10.
1 Cellular Respiration: pyruvate and other oxidizable substrates, they can then trans-
Maximizing ATP Yields
Cellular respiration, or respiration for short, dramatically
improves the metabolic energy yield per molecule of glucose.
With an external electron acceptor available, complete oxida-
tion of substrates to CO2 becomes possible, and ATP yields are
much higher. By “external” we mean an electron acceptor that
is not a by-product of glucose catabolism. When an “internal”
electron acceptor such as pyruvate or acetaldehyde is used
(as during fermentation) to accept electrons from NADH (see
Figure 9-8), the by-products are not completely oxidized to CO2.
As a formal definition, cellular respiration is the flow of
electrons, through or within a membrane, from reduced coenzymes
to an external electron acceptor, usually accompanied by the gen-
eration of ATP. We have already encountered NADH as the
reduced coenzyme generated by the glycolytic catabolism of
sugars or related compounds. As we will see shortly, two other
coenzymes, FAD (for flavin adenine dinucleotide) and coenzyme
Q (or ubiquinone), also collect the electrons that are removed
from oxidizable organic substrates and pass them to the termi-
nal electron acceptor via a series of electron carriers, generat-
ing ATP in the process.
For many organisms, including us, the terminal electron
acceptor is oxygen, which is reduced to water and, therefore,
the overall oxygen-requiring process is known as aerobic
respiration. (Note that, in medical terminology at an or-
ganismal level, respiration refers to breathing, the uptake of
oxygen.) But although many chemotrophs on Earth carry out
aerobic respiration, other organisms, especially some bacteria
and archaea, can use a variety of terminal electron acceptors
other than molecular oxygen and thus carry out anaerobic
respiration. Examples of alternative electron acceptors and
their reduced forms include elemental sulfur (S/H2S), protons
(H+/H2), and ferric ions (Fe3+ /Fe2+). Respiratory processes
that involve electron acceptors such as these require no mo-
lecular oxygen and are therefore anaerobic. These anaerobic
respiratory processes play important roles in nutrient cycling
and in the overall energy economy of the biosphere.
Here, however, we will concentrate on aerobic respira-
tion because it is the basis of energy metabolism in the aerobic
world of which we and most other familiar organisms are a
part. We will focus much of our attention on the mitochon-
drion (Figure 10-1) because most aerobic ATP production in
eukaryotic cells takes place within this organelle. In bacterial
cells, the roles of the plasma membrane and cytoplasm in en-
ergy production are analogous to the roles of the mitochon-
drial inner membrane and matrix, respectively.
Aerobic Respiration Yields Much More Energy
than Fermentation Does
With oxygen available as the terminal electron acceptor, pyru-
vate can be oxidized completely to CO2 instead of being used to
accept electrons from NADH. Therefore, aerobic respiration has
the potential of generating up to 38 ATP molecules per glucose.
Oxygen makes all of this possible by serving as the termi-
nal electron acceptor, thereby providing a means for the con-
tinuous reoxidation of NADH and other reduced coenzymes.
After these coenzyme molecules accept electrons from
M10_HARD7694_09_GE_C10.indd 267
fer these electrons to oxygen. Aerobic respiration therefore
involves oxidative pathways in which electrons are removed
from organic substrates and transferred to coenzyme carriers,
which then transfer these electrons to oxygen, accompanied
by the generation of ATP.
Respiration Includes Glycolysis, Pyruvate
Oxidation, the Citric Acid Cycle, Electron
Transport, and ATP Synthesis
We will consider aerobic respiration in five stages, as shown
in Figure 10-1. The first three stages involve substrate oxida-
tion and the simultaneous reduction of coenzymes, and the
second two involve coenzyme reoxidation and the generation
of ATP. In cells, of course, all five stages occur continuously
and simultaneously.
Stage ●1 is the glycolytic pathway (which we encountered
in Chapter 9). The steps of glycolysis are the same under
aerobic and anaerobic conditions and result in the oxidation
of glucose to pyruvate. However, in the presence of oxygen,
the fate of the pyruvate is different (see Figure 9-8). Instead
of serving as an electron acceptor, as occurs in anaerobic fer-
Chapter 10
mentation, pyruvate is further oxidized. In stage ● 2 , pyruvate
is oxidized to generate acetyl coenzyme A (acetyl CoA), which
enters the citric acid cycle (stage ●
3 ). The citric acid cycle com-
pletely oxidizes acetyl CoA to CO2 and conserves most of the
|
energy as high-energy reduced coenzyme molecules.
Chemotrophic Energy Metabolism: Aerobic Respiration
Stage ●4 involves electron transport, or the transfer of elec-
trons from reduced coenzymes to oxygen, coupled to the active
transport (pumping) of protons across a membrane. The trans-
fer of electrons from coenzymes to oxygen is exergonic and
provides the energy that drives the pumping of protons across
the membrane containing the carriers. This generates an elec-
trochemical proton gradient across the membrane. In stage ● 5,
the energy of this proton gradient is used to drive ATP synthe-
sis in a process known as oxidative phosphorylation.
Our goal in this chapter is to understand the processes fol-
lowing glycolysis—the complete oxidation of pyruvate in the
citric acid cycle, electron transport from pyruvate via coen-
zymes to oxygen, and the formation of a proton gradient that
drives ATP synthesis. We will begin our discussion of aerobic
energy metabolism with a close look at the mitochondrion be-
cause of its prominent role in eukaryotic energy metabolism.
CONCEPT CHECK 10.1
Aerobic respiration uses an external electron acceptor rather
than an internal electron acceptor, as is used in anaerobic
fermentation. Explain what this means. Why does using an
external electron acceptor provide so much more energy to
the cell?
10.2 The Mitochondrion: Where
the Action Takes Place
Our discussion of aerobic respiration starts with a description
of the mitochondrion because most of aerobic energy me-
tabolism in eukaryotic cells takes place within this organelle.
Because of this, the mitochondrion is often called the “energy
267
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 Mitochondrion

Triglycerides

CO2 2 PYRUVATE
ATP OXIDATION Hydrolysis

Glucose Pyruvate Pyruvate Acetyl CoA Fatty acids

b oxidation
1 GLYCOLYSIS
NADH CO2
2 NADH 3
CITRIC CO2
3 NADH
CYTOSOL ACID
- CYCLE
2e
FADH2 ATP

1/2 O2
MATRIX H2O
H+ H+ H+ 4 ELECTRON
+
2 e- 2H TRANSPORT
Inner membrane +
H and
Intermembrane space PROTON
H+ H+ H+ H+
+
H+ H + H+
H+ H+ H+ H+ H+ PUMPING
Outer membrane H+ H+ H+ H H+
H+ H+ H+
H+ H+ H + H+ +
H
H+
H+ Crista

H+
ADP H+ ADP
H+ ADP
+ Pi ATP + Pi H+
H+ ATP + Pi
ATP
H+
H+ 5 ATP SYNTHESIS
H+ H+ H+
H+ H+

Figure 10-1 The Role of the Mitochondrion in Aerobic Respiration. The mitochondrion plays a central
role in aerobic respiration. Most respiratory ATP production in eukaryotic cells occurs in this organelle. Oxida-
tion of glucose and other sugars begins in the cytosol with glycolysis (stage ● 1 ), producing pyruvate. Pyruvate
is transported into the mitochondrion, where it is oxidized within the matrix to acetyl CoA (stage ● 2 ), the
primary substrate of the citric acid cycle (stage ●3 ). Acetyl CoA can also be formed by b oxidation of fatty acids.
Electron transport is coupled to proton pumping (stage ● 4 ) to produce an electrochemical proton gradient
across the inner membrane of the mitochondrion. The energy of the proton gradient drives the synthesis of ATP
from ADP and inorganic phosphate (stage ● 5 ).

powerhouse” of the eukaryotic cell. Defects in mitochondrial
function can lead to human diseases known as mitochon-
drial myopathies. In later chapters, we will discuss additional
aspects of mitochondrial biology, such as protein import into
mitochondria, destruction of damaged mitochondria by au-
tophagy, their role in programed cell death, and the mitochon-
drial genome. Here, however, we will focus on the key role of
mitochondria in cellular energy production.
268
As early as 1850, the German biologist Rudolph Kölliker
described the presence of what he called “ordered arrays of
particles” in muscle cells. Isolated particles were found to swell
in water, leading Kölliker to conclude that each particle was
surrounded by a semipermeable membrane. These particles
are now called mitochondria (singular: mitochondrion) and are
believed to have arisen when bacterial cells were engulfed by
larger cells but survived, taking up permanent residence in

M10_HARD7694_09_GE_C10.indd 268 13/02/17 6:28 pm

 the host cell cytoplasm via endosymbiosis (as discussed in
Chapter 4).
Evidence suggesting a role for this organelle in oxidative
events began to accumulate almost a century ago. In 1913,
for example, Otto Warburg showed that these particles could
consume oxygen. However, most of our understanding of the
role of mitochondria in energy metabolism came since the
development of differential centrifugation, pioneered by Al-
bert Claude (see Key Technique in Chapter 4, page 120).
Intact, functionally active mitochondria were first isolated by
this technique in 1948 and were subsequently shown by Eu-
gene Kennedy, Albert Lehninger, and others to be capable of
carrying out not only all the reactions of the citric acid cycle,
but also electron transport and oxidative phosphorylation.
Mitochondria Are Often Present Where
the ATP Needs Are Greatest
Mitochondria are found in virtually all aerobic cells of eukary-
otes and are prominent features of many cell types when ex-
amined by electron microscopy (Figure 10-2). Mitochondria
are present in both chemotrophic and phototrophic cells and
are therefore found not only in animals but also in plants. Their
occurrence in phototrophic cells reminds us that even photo-
synthetic organisms rely on respiration to meet energy needs.
The crucial role of the mitochondrion in meeting cellular
ATP needs is often reflected in the localization of mitochon-
dria within the cell. Frequently, mitochondria are clustered
in regions of cells with the most intense metabolic activity
and the greatest need for ATP. An especially good example is
Mitochondria
0.5 mm
Figure 10-2 Mitochondria: One or Many? Mitochondria are
prominent features in this cross section of a rat liver cell and were
traditionally thought to be discrete oval structures. However, current
research suggests that these may be separate slices through one or
more large, multilobed mitochondria (TEM).
M10_HARD7694_09_GE_C10.indd 269
in muscle cells (see Figure 4-14 and chapter-opening figure).
The mitochondria in these muscle cells are organized in rows
along the fibrils responsible for contraction. A similar strate-
gic localization of mitochondria occurs in flagella and cilia, as
well as in sperm tails (see Figure 4-13).
Are Mitochondria Interconnected Networks
Rather than Discrete Organelles?
When seen in an electron micrograph such as that in
Figure 10-3a, mitochondria typically appear in outline as oval
structures measuring one or more micrometers in length and
0.5–1.0 mm across. This appearance has fostered the widely
accepted view that mitochondria are discrete oval-shaped en-
tities, as depicted in Figure 10-3b. However, depending on the
cell type, mitochondria can be seen in various shapes and siz-
es. Recent work using electron microscopy (EM) tomography,
which allows reconstruction of a three-dimensional model
of a cellular structure from a parallel series of cross sections,
shows that some mitochondria are not simple ovals, but have
much more complex shapes, as shown in Figure 10-4. (The
technique of EM tomography is discussed in more detail in
Key Technique, pages 272–273.)
Chapter 10
Calculations based on cross-sectional views of mitochon-
dria in electron micrographs indicate that the number of mi-
tochondria per cell is highly variable, ranging from one or a
few per cell in many protists, fungi, and algae (and in some
|
mammalian cells as well) to a few thousand per cell in some
Chemotrophic Energy Metabolism: Aerobic Respiration
tissues of higher plants and animals. Mammalian liver cells,
for example, are thought to contain about 500–1000 mito-
chondria each, though only a small fraction of these are seen
in a typical thin section prepared for electron microscopy, such
as that shown in Figure 10-2.
The notion that the mitochondrial profiles seen in elec-
tron micrographs represent discrete organelles of known size
and abundance has been challenged by the work of Hans-
Peter Hoffmann and Charlotte Avers. After examining a com-
plete series of thin sections through an entire yeast cell, these
investigators concluded that the oval profiles observed in indi-
vidual micrographs all represent slices through a single large,
extensively branched mitochondrion. Such results suggest
that the number of mitochondria present in a cell may be con-
siderably smaller than generally believed—and that the size of
a mitochondrion may be much larger than can be calculated
from individual cross-sectional profiles seen in thin-section
electron micrographs.
Further support for the concept of mitochondria as inter-
connected networks (rather than many separate organelles)
comes from studies in which intact living cells were examined
by phase-contrast microscopy or by fluorescence microscopy
using mitochondria-specific fluorescent dyes. Such investiga-
tions revealed that living cells contain large branched mito-
chondria in a dynamic state of flux, with segments of one mi-
tochondrion frequently pinching off and fusing with another
mitochondrion.
Most of the discussion and illustrations in this chapter
will presume the conventional view of mitochondria as dis-
crete entities, but keep in mind that what we regard as indi-
vidual mitochondria may well be parts of a large, dynamic
network instead, at least in some types of cells.
269
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 Inner and outer Rough endoplasmic
membranes reticulum

Cristae

Outer membrane

Intermembrane
space

Inner membrane

Matrix

Cristae junction

Matrix Intracristal spaces

Cristae

(a) Electron micrograph 1 mm (b) Schematic diagram

Figure 10-3 Mitochondrial Structure. (a) A mitochondrion of a bat pancreas cell as seen by electron mi-
croscopy (TEM). The cristae are formed by infoldings of the inner membrane. (b) A mitochondrion is illustrated
schematically in this cutaway view that shows the traditional “baffle” model of cristae structure. As noted in the
text, however, this model is currently being reconsidered.

The Outer and Inner Membranes Define Two
Separate Compartments and Three Regions
Figure 10-3 shows both an electron micrograph and an il-
lustration of a mitochondrion. A distinctive feature is the
presence of two membranes, called the outer and inner
Figure 10-4 EM Tomography of an Extended
Mitochondrion. This tomogram of a mouse embryonic fibroblast
shows the extended shape of the intact mitochondrion. Individual
cristae, mostly lamellar, are colored separately. Note the tubular
crista (light blue) at the bottom left.
270
membranes. The outer membrane is not a significant
permeability barrier for ions and small molecules because
it contains transmembrane channel proteins called porins
that permit the passage of solutes with molecular weights
up to about 5000 (see Figure 8-9). Similar proteins are
found in the outer membrane of gram-negative bacteria.
Because porins allow the free movement of small molecules
and ions across the outer membrane, the intermembrane
space between the inner and outer membranes of the mi-
tochondrion is continuous with the cytosol with respect to
small solutes. However, enzymes targeted to the intermem-
brane space are effectively confined there because enzymes
and other soluble proteins are too large to pass through the
porin channels.
In contrast to the outer membrane, the inner
membrane of the mitochondrion presents a permeability
barrier to most solutes, thereby partitioning the mitochon-
drion into two separate compartments—the intermembrane
space and the interior of the organelle (the mitochondrial ma-
trix). The inner and outer membranes are each about 7 nm
thick and are typically separated by an intermembrane space
of about 7 nm. However, in certain spots the membranes are
in contact, and it is in these regions that proteins destined for
the mitochondrial matrix pass through the two membranes
(see Figure 19-26).
At the perimeter of the mitochondrion, the portion of
the inner membrane adjacent to the intermembrane space is
known as the inner boundary membrane. In addition, the inner
membrane of most mitochondria has many distinctive infold-
ings called cristae (singular: crista) that greatly increase its
surface area. In a typical liver mitochondrion, for example,
the area of the inner membrane (including the cristae) is

M10_HARD7694_09_GE_C10.indd 270 13/02/17 6:29 pm

 about five times greater than that of the outer membrane.
Because of its large surface area, the inner membrane can ac-
commodate large numbers of the protein complexes needed
for electron transport and ATP synthesis, thereby enhancing
the mitochondrion’s capacity for ATP generation. The inner
membrane is about 75% protein by weight, which is a higher
proportion of protein than in any other cellular membrane.
These inner membrane proteins include the transmembrane
portions of proteins involved in solute transport, electron
transport, and ATP synthesis. The cristae also provide numer-
ous localized regions, the intracristal spaces, where protons can
accumulate between the folded inner membranes during the
electron transport process, which we will discuss later in the
chapter.
It was long thought that cristae were wide, flattened
structures with broad connections to the inner bound-
ary membrane, giving rise to the “baffle” model of cristae
structure shown in Figure 10-3b. Thanks to EM tomogra-
phy, however, we know that cristae can adopt a variety of
shapes. It is now believed that cristae in many tissues may be
tubular structures that associate in layers to form lamellar
(layered) cristae of irregular size and shape (Figure 10-5).
Cristae appear to have only limited connections to the in-
ner boundary membrane through small tubular openings
known as crista junctions. The small size of these openings
(approximately 20 nm) is thought to limit diffusion of mate-
rials between the intracristal space and the intermembrane
0.2 mm
Figure 10-5 Structure of Individual Cristae. EM tomography
was used to create this three-dimensional reconstruction of three
cristae (green, light blue, dark blue) in a mitochondrion. White ar-
rows point to crista junctions connecting two of the cristae with the
inner membrane (gold), making the intracristal spaces contiguous
with the intermembrane space. The outer membrane is not shown.
M10_HARD7694_09_GE_C10.indd 271
space, effectively creating a third, nearly enclosed region in
the mitochondrion.
The relative prominence of cristae within the mitochon-
drion frequently reflects the relative metabolic activity of the
cell or tissue in which the organelle is located. Heart, kidney,
and muscle cells have high respiratory activities, and thus
their mitochondria have correspondingly large numbers of
prominent cristae. The flight muscles of birds are especially
high in respiratory activity and have mitochondria that are
exceptionally abundant in cristae. Plant cells, by contrast,
have lower rates of respiratory activity than do most animal
cells and have correspondingly fewer cristae within their
mitochondria.
The interior of the mitochondrion is filled with a semi-
fluid matrix. Within the matrix are many of the enzymes
involved in mitochondrial function as well as DNA molecules
and ribosomes. In most mammals, the mitochondrial genome
consists of a circular DNA molecule of about 15,000–20,000
base pairs that code for ribosomal RNAs, transfer RNAs, and
about a dozen polypeptide subunits of inner-membrane pro-
teins. Mutations in mitochondrial DNA can lead to human
disease and have been associated with aging and neurodegen-
eration. Besides the proteins encoded by the mitochondrial
Chapter 10
genome, mitochondria contain numerous nuclear-encoded
proteins that are synthesized on cytoplasmic ribosomes and
then imported into mitochondria (see Chapter 19).
|Chemotrophic Energy Metabolism: Aerobic Respiration
Mitochondrial Functions Occur in or on
Specific Membranes and Compartments
Specific functions and pathways have been localized within
the mitochondrion by disruption of the organelle and frac-
tionation of the various components. Table 10-1 lists some
of the main functions localized to each compartment of the
mitochondrion.
Most of the mitochondrial enzymes involved in pyruvate
oxidation, in the citric acid cycle, and in the catabolism of fatty
Table 10-1 Localization of Metabolic Functions
Within the Mitochondrion
Membrane or Compartment Metabolic Functions
Outer membrane Phospholipid synthesis
Fatty acid desaturation
Fatty acid elongation
Inner membrane Electron transport
Proton translocation for ATP
synthesis
Oxidative phosphorylation
Pyruvate import
Fatty acyl CoA import
Metabolite transport
Matrix Pyruvate oxidation
Citric acid cycle
ATP synthesis
b oxidation of fats
DNA replication
RNA synthesis (transcription)
Protein synthesis (translation)
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