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Chapter 10
Chemotrophic
Energy
Metabolism:
Aerobic Respiration
Lectures by
Kathleen Fitzpatrick
Simon Fraser University
© 2012 Pearson Education, Inc.
Chemotrophic Energy Metabolism:
Aerobic Respiration
• Some cells meet their energy needs through
anaerobic fermentation
• However, fermentation yields only modest
amounts of energy due to the absence of
electron transfer
• ATP yield is much higher in cellular
respiration
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Cellular Respiration: Maximizing
ATP Yields
• Cellular respiration (or respiration) uses an
external electron acceptor to oxidize substrates
completely to CO2
• External electron acceptor: one that is not a
by-product of glucose catabolism
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Cellular respiration defined
• Respiration is the flow of electrons through or
within a membrane, from reduced coenzymes
to an external electron acceptor usually
accompanied by the generation of ATP
• Coenzymes such as FAD (flavin adenine
dinucleotide) and coenzyme Q (ubiquinone)
are involved
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The terminal electron acceptor
• In aerobic respiration, the terminal electron
acceptor is oxygen and the reduced form is
water
• Other terminal electron acceptors (sulfur,
protons, and ferric ions) are used by other
organisms, especially bacteria and archaea
• These are examples of anaerobic respiration
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Mitochondria
• Most aerobic ATP production in eukaryotic
cells takes place in the mitochondrion
• In bacteria, the plasma membrane and
cyotoplasm are analogous to the
mitochondrial inner membrane and matrix
with respect to energy metabolism
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Figure 10-1
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Bioflix: Cellular Respiration
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Aerobic Respiration Yields Much More
Energy than Fermentation Does
• With O2 as the terminal electron acceptor, pyruvate
can be oxidized completely to CO2
• Aerobic respiration has the potential of generating
up to 38 ATP molecules per glucose
• Oxygen provides a means of continuous
reoxidation of NADH and other reduced coenzymes
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Respiration Includes Glycolysis,
Pyruvate Oxidation, the TCA Cycle,
Electron Transport, and ATP Synthesis
• Respiration will be considered in five stages
– Stage 1: the glycolytic pathway
– Stage 2: pyruvate is oxidized to generate acetyl CoA
– Stage 3: acetyl Co A enters the tricarboxylic acid
cycle (TCA cycle), where it is completely oxidized to
CO2
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The five stages, continued
• Respiration will be considered in five stages
– Stage 4: electron transport, the transfer of electrons
from reduced coenzymes to oxygen coupled to
active transport of protons across a membrane
– Stage 5: The electrochemical proton gradient
formed in step 4 is used to drive ATP synthesis
(oxidative phosphorylation)
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The Mitochondrion: Where the Action
Takes Place
• The mitochondrion is called the “energy
powerhouse” of the eukaryotic cell
• These organelles are thought to have arisen from
bacterial cells
• Mitochondria have been shown to carry out all the
reactions of the TCA cycle, electron transport, and
oxidative phosphorylation
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Mitochondria Are Often Present Where
the ATP Needs Are Greatest
• Mitochondria are found in virtually all aerobic cells
of eukaryotes
• They are present in both chemotrophic and
phototrophic cells
• Mitochondria are frequently clustered in regions of
cells with the greatest need for ATP, e.g., muscle
cells
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Figure 10-2
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Are Mitochondria Interconnected
Networks Rather than Discrete
Organelles?
• In electron micrographs, mitochondria usually
appear as oval structures
• However they can take various shapes and sizes,
depending on the cell type
• Their appearance under EM suggests that they are
large, and numerous discrete entities
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A challenge to the accepted view
• The work of Hoffman and Avers suggests that the
oval profiles in electron micrographs of yeast cells
reflect slices through a single large branched
mitochondrion
• This work suggests that mitochondria may be larger
than previously thought, and less numerous
• There is additional support for this idea
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The Outer and Inner Membranes
Define Two Separate Compartments
and Three Regions
• A distinctive feature of mitochondria is the presence
of both outer and inner membranes
• The outer membrane contains porins that allow
passage of solutes with molecular weights up
to 5000
• The intermembrane space between the inner and
outer membranes is thus continuous with the cytosol
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The inner membrane
• The inner membrane of the mitochondria is
impermeable to most solutes, partitioning the
mitochondrion into two separate compartments
– The intermembrane space
– The interior of the organelle, or mitochondrial
matrix
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Figure 10-3
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Figure 10-3A
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Figure 10-3B
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Video: Mitochondria in 3-D
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Figure 10-4
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The inner boundary membrane and
cristae
• The portion of the inner membrane adjacent to
the intermembrane space is called the inner
boundary membrane
• The inner membrane is about 75% protein by
weight; the proteins include those involved in
solute transport, electron transport, and ATP
synthesis
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The cristae
• The inner membrane of most mitochondria has
many infoldings called cristae
• They increase surface area of the inner
membrane, and provide more space for electron
transport to take place
• The cristae provide localized regions, intracristal
spaces, where protons can accumulate during
electron transport
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The cristae (continued)
• The cristae are thought to be tubular structures
that associate in layers
• They have limited connections to the inner
boundary membrane through small openings,
crista junctions
• Cells with high metabolic activity seem to have
more cristae in their mitochondria
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The mitochondrial matrix
• The interior of the mitochondrion is filled with a
semi-fluid matrix
• The matrix contains many enzymes involved in
mitochondrial function as well as DNA molecules
and ribosomes
• Mitochondria contain proteins encoded by their own
DNA as well as some that are encoded by nuclear
genes
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Mitochondrial Functions Occur in
or on Specific Membranes and
Compartments
• Specific functions and pathways have been
localized within mitochondria by fractionation
studies
• Most of the enzymes involved in pyruvate
oxidation, the TCA cycle, and catabolism of fatty
acids and amino acids are found in the matrix
• Most electron transport intermediates are
integral inner membrane components
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Table 10-1
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Localization of Specific Mitochondrial
Functions
• Knoblike spheres called F1 complexes protrude
from the inner membrane into the matrix
• These are involved in ATP synthesis
• Each complex is an assembly of several
different polypeptides, and can be seen in an
electron micrograph using negative staining
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Figure 10-5
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Figure 10-5A
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Figure 10-5B
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Figure 10-5C
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F1 complexes
• Each F1 complex is attached by a short protein
stalk to an Fo complex
• This is an assembly of hydrophobic polypeptides
embedded in the mitochondrial inner membrane
• This FoF1 complex is an ATP synthase that is
responsible for most of the ATP generation in the
mitochondria (and in bacterial cells as well)
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In Bacteria, Respiratory Functions Are
Localized to the Plasma Membrane
and the Cytoplasm
• Bacteria do not have mitochondria but are capable
of aerobic respiration
• Their plasma membrane and cytoplasm perform
the same functions as the inner membrane and
matrix of mitochondria
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The Tricarboxylic Acid Cycle:
Oxidation in the Round
• In the presence of oxygen pyruvate is oxidized fully
to carbon dioxide with the released energy used to
drive ATP synthesis
• This involves the TCA (tricarboxylic acid) cycle, in
which citrate is an important intermediate
• The TCA cycle is also called the Krebs cycle after
Hans Krebs, whose lab played a key role in
elucidating the cycle
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The Tricarboxylic Acid Cycle
• The TCA cycle metabolized acetyl CoA, produced
from pyruvate decarboxylation
• Acetyl CoA can also arise from fatty acid oxidation
• Acetyl CoA transfers its acetate group to a fourcarbon acceptor called oxaloacetate, generating
citrate
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The fate of citrate
• After its formation, citrate undergoes two successive
decarboxylations
• It also goes through several oxidation steps
• Eventually oxaloacetate is regenerated, and can
accept two more carbons from acetyl CoA and the
cycle begins again
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The overall cycle
• Each round of the TCA cycle involves the entry of
two carbons, the release of two CO2, and the
regeneration of oxaloacetate
• Oxidation occurs at five steps, four in the cycle itself
and one when pyruvate is converted to acetyl CoA
• In each case, electrons are accepted by coenzymes
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Figure 10-6
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Pyruvate Is Converted to Acetyl
Coenzyme A by Oxidative
Decarboxylation
• The glycolytic pathway ends with pyruvate, which is
small enough to enter the intermembrane space of
the mitochondrion
• At the inner mitochondrial membrane, a specific
symporter transports pyruvate into the matrix, along
with a proton
• Then, pyruvate is converted to acetyl CoA by
pyruvate dehydrogenase complex (PDH)
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Conversion of pyruvate
• The conversion is a decarboxylation because one
carbon is liberated as CO2
• It is also an oxidation because two electrons (and
one proton) are transferred to NAD+ to form NADH
• Coenzyme A contains the B vitamin pantothenic
acid
• Coenzyme A has a SH group that makes it a good
carrier of acetate (and other organic acids)
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Conversion of pyruvate
• The conversion of pyruvate to acetyl CoA can be
summarized as follows:
–
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Figure 10-7
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The TCA Cycle Begins with the
Entry of Acetate as Acetyl CoA
• With each round of the TCA cycle, two carbon
atoms enter in organic form as acetate and leave in
inorganic form as carbon dioxide
• In the first reaction, TCA-1, the two-carbon acetate
group is transferred from acetyl CoA to oxaloacetate
(4C) to form citrate (6C)
• This reaction is catalyzed by citrate synthetase
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TCA-2
• Citrate is converted to isocitrate in the second step
of the TCA cycle
• The enzyme aconitase catalyzes the reaction
• Isocitrate has a hydroxyl group that is easily
oxidized (or dehydrogenated) in step TCA-3
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Figure 10-8
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Two Oxidative Decarboxylations Then
Form NADH and Release CO2
• Four of the eight steps in the TCA cycle are
oxidations (TCA-3, TCA-4, TCA-6, TCA-8)
• These all involve coenzymes that enter in the
oxidized form and leave in the reduced form
• The first two of these are decarboxylation steps,
releasing one CO2 in each step
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TCA-3, TCA-4
• Isocitrate is oxidized by isocitrate dehydrogenase to
oxalosuccinate, with NAD+ as the electron acceptor
• Oxalosuccinate immediately undergoes
decarboxylation to form a-ketoglutarate (5C)(TCA3)
• a-ketoglutarate is oxidized to succinyl CoA, by
a-ketoglutarate dehydrogenase (TCA-4)
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Direct Generation of GTP (or ATP)
Occurs at One Step in the TCA Cycle
• So far in the cycle, two carbon atoms have entered
and two have left (but not the same two carbons),
and two molecules of NADH have been generated
• Succinyl CoA has been generated; like acetyl CoA it
has a high-energy thioester bond
• The energy from hydrolysis of this bond is used to
generate one ATP (bacteria, plants) or
GTP(animals) (TCA-5)
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The Final Oxidative Reactions of the
TCA Cycle Generate FADH2 and NADH
• Of the remaining three steps in the cycle, two are
oxidations
• Succinate is oxidized to fumarate (TCA-6); this
transfers electrons to FAD, a lower-energy
coenzyme than NAD+
• In the next step, fumarate is hydrated to produce
malate (TCA-7) by fumarate hydratase
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Figure 10-9
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TCA-8
• In the final step of the TCA cycle, the hydroxyl
group of malate becomes the target of the final
oxidation in the cycle
• Electrons are transferred to NAD+, producing
NADH as malate is converted to oxaloacetate
(TCA-8)
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Summing Up: The Products of the TCA
Cycle Are CO2, ATP, NADH, and FADH2
• The TCA cycle accomplishes the following:
1. Two carbons enter the cycle as acetyl CoA, which
joins oxaloacetate to form the six-carbon citrate
2. Decarboxylation occurs at two steps to balance the
input of two carbons by releasing two CO2
3. Oxidation occurs at four steps, with NAD+ the
electron acceptor in three steps and FAD in one
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The Products of the TCA Cycle Are CO2,
ATP, NADH, and FADH2 (continued)
• The TCA cycle accomplishes the following:
4. ATP is generated at one point, with GTP as an
intermediate in the case of animal cells
5. One turn of the cycle is completed as oxaloacetate,
the original 4C acceptor, is regenerated
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Summing up the TCA cycle
• The TCA cycle can be summarized as follows:
• Including glycolysis, pyruvate decarboxylation, and
the TCA cycle
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Several TCA Cycle Enzymes Are
Subject to Allosteric Regulation
• Like all metabolic pathways, the TCA cycle must be
carefully regulated to meet cellular needs
• Most of the control of the cycle involves allosteric
regulation of four key enzymes by specific effector
molecules
• Effector molecules may be activators or inhibitors
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Regulation of PDH
• PDH is reversibly inactivated by phosphorylation
and activated by dephosphorylation of one of its
protein components
• PDH is inhibited by ATP, which is abundant where
energy is plentiful
• PDH and isocitrate dehydrogenase are activated
by AMP and ADP, which are more abundant when
energy is needed
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Regulation of acetyl CoA
• Overall availability of acetyl CoA is determined
mainly by the activity of the PDH complex
• PDH is allosterically inhibited by ATP, NADH, and
acetyl CoA and high [ATP/ADP] (enzyme: PDH
kinase)
• It is activated by AMP, NAD+, and free CoA and
low [ATP/ADP] (enzyme: PDH phosphatase)
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Figure 10-10
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The TCA Cycle Also Plays a Central
Role in the Catabolism of Fats and
Proteins
• The TCA cycle represents the main conduit of
aerobic energy metabolism for a variety of
substrates besides sugar, in particular, fats and
proteins
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Fat as a Source of Energy
• Fats are highly reduced compounds that liberate
more energy per gram upon oxidation than do
carbohydrates
• They are a long-term energy storage form for many
organisms
• Most fat is stored as deposits of triacylglycerols,
neutral triesters of glycerol and long-chain fatty acids
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Catabolism of triacylglycerols
• Triacylglycerol catabolism begins with their
hydrolysis to glycerol and free fatty acids
• The glycerol is channeled into the glycolytic
pathway by oxidative conversion to
dihydroxyacetone phosphate
• Fatty acids are linked to coenzyme A, to form fatty
acyl CoAs, then degraded by b-oxidation
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b-oxidation
• b-oxidation is a catabolic process that generates
acetyl CoA and the reduced coenzymes NADH
and FADH2
• b-oxidation occurs in different compartments in
different organisms
• Here, we will focus on the mitochondrion of animals
using saturated fatty acids with an even number of
carbons as an energy source
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Fatty acid degradation
• Most fatty acids are oxidatively converted to acetyl
CoA in the mitochondrion
• These can be further catabolized in the TCA cycle
• The fatty acids are degraded in a series of
repetitive cycles, which removes two carbons at a
time until the fatty acid is completely degraded
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Steps of b-oxidation
• Each cycle involves
–
–
–
–
1.Oxidation
2. Hydration
3. Reoxidation
4. Thiolysis
• The result is the production of one FADH2, one
NADH, and one acetyl CoA per cycle
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Formation of fatty acyl CoA
• b-oxidation of a fatty acid begins with an activation
step in the cytosol that requires the energy of ATP
hydrolysis
• This drives the attachment of a CoA molecule to
the fatty acid (FA-1)
• The fatty acyl CoA is then transported into the
mitochondrion by a translocase in the inner
membrane
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Degradation of fatty acyl CoA
• An integral membrane dehydrogenase oxidizes the
fatty acyl CoA, forming a double bond between the
a and b carbons (FA-2)
• The two electrons and protons removed are
transferred to FAD, forming FADH2
• Water is added across the double bond by a
hydratase (FA-3)
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Figure 10-11
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Degradation of fatty acyl CoA (continued)
• Another dehydrogenase oxidizes the b-carbon,
converting the hydroxyl group to a keto group (FA4)
• Two electrons and one proton are transferred to
NAD+ to form NADH
• The bond between the a and b carbons is broken by
a thiolase and a two-carbon fragment is transferred
to a second acetyl CoA (FA-5)
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Degradation of fatty acyl CoA (continued)
• The steps FA-2 to FA-5 are repeated until the
original fatty acid is completely degraded
• Most fatty acids have an even number of carbons
and are completely degraded; those with an uneven
number of carbons require extra steps before
feeding into the TCA cycle
• Unsaturated fatty acids require one or two additional
enzymes
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Protein as a Source of Energy and
Amino Acids
• Besides their other numerous functions, proteins
can be catabolized to produce ATP if necessary
when carbohydrate and lipid stores are depleted
• Eventually cells undergo turnover of proteins and
protein-containing structures
• The resulting amino acids can be used to generate
new proteins or degraded for energy
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Protein catabolism
• Protein catabolism begins with hydrolysis of the
peptide bonds that link amino acids together
• This is called proteolysis and the enzymes
responsible for it are called proteases
• The products of proteolysis are small peptides
and free amino acids
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Endopeptidases and exopeptidases
• Further digestion of peptides is catalyzed by
peptidases
• Endopeptidases hydrolyze internal peptide bonds
• Exopeptidases remove successive amino acids
from the end of the peptide
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Amino acid catabolism
• Free amino acids can be catabolized for energy
• These are converted into intermediates of
mainstream catabolism in as few steps as possible
• The pathways differ for individual amino acids, but
all eventually lead to acetyl CoA, pyruvate, or a few
key TCA cycle intermediates
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Figure 10-12
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The TCA Cycle Serves as a Source of
Precursors for Anabolic Pathways
• In addition to the catabolic function of the TCA
cycle, there is a flow of four-, five- and six-carbon
intermediates into and out of it in most cells
• These side reactions can use intermediates for the
synthesis of needed compounds
• The TCA cycle is a link between anabolic and
catabolic pathways and is therefore amphibolic
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The TCA cycle has a central role in
some anabolic processes
• a-keto intermediates of the TCA cycle can be
converted into the amino acids alanine, aspartate,
and glutamate
• Succinyl CoA is the starting point for heme
synthesis
• Citrate can be transported out of the
mitochondrion and used as an acetyl CoA source
for fatty acid synthesis
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The Glyoxylate Cycle Converts
Acetyl CoA to Carbohydrates
• The glyoxylate cycle has several intermediates in
common with the TCA cycle but performs a
specialized function in some plant cells and fungi
• This occurs in a specialized peroxisome called the
glyoxosome
• Two acetate molecules (from acetyl CoA) form
succinate which is converted to PEP, from which
sugars can be synthesized
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Figure 10-10A-1
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Figure 10-10A-2
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Electron Transport: Electron Flow
from Coenzymes to Oxygen
• Chemotrophic energy metabolism through the TCA
cycle accounts for synthesis of 4 ATP per glucose
(2 from glycolysis and 2 from the TCA cycle)
• This accounts for only a small portion of the energy
in the original glucose molecule
• The remainder is stored in NADH and FADH2
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The Electron Transport System
Conveys Electrons from Reduced
Coenzymes to Oxygen
• Coenzyme reoxidation by transfer of electrons to
oxygen is called electron transport
• Electron transport and ATP generation are not
independent processes; they are functionally
linked to each other
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Electron Transport and Coenzyme
Oxidation
• Electron transport involves the highly exergonic
oxidation of NADH and FADH2 with O2 as the
terminal electron acceptor and so accounts for the
formation of water
•
•
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The Electron Transport System
• Electron transfer is carried out as a multistep
process involving an ordered series of reversibly
oxidized electron carriers functioning together
• This is called the electron transport system, ETS
• The ETS contains a number of integral membrane
proteins that are found in the inner mitochondrial
membrane (or plasma membrane of bacteria)
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The Electron Transport System
Consists of Five Kinds of Carriers
• Flavoproteins
• Iron-sulfur proteins
• Cytochromes
• Copper-containing cytochromes
• Coenzyme Q
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Features of electron carriers
• All except coenzyme Q are proteins with prosthetic
groups capable of being reversibly oxidized and
reduced
• Most are hydrophobic
• Most of these intermediates occur in the membrane
as large assemblies of proteins called respiratory
complexes
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Flavoproteins
• Membrane-bound flavoproteins use either (FAD)
or flavin adenine mononucleotide (FMN) as the
prosthetic group
– For example, NADH dehyrogenase and succinate
dehydrogenase
• Flavoproteins transfer both electrons and protons
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Iron-Sulfur Proteins
• Iron-sulfur proteins are also called nonheme iron
proteins and have an iron-sulfur (Fe-S) center
• The iron atoms in the center of the proteins are the
actual electron carriers; these alternate between
the Fe2+ (oxidized) and Fe3+ (reduced) states
• They transfer one electron at a time, and no
protons
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Cytochromes
• Cytochromes also contain iron, but as part of a
porphyrin prosthetic group, heme
• There are five types: b, c, c1, a, and a3
• The iron atom of the heme group serves as the
electron carrier and transfers one electron at a
time and no protons
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Figure 10-13
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Copper-Containing Cytochromes
• In addition to their iron atoms, cytochromes a and
a3 contain a single copper atom bound to their
heme group
• It associates with the iron atom to form a bimetallic
iron-copper (Fe-Cu) center
• Copper ions can be reversibly converted from the
oxidized (Cu2+) to the reduced form (Cu+) by
accepting or donating single electrons
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Copper-Containing Cytochromes
(continued)
• The iron-copper center plays a critical role in
keeping an O2 molecule bound to the cytochrome
oxidase complex
• The oxygen is held there until it has picked up four
electrons and four protons, at which point two water
molecules are released
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Coenzyme Q
• The only nonprotein component of the ETS is
coenzyme Q (CoQ), a quinone
• Because of its ubiquitous occurrence in nature, it is
also called ubiquinone
• CoQ is reduced in two successive (one electron
plus one proton) steps to semiquinone (CoQH) and
then dihydroquinone (CoQH2)
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Coenzyme Q (continued)
• Unlike the proteins of the ETS, most of the CoQ is
freely mobile in the inner mitochondrial membrane
• They serve as a collection point for electrons from
the reduced FMN and FAD-linked dehydrogenases
in the membrane
• A portion of the CoQ is tightly bound to specific
respiratory complexes
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Coenzyme Q (continued)
• CoQ accepts both protons and electrons when it is
reduced and releases both protons and electrons
when it is oxidized
• This is vital to its role in the active transport of
protons across the inner mitochondrial membrane
• It accepts protons on one side of the membrane,
diffuses across and releases the protons
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Figure 10-14
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The Electron Carriers Function in a
Sequence Determined by Their
Reduction Potentials
• The standard reduction potential (E0) for an
electron carrier is a measure of its affinity for
electrons.
• For example, O2 accepting electrons is very
favorable with E0 of +0.816 V
•
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The Electron Carriers Function in
Sequence (continued)
• NAD+ accepting electrons is favorable, too, but less
so, with E0 of 0.32 V.
•
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Understanding Standard Reduction
Potentials
• Arrange pairs of oxidized/reduced forms of the
same molecule with the most negative reduction
potential on the top and the rest in order of
reduction potential
• Under standard conditions, the reduced form of any
redox pair can reduce the oxidized form of any
redox pair below it in the list
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Table 10-2
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Energetics of redox reactions
• The tendency of the reduced form of one pair to
reduce the oxidized form of another pair can be
quantified by determining the difference in Eo
values between the two pairs
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Energetics of redox reactions (continued)
• For example, for transfer of electrons from
NADH to O2
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The relationship between DGo and DEo
• DEo is a measure of thermodynamic spontaneity
for the redox reaction between any two redox pairs
under standard conditions
•
• Where n = number of electrons transferred, F is
the Faraday constant (23,062cal/mol*V)
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Example: the reaction of NADH with
oxygen
• Two electrons are transferred, so we calculate DG0
• The value is highly negative, so transfer of electrons
this way is thermodynamically spontaneous
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Ordering of the Electron Carriers
• The position of each carrier is determined by its
standard reduction potential
• This means that electron transfer from NADH or
FADH2 at the top to O2 at the bottom is
spontaneous and exergonic
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Most of the Carriers Are Organized into
Four Large Respiratory Complexes
• Although many electron carriers are part of the
ETS, most are organized into multiprotein
complexes
• Most are thought to be organized into four different
kinds of respiratory complexes
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Figure 10-15
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Properties of the Respiratory
Complexes
• Each respiratory complex consists of distinctive
assembly of polypeptides and prosthetic groups
• Complex I transfers electrons from NADH to CoQ
and is called the NADH-coenzyme Q oxidation
complex (or NADH dehydrogenase complex)
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The respiratory complexes
• Complex II transfers to CoQ the electrons derived
from succinate in Reaction TCA-6 and it is called
the succinate-coenzyme Q oxidoreductase complex,
or succinate dehydrogenase
• Complex III is called the coenzyme Q-cytochrome
oxidoreductase complex because it accepts
electrons from coenzyme Q and passes them to
cytochrome c
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The respiratory complexes (continued)
• Complex III is also called cytochrome b/c1 complex
• Complex IV transfers electrons from cytochrome c
to oxygen and is called cytochrome c oxidase
• For each pair of electrons transported through
complexes I through IV, 10 protons are pumped
from the matrix to the intermembrane space
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Table 10-3
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Figure 10-16A
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Figure 10-16B
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Figure 10-16C
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Figure 10-16D
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The Role of Cytochrome c Oxidase
• Cytochrome c oxidase (complex IV) is the terminal
oxidase, transferring electrons directly to oxygen
• Cyanide and azide are toxic to most aerobic cells
because they bind the Fe-Cu center of cytochrome
c oxidase, blocking electron transport
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Incomplete reduction of oxygen
• Complexes I and III can also transfer electrons to
oxygen, resulting in its incomplete reduction
• This can generate toxic superoxide anion (O2) or
hydrogen peroxide (H2O2), both of which contribute
to cellular aging
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The Respiratory Complexes Move
Freely Within the Inner Membrane
• The protein complexes of the mitochondrial inner
membrane are mobile, free to diffuse within the
membrane
• Freeze-fracture analysis of membranes exposed
to an electrical field demonstrate the free
diffusion of the proteins
• The inner membrane has no cholesterol and is
very fluid, so the protein mobility is high
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Respirasomes
• Recent work suggests that the multiprotein
respiratory complexes are organized into
supercomplexes called respirasomes
• The association of several of the TCA cycle
dehydrogenases within the respirasomes suggests
that they function to minimize diffusion distances
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Coenzyme Q and cytochrome c
• Coenzyme Q is the “funnel” that collects electrons
from virtually every oxidation reaction in the cell
• Coenzyme Q and cytochrome c are both small
molecules that can diffuse rapidly within the
membrane (coenzyme Q) or on its surface
(cytochrome c)
• They are both quite numerous, which accounts for
observed rates of electron transfer
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Electron Transport and ATP Synthesis
Are Coupled Events
• The crucial link between electron transport and ATP
production is an electrochemical proton gradient
• It is established by the directional pumping of
protons across the membrane in which electron
transport is occurring
• ATP synthesis is coupled to electron transport
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ATP synthesis is dependent on
electron transport
• Certain chemicals known as uncouplers can abolish
the interdependence of the two processes
• These allow continued electron transport and O2
consumption in the absence of ATP synthesis
• Treatments that stop electron transport also inhibit
ATP synthesis, so ATP synthesis is dependent on
electron transport but the reverse is not true
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Respiratory Control of Electron
Transport
• The availability of ADP regulates the rate of
oxidative phosphorylation and thus of electron
transport
• This is called respiratory control
• Electron transport and ATP generation will be
favored when ADP concentration is high and
inhibited when ADP concentration is low
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The Chemiosmotic Model: The
“Missing Link” Is a Proton Gradient
• In 1961 Peter Mitchell proposed the chemiosmotic
coupling model
• The essential feature of the model is that the link
between electron transport and ATP formation is the
electrochemical potential across a membrane
• The electrochemical potential is created by the
pumping of protons across a membrane as electrons
are transferred through the respiratory complexes
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Coenzyme Oxidation Pumps Enough
Protons to Form 3 ATP per NADH and
2 ATP per FADH2
•
The transfer of two electrons from NADH is
accompanied by the pumping of a total of 10
protons (12 if the Q cycle is operating)
•
The number of protons required per molecule of
ATP is thought to be 3 or 4, with 3 regarded as
most likely
•
So, about 3 molecules of ATP are synthesized per
NADH oxidized
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Number of ATP generated is an estimate
• FADH2 donates electrons to complex II with higher
reduction potential, pumping 6 protons (8 if the
Q cycle is operating)
• So about 2 ATP are synthesized per FADH2
• These values are estimates, affected by an
organism’s specific ATP synthase and other factor
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The Chemiosmotic Model Is Affirmed by
an Impressive Array of Evidence
• Since its initial formulation, the chemiosmotic model
has become universally accepted as the link
between electron transport and ATP synthesis
• Several lines of evidence support the mode
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1. Electron Transport Causes Protons
to Be Pumped Out of the Mitochondrial
Matrix
• Mitchell and Moyle demonstrated experimentally that
the flow of electrons through the ETS is
accompanied by the unidirectional pumping of
protons across the inner mitochondrial membrane
• One mechanism involves allosteric changes in
protein conformation as electrons are transferred,
allowing protons to be moved to the outer surface of
the membrane
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Figure 10-17
© 2012 Pearson Education, Inc.
2. Components of the Electron Transport
System Are Asymmetrically Oriented
Within the Inner Mitochondrial Membrane
• Electron carriers of the ETS must be asymmetrically
oriented in the membrane
• Otherwise, protons would be pumped randomly in
both directions
• Labeling studies show that some parts of the
respiratory complexes face inward, some are
transmembrane proteins, others face outward
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3. Membrane Vesicles Containing
Complexes I, III, or IV Establish Proton
Gradients
• Vesicles can be reconstituted from various
components of the ETS
• Vesicles that contain complex I, III, or IV are able to
pump protons out of the vesicle, if provided with an
appropriate oxidizable substrate
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4. Oxidative Phosphorylation Requires
a Membrane-Enclosed Compartment
• Without the mitochondrial membrane, the protein
gradient that drives ATP synthesis could not be
maintained
• Electron transfer carried out by isolated complex
cannot be coupled to ATP synthesis unless the
complexes are incorporated into membranes that
form enclosed vesicles
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5. Uncoupling Agents Abolish Both the
Proton Gradient and ATP Synthesis
• Dinitrophenol (DNP) is known to uncouple ATP
synthesis from electron transport
• When membranes are treated with DNP, they allow
protons to cross the membrane freely, so that no
proton gradient can be formed
• ATP synthesis is abolished as well
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6. The Proton Gradient Has Enough
Energy to Drive ATP Synthesis
• The electrochemical proton gradient across the
membrane involves both a membrane potential
and a concentration gradient
• A mitochondrion actively respiring has a membrane
potential of about 0.16V (positive on the
intermembrane space side) and a pH gradient of
about 1.0 (higher on the matrix side)
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Proton motive force
• The electrochemical gradient exerts a proton
motive force (pmf), that tends to drive protons
back down their concentration gradient (back into
the matrix)
•
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Proton motive force in a mitochondrion
• In a mitochondrion that is undergoing aerobic
respiration, the pmf can be calculated:
• Note that the membrane potential accounts
for more than 70% of the pm
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Pmf can be used to calculate D Go
• The pmf can be used to calculate D Go as follows:
• D G for phosphorylation of ADP is about
10–14 kcal/mol, so 3 or 4 protons would produce
enough free energy to produce ATP
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7. Artificial Proton Gradients Are Able
to Drive ATP Synthesis in the Absence
of Electron Transport
• Direct evidence for the model comes from work
using vesicles or mitochondria that are exposed to
artificial pH gradients
• When the external proton concentration is increased,
ATP is generated in response
• So, ATP can be generated by a proton gradient,
even when electron transport is not taking place
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ATP Synthesis: Putting It All Together
• Some of the energy of glucose is transferred to
reduced coenzymes during glycolysis and the
TCA cycle
• This energy is used to generate an electrochemical
proton gradient across the inner mitochondrial
membrane
• The pmf of that gradient is harnessed to make ATP
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F1 Particles Have ATP Synthase Activity
• Racker and colleagues took intact mitochondria and
disrupted the membrane to form small vesicles
called submitochondrial particles
• These particles could carry out electron transport
and ATP synthesis
• By mechanical agitation or protease treatment, the
F1 structures were isolated from the membranes
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Figure 10-18A
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Figure 10-18B
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Figure 10-18C
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Uncoupling synthesis of ATP and electron
transport
• F1 particles and membranous vesicles were
separated by centrifugation
• The membranes could still carry out electron
transport, but could not synthesize ATP
• The isolated F1 particles could synthesize ATP but
could not carry out electron transport
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Figure 10-18D
© 2012 Pearson Education, Inc.
Figure 10-18E
© 2012 Pearson Education, Inc.
Reconnecting synthesis of ATP and
electron transport
• Separated F1 particles and membranous vesicles
were reconstituted
• The reconstituted structures were capable of both
electron transport and ATP synthesis
• The F1 particles were called coupling factors and
are now known to have ATP synthesizing activity
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Figure 10-18E
© 2012 Pearson Education, Inc.
Figure 10-18F
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The FoF1 Complex: Proton
Translocation Through Fo Drives ATP
Synthesis by F1
• The F1 complex is not directly membranebound, but is attached to the Fo complex that
is embedded in the inner membrane
• Fo acts as a proton translocator, the
channel through which protons flow across
the membrane
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The FoF1 ATP synthase
• Fo provides a channel for exergonic flow of protons
across the membrane
• F1 carries out the ATP synthesis, driven by the
energy of the proton gradient
• Together, they form a complete ATP synthase
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Figure 10-19
© 2012 Pearson Education, Inc.
Figure 10-19A
© 2012 Pearson Education, Inc.
Figure 10-19B
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Figure 10-19C
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Figure 10-19D
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Video: Rotation of ATP Synthase
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ATP Synthesis by FoF1 Involves
Physical Rotation of the Gamma
Subunit
• Paul Boyer proposed the binding change model
in 1979
• He proposed that each of the three b subunits of the
F1 complex progresses through three different
conformations
• Each conformation has distinct affinities for the
substrates, ADP and Pi, and the product, ATP
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The three conformations
• Boyer named the conformations
- L (for loose), which binds ADP and Pi loosely
- T (for tight), which binds ADP and Pi tightly and
catalyzes the formation of ATP
• O (for open), with little affinity for either substrates
or product
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Rotation of a and b with respect to g
• Boyer proposed that at any time, each of the active
sites is in a different conformation and the
hexagonal ring of a and b subunits rotates relative
to the central stalk containing the g subunit
• The rotation was thought to be driven by the flow of
protons through Fo
• It is now known that it is the g subunit that actually
rotates
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The Binding Change Model in Action
• Each b subunit passes through the O, L, and T
conformations as the g subunit rotates 360 degrees
• In Fo, the 10 c subunits each have an asp residue
with an ionic bond to an arg residue on the
immobile a subunit
• When taken up, a proton neutralizes the asp,
disrupting the ionic bond, and rotating the c10 ring
(and attached g subunit) 1/10th turn
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The binding change model (continued)
• As the ring turns, the asp in the adjacent residue
loses a proton and forms an ionic bond to arg in
the a subunit
• As 10 protons pass through the membrane via the
a subunit, the ring goes through one complete
rotation
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Steps of the model
• Step 1a: The b1 subunit in the O conformation shifts
to the L conformation (loosely binding ADP and Pi)
when the flow of protons through Fo causes a
120-degree rotation of the g subunit
• Step 1b: ATP is synthesized by b3
• Step 2a: The g subunit rotates another 120 degrees,
inducing b1 to shift to the T conformation
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Steps of the model (continued)
• Step 2b: The b1 subunit has an increased affinity for
ADP and Pi, allowing spontaneous formation of ATP
• Step 3a: The g subunit rotates another 120 degrees,
inducing b1 to shift to the O conformation, releasing
ATP
• Following ATP generation by the b2 unit (step 3b),
the cycle is complete
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Figure 10-20
© 2012 Pearson Education, Inc.
Video: ATP Synthase 3-D Structure
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Spontaneous Synthesis of ATP?
• Each of the three steps at which ATP is formed
occurs without a direct input of energy
• However, ATP synthesis is highly endergonic (in
dilute aqueous solution)
• The catalytic site of a b subunit is such that the
charge repulsion between ADP and Pi is minimized,
favoring ATP formation with a D Go close to zero
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Synthesis of ATP without thermodynamic
cost?
• ATP synthesis does not proceed without
thermodynamic cost
• The needed input of energy occurs elsewhere in
the cycle
• Energy comes from the proton gradient generated
by electron transport and transmitted through
rotation of the g subunit
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Synthesis of ATP without thermodynamic
cost?
• ATP synthesis does not proceed without
thermodynamic cost
• The needed input of energy occurs elsewhere in
the cycle
• Energy comes from the proton gradient generated
by electron transport and transmitted through
rotation of the g subunit
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Figure 10-21
© 2012 Pearson Education, Inc.
The Chemiosmotic Model Involves
Dynamic Transmembrane Proton Traffic
• There is continuous, dynamic two-way proton traffic
across the inner membrane
• NADH sends 10 protons across via complexes I, III,
and IV; FADH2 sends 6 across, via complexes II, III,
and IV
• Assuming that 3 protons must return through FoF1
per ATP generated, this means 3 ATP per NADH
and 2 per FADH2 are generated
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Figure 10-22
© 2012 Pearson Education, Inc.
Aerobic Respiration: Summing It All Up
• As carbohydrates and fats are oxidized to generate
energy, coenzymes are reduced
• These reduced coenzymes represent a storage
form of the energy released during oxidation
• This energy can be used to drive ATP synthesis as
the enzymes are reoxidized by the ETS
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Summing it all up (continued)
• As electrons are transported from NADH or FADH2
to O2, they pass through respiratory complexes
where proton pumping is coupled to electron
transport
• The resulting electrochemical gradient exerts a pmf
that serves as the driving force for ATP synthesis
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The Maximum Yield of Aerobic
Respiration Is 38 ATPs per glucose
• The maximum ATP yield per glucose under
aerobic conditions:
• Including the summary reactions of glycolysis and
the TCA cycle to this gives:
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1. Why Does the Maximum ATP Yield
in Eukaryotic Cells Vary Between 36
and 38 ATPs Per Glucose?
• Glycolysis produces two NADH per glucose in the
cytosol, and catabolism of pyruvate produces eight
more in the mitochondrial matrix
• NADH in the cytosol cannot enter the matrix to
deliver its electrons to complex I
• Instead the electrons and H+ ions are passed
inward by an electron shuttle system
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Electron shuttle systems
• There are several electron shuttle systems that
differ in the number of ATP molecules formed per
NADH molecule oxidized
• Electron shuttle systems consist of one or more
electron carriers that can be reversibly reduced, with
transport proteins in the membrane for both reduced
and oxidized forms of carrier
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The glycerol phosphate shuttle
• The glycerol phosphate shuttle is one type of
electron shuttle
• NADH in the cytosol reduces dihydroxyacetone
phosphate (DHAP) to glycerol-3-phosphate,
which is transported into the mitochondrion
• There it is reoxidized to DHAP using FAD, so the
electrons bypass complex I, generating 2 ATP
instead of 3
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Figure 10-23
© 2012 Pearson Education, Inc.
2. Why Is the ATP Yield of Aerobic
Respiration Referred to as the
“Maximum Theoretical ATP Yield”?
• Yields of 36 to 38 ATP per glucose are possible,
only if the energy of the electrochemical gradient is
only used for ATP synthesis
• This is not realistic, because the pmf of the proton
gradient is used for other processes too, as
needed by the cell
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Figure 10-24
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Figure 10-24A
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Figure 10-24B
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Figure 10-24C
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Figure 10-24D
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Figure 10-24E
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Aerobic Respiration Is a Highly
Efficient Process
• To determine efficiency of respiration, we need to
determine how much of the energy of glucose is
preserved in the resulting 36–38 ATP
• DGo for glucose  CO2 + H2O is 686 kcal/mol
• ATP hydrolysis under cellular conditions is about
10 to 14 kcal/mol
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Efficiency of aerobic respiration
• For 36–38 ATP, assuming a value of 10 kcal/mol,
the energy per mole of glucose is about
360–380 kcal conserved
• This efficiency of 52–55% is well above that
obtainable from the most efficient machines created
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