CHAPTER 9

Mitochondrial Structure and Function

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 Keys
• Clarify the overall mitochondrial structure and function.
• Describe the inner/outer mitochondrial membranes and matrix.
• Review the processes of glycolysis and anaerobic fermentation.
• Emphasize the electron transport system in forming a proton gradient.
• Describe the chemiosmotic mechanism and cellular energy transduction.
• Clarify mechanisms for transporting proton to the intermembrane space.
• Describe the mechanisms ATP synthase may convert ADP and Pi to ATP.
• Emphasize the roles of the proton-motive force other than ATP synthesis.
• Discuss the control of respiration.
• Discuss the role of mitochondria in heat production.
• Describe the mechanisms to import proteins into the mitochondria.
• Elucidate the structure and function of peroxisomes and glyoxysomes.

© 2013 John Wiley & Sons, Inc. All rights reserved.

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 Introduction

• The early Earth was populated by anaerobes, which captured

and utilized energy by oxygen-independent metabolism.
• Oxygen accumulated in the primitive atmosphere after
cyanobacteria appeared.
• Aerobes evolved to use oxygen to extract more energy from
organic molecules.
• In eukaryotes, aerobic respiration takes place in the
mitochondrion.

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(9.1) Mitochondrial Structure and Function

Elongated mitochondria Transmission electron Mitochondria in the

of fibroblast micrograph sperm mid-piece

• Mitochondria: characteristic morphologies despite variable appearance.

– Typical mitochondria are bean-shaped organelles but may be round or
threadlike.
– Size and number of mitochondria reflect the energy requirements of the cell.

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 Mitochondrial Structure and Function

Dynamic nature of
mitochondria
revealed in mouse
fibroblasts with a
fluorescent tagged
mitochondrial
protein.

• Mitochondria can fuse with one another, or split in two.

– The balance between fusion and fission is likely a major determinant
of mitochondrial number, length, and degree of interconnection.

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 Mitochondrial Structure and Function

3D model of contacts Model for mitochondrial fission: ER

between ER and tubules and Drp1 mediate mitochondrial
mitochondria constriction.

• Mitochondria can fuse with one another, or split in two.

– The balance between fusion and fission is likely a major determinant
of mitochondrial number, length, and degree of interconnection.

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 Mitochondrial Structure and Function
The structure of a mitochondrion
• Inner and outer mitochondrial Scanning
membranes enclose two electron
spaces: the matrix and micrograph of
a macerated
intermembrane space.
mitochondrion
– The outer mitochondrial
membrane serves as its outer
boundary.
– The inner mitochondrial 3D
membrane is subdivided into reconstruction of
two interconnected domains: a mitochondrion
based on a
• Inner boundary
micrographs
membrane
taken with a
• Cristae: where the high-voltage
machinery for ATP is electron
located microscope

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 Mitochondrial Structure and Function
The structure of a mitochondrion
• Outer mitochondrial
membrane: outer boundary.
• Inner mitochondrial
membrane has two
interconnected domains:
• Inner boundary membrane
• Cristae: where the
machinery for ATP is located
• The mitochondrial matrix
– Contains a circular DNA
molecule, ribosomes, and
enzymes.
– RNA and proteins can be Schematic diagrams showing the 3D
synthesized in the matrix. internal structure and a thin section of a
mitochondrion from bovine heart tissue

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 Mitochondrial Structure and Function

• Mitochondrial Membranes
– The outer membrane is about 50%
lipid; the inner membrane is more
than 75% protein.
– The inner membrane contains
cardiolipin but not cholesterol,
both are true of bacterial
membranes.
– The outer membrane contains a
large pore-forming protein called
porin.
– The inner membrane is
impermeable to even small
molecules; the outer membrane is Porin motif: a β-sheet barrel that
permeable to even some proteins. forms an opening for passage of
moderate-sized molecules

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 Mitochondrial Structure and Function

• Mitochondrial Membranes
– The outer membrane is about 50%
lipid; the inner membrane is more
than 75% protein.
– The inner membrane contains
cardiolipin but not cholesterol,
both are true of bacterial
membranes.
– The outer membrane contains a
large pore-forming protein called
porin.
– The inner membrane is
impermeable to even small
molecules; the outer membrane is Porin motif: a β-sheet barrel that
permeable to even some proteins. forms an opening for passage of
moderate-sized molecules

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 Mitochondrial Structure and Function
Carbohydrate metabolism in eukaryotic cells

Coupling cytosolic glycolysis and pyruvate production

to the mitochondrial TCA cycle and ATP formation
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 (9.2) Oxidative Metabolism in the
Mitochondrion
• The first steps in
oxidative metabolism are
carried out in glycolysis.
– Glycolysis produces
pyruvate, NADH, and
two molecules of ATP.
– Aerobic organisms use
O2 to extract more than
30 additional ATPs from
pyruvate and NADH.
– Pyruvate is transported
across the inner
membrane and
decarboxylated to form
Coupling cytosolic glycolysis and pyruvate production
acetyl CoA, which enters
to the mitochondrial TCA cycle and ATP formation
the next stage.

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Oxidative Metabolism in the Mitochondrion
An overview of glycolysis

Overview of glycolysis
showing some of the
key steps.

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Oxidative Metabolism in the Mitochondrion
The TCA cycle
• The tricarboxylic acid (TCA)
cycle
– It is a stepwise cycle where
substrate is oxidized and its
energy conserved.
– The two-carbon acetyl group
from acetyl CoA is condensed
with the four-carbon
oxaloacetate to form a six-
carbon citrate.
– During the cycle, two carbons
are oxidized to CO2, regenerating
the four-carbon oxaloacetate
needed to continue the cycle.

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Oxidative Metabolism in the Mitochondrion
The TCA cycle
• The TCA cycle
– Four reactions in the
cycle transfer a pair
of electrons to NAD+
to form NADH, or to
FAD+ to form FADH2.
– Reaction
intermediates in the
TCA cycle are
common compounds
generated in other
catabolic reactions
making the TCA cycle
the central metabolic
pathway of the cell. Catabolic pathways generate compounds
that are fed into the TCA cycle
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Oxidative Metabolism in the Mitochondrion
The glycerol phosphate shuttle

• The Importance of
Reduced Coenzymes in
the Formation of ATP
– The reduced coenzymes
FADH2 and NADH are the
primary products of the
TCA cycle.
– NADH formed during
glycolysis enters the
mitochondria via malate-
aspartate or glycerol
phosphate shuttles.
Electron transfer from NADH to DHAP to form
glycerol 3-phosphate, then to FAD to form FADH2

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Oxidative Metabolism in the Mitochondrion
Summary of oxidative phosphorylation
• Importance of Reduced Coenzymes
– As electrons move through the
electron-transport chain, H+ are
pumped out across the inner
membrane.
– ATP is formed by the controlled
movement of H+ back across the
membrane through the ATP-
synthesizing enzyme.
– Coupling of H+ translocation to ATP
synthesis is called chemiosmosis.
– Three molecules of ATP are formed
from each pair of electrons donated
by NADH; two molecules of ATP are Two step process of oxidative
formed from each pair of electrons phosphorylation: Formation and
donated by FADH2. harnessing of the proton gradient

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 The Role of Mitochondria in the
Formation of ATP

• Electron Transport
– Electrons move through
the inner membrane via a
series of carriers of
decreasing redox
potential.
– Electrons associated with
either NADH or FADH2 are
transferred through
specific electron carriers
that make up the
electron transport chain.

Redox potential of some reaction couples

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 The Role of Mitochondria in the
Formation of ATP
• Types of Electron Carriers
– Flavoproteins are
polypeptides bound to either
FAD or FMN.
– Cytochromes contain heme
groups bearing Fe or Cu metal
ions.
– Three cooper atoms are
located within a single protein
complex and alternate
between Cu2+/Cu3+
– Ubiquinone (coenzyme Q) is a
lipid-soluble molecule made
of five-carbon isoprenoid
units.
Structures of three electron carriers
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 The Role of Mitochondria in the
Formation of ATP

Iron-sulfur centers Arrangement of several carriers Inhibitors to determine

in the electron-transport chain the ETC carrier sequence

• Types of Electron Carriers

– Iron-sulfur proteins contain Fe in association with inorganic sulfur.
– These carriers are arranged in order of increasingly positive redox potential.
– Sequence of carriers determined by use if inhibitors.

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 The Role of Mitochondria in the
Formation of ATP

• Electron-Transport Complexes
– Complex I (NADH dehydrogenase) catalyzes transfer of electrons from
NADH to ubiquinone and transports four H+ per pair.
– Complex II (succinate dehydrogenase) catalyzes transfer of electrons from
succinate to FAD to ubiquinone without transport of H+.
– Complex III (cytochrome bc1) catalyzes the transfer of electrons from
ubiquinone to cytochrome c and transports four H+ per pair.
– Complex IV (cytochrome c oxidase) catalyzes transfer of electrons to O2
and transports H+ across the inner membrane.
– Cytochrome oxidase is a large complex that adds four electrons to O2 to
form two molecules of H2O.
– The metabolic poisons CO, N3–, and CN– bind catalytic sites in Complex IV.

© 2013 John Wiley & Sons, Inc. All rights reserved.

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The Role of Mitochondria in the
Formation of ATP

The electron-transport chain of the inner mitochondrial membrane

© 2013 John Wiley & Sons, Inc. All rights reserved.
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 The Role of Mitochondria in the
Formation of ATP

cytochrome oxidase:
A proton pump in
synthetic liposomes

Model of electron
flow through the
four redox centers

– Cytochrome oxidase is a large complex that adds four electrons to O2 to

form two molecules of H2O.
– Electrons are transferred one at a time.
– Energy released by O2 reduction is thought to drive conformational changes.
– These changes would promote H+ ions movement through the protein.

© 2013 John Wiley & Sons, Inc. All rights reserved.

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 (9.4) Translocation of Protons and the
Establishment of a Proton-Motive Force

• Two components of the proton gradient:

– Concentration gradient between matrix
and intermembrane space creates a pH
gradient (ΔpH).
– Separation of charge across the membrane
creates an electric potential (Ψ).
– Energy present in both components of the
gradients is proton-motive force (Δp).

Visualizing the proton-motive

force in active mitochondria
with the fluorescent, cationic
dye rhodamine

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 The Machinery for ATP Formation
The structure of the ATP synthase
Schematic
diagram and 3D
structure of the
bacterial ATP
synthase. The
enzyme consists
of two major
portions, called
F1 and F0

• The structure of the ATP synthase:

– The F1 particle is the catalytic subunit, and contains three catalytic sites for
ATP synthesis.
– The F0 particle attaches to the F1 and is embedded in the inner membrane.
– The F0 base contains a channel through which protons are conducted from the
intermembrane space to the matrix–demonstrated in experiments with sub-
mitochondrial particles.
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 The Machinery for ATP Formation
The structure of the ATP synthase
Schematic diagram of the
bacterial ATP synthase.

Atomic force
microscopy of a
“field” of c rings
from chloroplast
ATP synthases
with 14 subunits

• The structure of the ATP synthase:

– The number of subunits in the c ring is 10–14 because structural studies have
revealed that this number can vary depending on the source of the enzyme.
– Yeast mitochondrial and E. coli ATP synthase have 10 c subunits.
– The chloroplast ATP synthase has 14 c subunits.

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 The Machinery for ATP Formation
Binding Change Mechanism
A section through the F1
head shows the spatial
organization of three of
its subunits
Top view of the F1 head
with the six α and β
subunits around the
asymmetric γ subunit

• The Basis of ATP Formation According to the Binding Change Mechanism

– The binding change mechanism states the following:
• Movement of protons through ATP synthase alters the binding affinity of
the active site.
• Each active site goes through distinct conformations that have different
affinities for substrates and product.
• There is a structural basis of catalytic site conformation.

© 2013 John Wiley & Sons, Inc. All rights reserved.

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 The Machinery for ATP Formation
Binding Change Mechanism

The binding
change
mechanism
for ATP
synthesis.

• Binding change mechanism

– Binding sites on the catalytic subunit can be tight, loose, or open.
– ATP is synthesized through rotational catalysis where the stalk of ATP synthase
rotates relative to the head.
– There is structural and experimental evidence to support this mechanism.

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 The Machinery for ATP Formation

• Using the Proton Gradient to

Drive the Catalytic Machinery:
The Role of the F0 Portion of ATP
Synthase
– The c subunits of the F0 base
form a ring.
– The c ring is bound to γ subunit
of the stalk.
– Protons moving through
membrane rotate the ring.
– Rotation of the ring provides
twisting force that drives ATP
synthesis.
A model of the proton diffusion coupled
to rotation of c ring in the F0 complex

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 The Machinery for ATP Formation

• Other Roles for the Proton-

Motive Force in Addition to
ATP Synthesis
– The H+ gradient drives
transport of ADP into and
ATP out of the
mitochondrion.
– ADP is the most important
factor controlling the
respiration rate.
– Many factors influence
the rate of respiration, but
the pathways are poorly
understood.
Summary of the major activities during
aerobic respiration in a mitochondrion.

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