the membrane.
F1 harvests the free energy derived from proton movement down the electrochemical gradient by catalyzing the synthesis of ATP.
The F0 portion spans the inner membrane and provides a channel through which protons are able to flow back
from the intermembrane space to the matrix. The energetically favorable return of protons to the matrix is
coupled to ATP synthesis by the F1 subunit, which catalyzes the synthesis of ATP from ADP and phosphate
ions (Pi). Detailed structural studies have established the mechanism of ATP synthase action, which involves
mechanical coupling between the F0 and F1 subunits. In particular, the flow of protons through F0 drives the
rotation of F1, which acts as a rotary motor to drive ATP synthesis.
It appears that the flow of four protons back across the membrane through F0 is required to drive the synthesis
of one molecule of ATP by F1, consistent with the proton transfers at complexes I, III, and IV each contributing
sufficient free energy to the proton gradient to drive the synthesis of one ATP molecule. The oxidation of one
molecule of NADH thus leads to the synthesis of three molecules of ATP, whereas the oxidation of FADH2,
which enters the electron transport chain at complex II, yields only two ATP molecules.
Transport of Metabolites across the Inner Membrane
In addition to driving the synthesis of ATP, the potential energy stored in the electrochemical gradient drives
the transport of small molecules into and out of mitochondria. For example, the ATP synthesized within
mitochondria has to be exported to the cytosol, while ADP and Pi need to be imported from the cytosol for ATP
synthesis to continue. The electrochemical gradient generated by proton pumping provides energy required for
the transport of these molecules and other metabolites that need to be concentrated within mitochondria (Figure
10.12).
Figure 10.12. Transport of metabolites across the mitochondrial
inner membrane The transport of small molecules across the inner
membrane is mediated by membrane-spanning transport proteins and
driven by the electrochemical gradient. For example, ATP is
exported from mitochondria to the cytosol by a transporter that
exchanges it for ADP. The voltage component of the electrochemical
gradient drives this exchange: ATP carries a greater negative charge
(-4) than ADP (-3), so ATP is exported from the mitochondrial
matrix to the cytosol while ADP is imported to mitochondria. In
contrast, the transport of phosphate (P i) and pyruvate is coupled to an
exchange for hydroxyl ions (OH-); in this case, the pH component of
the electrochemical gradient drives the export of hydroxyl ions,
coupled to the transport of Pi and pyruvate into mitochondria.
The transport of ATP and ADP across the inner membrane is mediated by an integral membrane protein, the
adenine nucleotide translocator, which transports one molecule of ADP into the mitochondrion in exchange for
one molecule of ATP transferred from the mitochondrion to the cytosol. Because ATP carries more negative
charge than ADP (-4 compared to -3), this exchange is driven by the voltage component of the electrochemical
gradient. Since the proton gradient establishes a positive charge on the cytosolic side of the membrane, the
export of ATP in exchange for ADP is energetically favorable.
The synthesis of ATP within the mitochondrion requires phosphate ions (Pi) as well as ADP, so Pi must also be
imported from the cytosol. This is mediated by another membrane transport protein, which imports phosphate
(H2PO4-) and exports hydroxyl ions (OH-). This exchange is electrically neutral because both phosphate and
hydroxyl ions have a charge of -1. However, the exchange is driven by the proton concentration gradient; the
higher pH within mitochondria corresponds to a higher concentration of hydroxyl ions, favoring their
translocation to the cytosolic side of the membrane.
Energy from the electrochemical gradient is similarly used to drive the transport of other metabolites into
mitochondria. For example, the import of pyruvate from the cytosol (where it is produced by glycolysis) is
mediated by a transporter that exchanges pyruvate for hydroxyl ions. Other intermediates of the citric acid cycle
are able to shuttle between mitochondria and the cytosol by similar exchange mechanisms.
Chloroplasts and Other Plastids
Chloroplasts, the organelles responsible for photosynthesis, are in many respects similar to mitochondria. Both
chloroplasts and mitochondria function to generate metabolic energy, evolved by endosymbiosis, contain their
own genetic systems, and replicate by division. However, chloroplasts are larger and more complex than
mitochondria, and they perform several critical tasks in addition to the generation of ATP. Most importantly,
chloroplasts are responsible for the photosynthetic conversion of CO2 to carbohydrates. In addition, chloroplasts
synthesize amino acids, fatty acids, and the lipid components of their own membranes. The reduction of nitrite
(NO2-) to ammonia (NH3), an essential step in the incorporation of nitrogen into organic compounds, also
occurs in chloroplasts. Moreover, chloroplasts are only one of several types of related organelles (plastids) that
play a variety of roles in plant cells.
The Structure and Function of Chloroplasts
Plant chloroplasts are large organelles (5 to 10 m long)
that, like mitochondria, are bounded by a double
membrane called the chloroplast envelope (Figure 10.13).
Figure 10.13. Structure of a chloroplast In addition to the inner and
outer membranes of the envelope, chloroplasts contain a third internal
membrane system: the thylakoid membrane. These membranes divide
chloroplasts into three internal compartments
In addition to the inner and outer membranes of the
envelope, chloroplasts have a third internal membrane
system, called the thylakoid membrane. The thylakoid
membrane forms a network of flattened discs called thylakoids, which are frequently arranged in stacks called
grana. Because of this three-membrane structure, the internal organization of chloroplasts is more complex than
that of mitochondria. In particular, their three membranes divide chloroplasts into three distinct internal
compartments: (1) the intermembrane space between the two membranes of the chloroplast envelope; (2) the
stroma, which lies inside the envelope but outside the thylakoid membrane; and (3) the thylakoid lumen.
Despite this greater complexity, the membranes of chloroplasts have clear functional similarities with those of
mitochondria as expected, given the role of both organelles in the chemiosmotic generation of ATP. The outer
membrane of the chloroplast envelope, like that of mitochondria, contains porins and is therefore freely
permeable to small molecules. In contrast, the inner membrane is impermeable to ions and metabolites, which
are therefore able to enter chloroplasts only via specific membrane transporters. These properties of the inner
and outer membranes of the chloroplast envelope are similar to the inner and outer membranes of mitochondria:
In both cases the inner membrane restricts the passage of molecules between the cytosol and the interior of the
organelle. The chloroplast stroma is also equivalent in function to the mitochondrial matrix: It contains the
chloroplast genetic system and a variety of metabolic enzymes,
including those responsible for the critical conversion of CO2
to carbohydrates during photosynthesis.
The major difference between chloroplasts and mitochondria,
in terms of both structure and function, is the thylakoid
membrane. This membrane is of central importance in
chloroplasts, where it fills the role of the inner mitochondrial
membrane in electron transport and the chemiosmotic
generation of ATP (Figure 10.14).
Figure 10.14. Chemiosmotic generation of ATP in chloroplasts and
mitochondria In mitochondria, electron transport generates a proton
gradient across the inner membrane, which is then used to drive ATP
synthesis in the matrix. In chloroplasts, the proton gradient is generated
across the thylakoid membrane and used to drive ATP synthesis in the
stroma.
The inner membrane of the chloroplast envelope (which is not folded into cristae) does not function in
photosynthesis. Instead, the chloroplast electron transport system is located in the thylakoid membrane, and
protons are pumped across this membrane from the stroma to the thylakoid lumen. The resulting
electrochemical gradient then drives ATP synthesis as protons cross back into the stroma. In terms of its role in
generation of metabolic energy, the thylakoid membrane of chloroplasts is thus equivalent to the inner
membrane of mitochondria.
The Chloroplast Genome
Like mitochondria, chloroplasts contain their own genetic system, reflecting their evolutionary origins from
photosynthetic bacteria. The genomes of chloroplasts are similar to those of mitochondria in that they consist of
circular DNA molecules present in multiple copies per organelle. However, chloroplast genomes are larger and
more complex than those of mitochondria, ranging from 120 to 160 kb and containing approximately 120 genes.
The chloroplast genomes of several plants have been completely sequenced, leading to the identification of
many of the genes contained in the organelle DNAs. These chloroplast genes encode both RNAs and proteins
involved in gene expression, as well as a variety of proteins that function in photosynthesis. Both the ribosomal
and transfer RNAs used for translation of chloroplast mRNAs are encoded by the organelle genome. These
include four rRNAs (23S, 16S, 5S, and 4.5S) and 30 tRNA species. In contrast to the smaller number of tRNAs
encoded by the mitochondrial genome, the chloroplast tRNAs are sufficient to translate all the mRNA codons
according to the universal genetic code. In addition to these RNA components of the translation system, the
chloroplast genome encodes about 20 ribosomal proteins, which represent approximately a third of the proteins
of chloroplast ribosomes. Some subunits of RNA polymerase are also encoded by chloroplasts, although
additional RNA polymerase subunits and other factors needed for chloroplast gene expression are encoded in
the nucleus.
The chloroplast genome also encodes approximately 30 proteins that are involved in photosynthesis, including
components of photosystems I and II, of the cytochrome bf complex, and of ATP synthase. In addition, one of
the subunits of ribulose bisphosphate carboxylase (rubisco) is encoded by chloroplast DNA. Rubisco is the
critical enzyme that catalyzes the addition of CO2 to ribulose-1,5-bisphosphate during the Calvin cycle. Not
only is it the major protein component of the chloroplast stroma, but it is also thought to be the single most
abundant protein on Earth, so it is noteworthy that one of its subunits is encoded by the chloroplast genome.
Import and Sorting of Chloroplast Proteins
Although chloroplasts encode more of their own proteins than
mitochondria, about 90% of chloroplast proteins are still encoded
by nuclear genes. As with mitochondria, these proteins are
synthesized on cytosolic ribosomes and then imported into
chloroplasts as completed polypeptide chains. They must then be
sorted to their appropriate location within chloroplasts an even
more complicated task than protein sorting in mitochondria, since
chloroplasts contain three separate membranes that divide them
into three distinct internal compartments.
Protein import into chloroplasts generally resembles
mitochondrial protein import (Figure 10.15).
Figure 10.15. Protein import into the chloroplast stroma Proteins are
targeted for import into chloroplasts by a transit peptide at their amino terminus.
The transit peptide directs polypeptide translocation through the Toc complex in
the chloroplast outer membrane and the Tic complex in the chloroplast inner
membrane. This peptide is then removed by proteolytic cleavage within the
stroma. Both cytosolic and chloroplast chaperones (Hsp60 and Hsp70) are
required for protein import.
Proteins are targeted for import into chloroplasts by N-terminal sequences of 30 to 100 amino acids, called
transit peptides, which direct protein translocation across the two membranes of the chloroplast envelope and
are then removed by proteolytic cleavage. The transit peptides are recognized by the translocation complex of
the chloroplast outer member (the Toc complex), and proteins are transported through this complex across the
membrane. They are then transferred to the translocation complex of the inner membrane (the Tic complex) and
transported across the inner membrane to the stroma. As in mitochondria, molecular chaperones on both the
cytosolic and stromal sides of the envelope are required for protein import, which requires energy in the form of
ATP. In contrast to the presequences of mitochondrial import,
however, transit peptides are not positively charged and the
translocation of polypeptide chains into chloroplasts does not
require an electric potential across the membrane.
Proteins incorporated into the thylakoid lumen are transported to
their destination in two steps (Figure 10.16).
Figure 10.16. Import of proteins into the thylakoid lumen Proteins are
imported into the thylakoid lumen in two steps. The first step is import into the
chloroplast stroma, as illustrated in Figure 10.15. Cleavage of the transit
peptide then exposes a second hydrophobic signal sequence, which directs
protein translocation across the thylakoid membrane.
They are first imported into the stroma, as already described, and
are then targeted for translocation across the thylakoid
membrane by a second hydrophobic signal sequence, which is
exposed following cleavage of the transit peptide. The
hydrophobic signal sequence directs translocation of the
polypeptide across the thylakoid membrane and is finally
removed by a second proteolytic cleavage within the lumen.
The pathways of protein sorting to the other four compartments
of chloroplasts the inner and outer membranes, thylakoid membrane, and intermembrane space are less well
established. As with mitochondria, proteins appear to be inserted directly into the outer membrane of the
chloroplast envelope. In contrast, proteins destined for either the thylakoid membrane or the inner membrane of
the chloroplast envelope are initially targeted for import into the stroma by N-terminal transit peptides.
Following cleavage of the transit peptides, these proteins are then targeted for insertion into the appropriate
membrane by other sequences, which are not yet well characterized. Finally, neither the sequences that target
proteins to the intermembrane space nor the pathways by which they travel to that destination have been
identified.
Other Plastids
Chloroplasts are only one, albeit the most prominent, member of a larger family of plant organelles called
plastids. All plastids contain the same genome as chloroplasts, but they differ in both structure and function.
Chloroplasts are specialized for photosynthesis and are unique in that they contain the internal thylakoid
membrane system. Other plastids, which are involved in different aspects of plant cell metabolism, are bounded
by the two membranes of the plastid envelope but lack both the thylakoid membranes and other components of
the photosynthetic apparatus.
The different types of plastids are frequently classified according to the kinds of pigments they contain.
Chloroplasts are so named because they contain chlorophyll. Chromoplasts lack chlorophyll but contain
carotenoids; they are responsible for the yellow, orange, and red colors of some flowers and fruits, although
their precise function in cell metabolism is not clear. Leucoplasts are nonpigmented plastids, which store a
variety of energy sources in nonphotosynthetic tissues. Amyloplasts and elaioplasts are examples of
leucoplasts that store starch and lipids, respectively.
All plastids, including chloroplasts, develop from proplastids, small (0.5 to 1 m in diameter) undifferentiated
organelles present in the rapidly dividing cells of plant roots and shoots. Proplastids then develop into the
various types of mature plastids according to the needs of differentiated cells. In addition, mature plastids are
able to change from one type to another. Chromoplasts develop from chloroplasts, for example, during the
ripening of fruit (e.g., tomatoes). During this process, chlorophyll and the thylakoid membranes break down,
while new types of carotenoids are synthesized.
An interesting feature of plastids is that their development is
controlled both by environmental signals and by intrinsic
programs of cell differentiation. In the photosynthetic cells
of leaves, for example, proplastids develop into chloroplasts
(Figure 10.18).
Figure 10.18. Development of chloroplasts Chloroplasts develop from
proplastids in the photosynthetic cells of leaves. Proplastids contain only
the inner and outer envelope membranes; the thylakoid membrane is
formed by vesicle budding from the inner membrane during chloroplast
development. If the plant is kept in the dark, chloroplast development is
arrested at an intermediate stage (etioplasts). Etioplasts lack chlorophyll
and contain semicrystalline arrays of membrane tubules. In the presence
of light, they continue their development to chloroplasts.
During this process, the thylakoid membrane is formed by
vesicles budding from the inner membrane of the plastid
envelope and the various components of the photosynthetic
apparatus are synthesized and assembled. However,
chloroplasts develop only in the presence of light. If plants
are kept in the dark, the development of proplastids in leaves is arrested at an intermediate stage (called
etioplasts), in which a semicrystalline array of tubular internal membranes has formed but chlorophyll has not
been synthesized. If dark-grown plants are then exposed to light, the etioplasts continue their development to
chloroplasts. It is noteworthy that this dual control of plastid development involves the coordinated expression
of genes within both the plastid and nuclear genomes. The mechanisms responsible for such coordinated gene
expression are largely unknown, and their elucidation represents a challenging problem in plant molecular
biology
Photosynthesis
During photosynthesis, energy from sunlight is harvested and used to drive the synthesis of glucose from CO2
and H2O. By converting the energy of sunlight to a usable form of potential chemical energy, photosynthesis is
the ultimate source of metabolic energy for all biological systems. Photosynthesis takes place in two distinct
stages. In the light reactions, energy from sunlight drives the synthesis of ATP and NADPH, coupled to the
formation of O2 from H2O. In the dark reactions, so named because they do not require sunlight, the ATP and
NADPH produced by the light reactions drive glucose synthesis. In eukaryotic cells, both the light and dark
reactions of photosynthesis occur within chloroplasts the light reactions in the thylakoid membrane and the
dark reactions within the stroma. This section discusses the light reactions of photosynthesis, which are related
to oxidative phosphorylation in mitochondria.
Electron Flow through Photosystems I and II
Sunlight is absorbed by photosynthetic pigments, the
most abundant of which in plants are the chlorophylls.
Absorption of light excites an electron to a higher energy
state, thus converting the energy of sunlight to potential
chemical energy. The photosynthetic pigments are
organized into photocenters in the thylakoid membrane,
each of which contains hundreds of pigment molecules
(Figure 10.20).
Figure 10.20. Organization of a photocenter Each photocenter
consists of hundreds of antenna pigment molecules, which absorb
photons and transfer energy to a reaction center chlorophyll. The
reaction center chlorophyll then transfers its excited electron to an acceptor in the electron transport chain. The reaction center illustrated
is that of photosystem II, in which electrons are transferred from the reaction center chlorophyll to pheophytin and then to quinones (QA,
QB, and QH2).
The many pigment molecules in each photocenter act as antennae to absorb light and transfer the energy of
their excited electrons to a chlorophyll molecule that serves as a reaction center. The reaction center chlorophyll
then transfers its high-energy electron to an acceptor molecule in an electron transport chain. High-energy
electrons are then transferred through a series of membrane carriers, coupled to the synthesis of ATP and
NADPH.
The best characterized photosynthetic reaction center is that of the bacterium Rhodopseudomonas viridis, the
structure of which was determined by Johann Deisenhofer, Hartmut Michel, Robert Huber, and their colleagues
in 1985. The reaction center consists of three transmembrane polypeptides, bound to a c-type cytochrome on the
exterior side of the membrane. Energy from sunlight is captured by a pair of chlorophyll molecules known as
the special pair. Electrons are then transferred from the special pair to another pair of chlorophylls and from
there to other prosthetic groups (pheophytins and quinones). From there the electrons are transferred to a
cytochrome bc complex in which electron transport is coupled to the generation of a proton gradient. The
electrons are then transferred to the reaction center cytochrome and finally returned to the chlorophyll special
pair. The reaction center thus converts the energy of sunlight to high-energy electrons, the potential energy of
which is converted to a proton gradient by the cytochrome bc complex.
The proteins involved in the light reactions of photosynthesis in plants are organized into five complexes in the
thylakoid membrane (Figure 10.22).
Figure 10.22. Electron transport and ATP
synthesis during photosynthesis Five
protein complexes in the thylakoid
membrane function in electron transport and
the synthesis of ATP and NADPH. Photons
are absorbed by complexes of pigment
molecules associated with photosystems I
and II (PS I and PS II). At photosystem II,
energy derived from photon absorption is
used to split a water molecule within the
thylakoid lumen. Electrons are then carried
by plastoquinone (PQ) to the cytochrome bf
complex, where they are transferred to a
lower energy state and protons are pumped
into the thylakoid lumen. Electrons are then transferred to photosystem I by plastocyanin (PC). At photosystem I, energy derived from
light absorption again generates high-energy electrons, which are transferred to ferrodoxin (Fd) and used to reduce NADP + to NADPH in
the stroma. ATP synthase then uses the energy stored in the proton gradient to convert ADP to ATP.
Two of these complexes are photosystems (photosystems I and II), in which light is absorbed and transferred
to reaction center chlorophylls. High-energy electrons are then transferred through a series of carriers in both
photosystems and in a third protein complex, the cytochromebfcomplex. As in mitochondria, these electron
transfers are coupled to the transfer of protons into the thylakoid lumen, thereby establishing a proton gradient
across the thylakoid membrane. The energy stored in this proton gradient is then harvested by a fourth protein
complex in the thylakoid membrane, ATP synthase, which (like the mitochondrial enzyme) couples proton flow
back across the membrane to the synthesis of ATP.
One important difference between electron transport in chloroplasts and that in mitochondria is that the energy
derived from sunlight during photosynthesis not only is converted to ATP but also is used to generate the
NADPH required for subsequent conversion of CO2 to carbohydrates. This is accomplished by the use of two
different photosystems in the light reactions of photosynthesis, one to generate ATP and the other to generate
NADPH. Electrons are transferred sequentially between the two photosystems, with photosystem I acting to
generate NADPH and photosystem II acting to generate ATP.
The pathway of electron flow starts at photosystem II, which is homologous to the photosynthetic reaction
center of R. viridis already described. However, at photosystem II the energy derived from absorption of
photons is used to split water molecules to molecular oxygen and protons (see Figure 10.22). This reaction takes
place within the thylakoid lumen, so the release of protons from H2O establishes a proton gradient across the
thylakoid membrane. The high-energy electrons derived from this process are transferred through a series of
carriers to plastoquinone, a lipid-soluble carrier similar to coenzyme Q (ubiquinone) of mitochondria.
Plastoquinone carries electrons from photosystem II to the cytochrome bf complex, within which electrons are
transferred to plastocyanin and additional protons are pumped into the thylakoid lumen. Electron transport
through photosystem II is thus coupled to establishment of a proton gradient, which drives the chemiosmotic
synthesis of ATP.
From photosystem II, electrons are carried by plastocyanin (a peripheral membrane protein) to photosystem I,
where the absorption of additional photons again generates high-energy electrons. Photosystem I, however, does
not act as a proton pump; instead, it uses these high-energy electrons to reduce NADP+ to NADPH. The
reaction center chlorophyll of photosystem I transfers its excited electrons through a series of carriers to
ferrodoxin, a small protein on the stromal side of the thylakoid membrane. The enzyme NADP reductase then
transfers electrons from ferrodoxin to NADP+, generating NADPH. The passage of electrons through
photosystems I and II thus generates both ATP and NADPH, which are used by the Calvin cycle enzymes in the
chloroplast stroma to convert CO2 to carbohydrates.
Cyclic Electron Flow
A second electron transport pathway, called cyclic
electron flow, produces ATP without the synthesis of
NADPH, thereby supplying additional ATP for other
metabolic processes. In cyclic electron flow, light
energy harvested at photosystem I is used for ATP
synthesis rather than NADPH synthesis (Figure 10.23).
Figure 10.23. The pathway of cyclic electron flow Light energy
absorbed at photosystem I (PS I) is used for ATP synthesis rather
than NADPH synthesis. High-energy electrons generated by
photon absorption are transferred to the cytochrome bf complex
rather than to NADP+. At the cytochrome bf complex, electrons are
transferred to a lower energy state and protons are pumped into the thylakoid lumen. The electrons are then returned to photosystem I by
plastocyanin (PC).
Instead of being transferred to NADP+, high-energy electrons from photosystem I are transferred to the
cytochrome bf complex. Electron transfer through the cytochrome bf complex is then coupled, as in
photosystem II, to the establishment of a proton gradient across the thylakoid membrane. Plastocyanin then
returns these electrons to photosystem I in a lower energy state, completing a cycle of electron transport in
which light energy harvested at photosystem I is used to pump protons at the cytochrome bf complex. Electron
transfer from photosystem I can thus generate either ATP or NADPH, depending on the metabolic needs of the
cell.
ATP Synthesis
The ATP synthase of the thylakoid membrane is similar to the mitochondrial enzyme. However, the energy
stored in the proton gradient across the thylakoid membrane, in contrast to the inner mitochondrial membrane,
is almost entirely chemical in nature. This is because the thylakoid membrane, although impermeable to protons,
differs from the inner mitochondrial membrane in being permeable to other ions, particularly Mg2+ and Cl-. The
free passage of these ions neutralizes the voltage component of the proton gradient, so the energy derived from
photosynthesis is conserved mainly as the difference in proton concentration (pH) across the thylakoid
membrane. However, because the thylakoid lumen is a closed compartment, this difference in proton
concentration can be quite large, corresponding to a differential of more than three pH units between the stroma
and the thylakoid lumen. Because of the magnitude of this pH differential, the total free energy stored across the
thylakoid membrane is similar to that stored across the inner mitochondrial membrane.
For each pair of electrons transported, two protons are transferred across the thylakoid membrane at
photosystem II and two to four protons at the cytochrome bf complex. Since four protons are needed to drive the
synthesis of one molecule of ATP, passage of each pair of electrons through photosystems I and II by noncyclic
electron flow yields between 1 and 1.5 ATP molecules. Cyclic electron flow has a lower yield, corresponding to
between 0.5 and 1 ATP molecules per pair of electrons.
Peroxisomes
Peroxisomes are small, membrane-enclosed organelles (Figure 10.24) that
contain enzymes involved in a variety of metabolic reactions, including
several aspects of energy metabolism. Although peroxisomes are
morphologically similar to lysosomes, they are assembled, like mitochondria
and chloroplasts, from proteins that are synthesized on free ribosomes and then imported into peroxisomes as
completed polypeptide chains. Although peroxisomes do not contain their own genomes, they are similar to
mitochondria and chloroplasts in that they replicate by division.
Functions of Peroxisomes
Peroxisomes contain at least 50 different enzymes, which are involved in a variety of biochemical pathways in
different types of cells. Peroxisomes originally were defined as organelles that carry out oxidation reactions
leading to the production of hydrogen peroxide. Because hydrogen peroxide is harmful to the cell, peroxisomes
also contain the enzyme catalase, which decomposes hydrogen peroxide either by converting it to water or by
using it to oxidize another organic compound. A variety of substrates are broken down by such oxidative
reactions in peroxisomes, including uric acid, amino acids, and fatty acids. The oxidation of fatty acids (Figure
10.25) is a particularly important example, since it provides a major source of metabolic energy. In animal cells,
fatty acids are oxidized in both peroxisomes and
mitochondria, but in yeasts and plants fatty acid
oxidation is restricted to peroxisomes.
Figure 10.25. Fatty acid oxidation in peroxisomes The oxidation
of a fatty acid is accompanied by the production of hydrogen
peroxide (H2O2) from oxygen. The hydrogen peroxide is
decomposed by catalase, either by conversion to water or by
oxidation of another organic compound (designated AH2).
In addition to providing a compartment for oxidation reactions, peroxisomes are involved in lipid biosynthesis.
In animal cells, cholesterol and dolichol are synthesized in peroxisomes as well as in the ER. In the liver,
peroxisomes are also involved in the synthesis of bile acids, which are derived from cholesterol. In addition,
peroxisomes contain enzymes required for the synthesis of plasmalogens a family of phospholipids in which
one of the hydrocarbon chains is joined to glycerol by an ether bond rather than an ester bond (Figure 10.26).
Plasmalogens are important membrane components in some tissues, particularly
heart and brain, although they are absent in others.
Figure 10.26. Structure of a plasmalogen The plasmalogen shown is analogous to
phosphatidylcholine. However, one of the fatty acid chains is joined to glycerol by an ether, rather
than an ester, bond.
Peroxisomes play two particularly important roles in
plants. First, peroxisomes in seeds are responsible
for the conversion of stored fatty acids to
carbohydrates, which is critical to providing energy
and raw materials for growth of the germinating
plant. This occurs via a series of reactions termed
the glyoxylate cycle, which is a variant of the citric
acid cycle(Figure 10.27). The peroxisomes in which
this takes place are sometimes called glyoxysomes.
Figure 10.27. The glyoxylate cycle Plants are capable of
synthesizing carbohydrates from fatty acids via the glyoxylate
cycle, which is a variant of the citric acid cycle (see Figure 2.34).
As in the citric acid cycle, acetyl CoA combines with
oxaloacetate to form citrate, which is converted to isocitrate.
However, instead of being degraded to CO2 and -ketoglutarate,
isocitrate is converted to succinate and glyoxylate. Glyoxylate
then reacts with another molecule of acetyl CoA to yield malate,
which is converted to oxaloacetate and used for glucose
synthesis.
Second, peroxisomes in leaves are involved in photorespiration, which serves to metabolize a side product
formed during photosynthesis (Figure 10.28). CO2 is converted to carbohydrates during photosynthesis via a
series of reactions called the Calvin cycle. The first step is the addition of CO2 to the five-carbon sugar ribulose1,5-bisphosphate, yielding two molecules of 3-phosphoglycerate (three carbons each). However, the enzyme
involved (ribulose bisphosphate carboxylase or rubisco) sometimes catalyzes the addition of O2 instead of CO2,
producing one molecule of 3phosphoglycerate and one
molecule of phosphoglycolate
(two carbons).
Figure 10.28. Role of peroxisomes in
photorespiration During photosynthesis,
CO2 is converted to carbohydrates by the
Calvin cycle, which initiates with the
addition of CO2 to the five-carbon sugar
ribulose-1,5-bisphosphate. However, the
enzyme involved sometimes catalyzes the
addition of O2 instead, resulting in
production of the two-carbon compound
phosphoglycolate. Phosphoglycolate is
converted to glycolate, which is then
transferred to peroxisomes, where it is
oxidized and converted to glycine.
Glycine is then transferred to
mitochondria and converted to serine. The serine is returned to peroxisomes and converted to glycerate, which is transferred back to
chloroplasts.
This is a side reaction, and phosphoglycolate is not a useful metabolite. It is first converted to glycolate and
then transferred to peroxisomes, where it is oxidized and converted to glycine. Glycine is then transferred to
mitochondria, where two molecules of glycine are converted to one molecule of serine, with the loss of CO2 and
NH3. The serine is then returned to peroxisomes, where it is converted to glycerate. Finally, the glycerate is
transferred back to chloroplasts, where it reenters the Calvin cycle. Photorespiration does not appear to be
beneficial for the plant, since it is essentially the reverse of photosynthesis O2 is consumed and CO2 is
released without any gain of ATP. However, the occasional utilization of O2 in place of CO2 appears to be an
inherent property of rubisco, so photorespiration is a general accompaniment of photosynthesis. Peroxisomes
thus play an important role by allowing most of the carbon in glycolate to be recovered and utilized.
Peroxisome Assembly
As already noted, the assembly of peroxisomes is fundamentally similar to that
of mitochondria and chloroplasts, rather than to that of the endoplasmic
reticulum, Golgi apparatus, and lysosomes. Proteins destined for peroxisomes are
translated on free cytosolic ribosomes and then transported into peroxisomes as
completed polypeptide chains (Figure 10.29).
Figure 10.29. Assembly of peroxisomes Proteins destined for peroxisomes are synthesized on free
ribosomes and imported into preexisting peroxisomes as completed polypeptide chains. Protein
import results in peroxisome growth and the formation of new peroxisomes by division of old ones.
Phospholipids are also imported to peroxisomes, via phospholipid transfer
proteins, from their major site of synthesis in the ER. The import of proteins and
phospholipids results in peroxisome growth, and new peroxisomes are then
formed by division of old ones.
Proteins are targeted to the interior of peroxisomes by at least two pathways,
which are conserved from yeasts to humans. Most proteins are targeted to
peroxisomes by the simple amino acid sequence Ser-Lys-Leu at their carboxy
terminus (peroxisome targeting signal 1, or PTS1). Other proteins are targeted by
a sequence of nine amino acids (PTS2) at their amino terminus, and some
proteins may be targeted by alternative signals that have not yet been well
defined.
PTS1 and PTS2 are recognized by distinct receptors and then transferred to a
translocation complex that mediates their transport across the peroxisome
membrane. However, the mechanism of protein import into peroxisomes is less
well characterized than the mechanisms of protein translocation across the membranes of other subcellular
organelles. In contrast to the translocation of polypeptide chains across the membranes of the endoplasmic
reticulum, mitochondria, and chloroplasts, targeting signals are usually not cleaved during the import of
proteins into peroxisomes. Cytosolic Hsp70 has been implicated in protein import to peroxisomes, but the
possible role of molecular chaperones within peroxisomes is unclear. Moreover, it appears that proteins can be
transported into peroxisomes in at least partially folded conformations, rather than as extended polypeptide
chains.
Some peroxisome membrane proteins are similarly synthesized on cytosolic ribosomes and targeted to the
peroxisome membrane by distinct internal signals. However, other experiments suggest that some peroxisomal
membrane proteins may be synthesized on membrane-bound polysomes of the endoplasmic reticulum and then
transported to peroxisomes, suggesting a role for the endoplasmic reticulum in peroxisome maintenance. The
import of proteins into peroxisomes thus appears to have several novel features, making it an active area of
investigation.
Interestingly, some components of peroxisome import pathways have been identified not only as mutants of
yeasts but also as mutations associated with serious human diseases involving disorders of peroxisomes. In
some such diseases, only a single peroxisomal enzyme is deficient. However, in other diseases resulting from
defects in peroxisome function, multiple peroxisomal enzymes fail to be imported to peroxisomes, instead being
localized in the cytosol. The latter group of diseases results from deficiencies in the PTS1 or PTS2 pathways
responsible for peroxisomal protein import. The prototypical example is Zellweger syndrome, which is lethal
within the first ten years of life. Zellweger syndrome can result from mutations in at least ten different genes
affecting peroxisomal protein import, one of which has been identified as the gene encoding the receptor for the
peroxisome targeting signal PTS1.