Cellular

Respiration
Doreen Alexis Villanueva
Introduction to Physiology

ATP provides the energy for cellular work

The ATP Cycle

ATP powers cellular work by coupling energy releasing to

energy using reactions

ATP + H2O ADP + P

ATP and Cellular Work

Relationship of Cellular Respiration to Breathing

Overall Equation for Cellular Respiration

Cellular respiration breaks down organic molecules to

yield energy.

"Falling" Electrons as an Energy Source

Food represents a source of high energy electrons similar to the
potential energy of being on top of a slide.
When the electrons pass from the high potential state of food to
oxygen, released energy is converted to other forms of energy.

How does burning

compare to cellular
respiration?

Burning Compared to Cell Respiration: The energy release is

controlled by enzymes and carrier molecules in a series of steps.

Electron Transport Chains

Compared with burning, cellular respiration is a more controlled.
Energy is released from glucose in small amounts that cells can
put to productive usethe formation of ATP molecules.

In Eukaryotic Cells, the reaction of Aerobic Respiration

occur
Inside MITOCHONDRIA.

Structure of Mitochondria
Mitochondria are found in almost all eukaryotic cells. Its structure is
key to its role in cellular respiration.

Its complex folding pattern of membranes and spaces allows for

many sites where reactions can occur.

Respiration involves glycolysis, the Krebs cycle,

and electron transport

Stage I: Glycolysis
The first stage in breaking down a glucose molecule, called glycolysis
(splitting sugar), takes place outside the mitochondria in the
cytoplasm of the cell.

Glycolysis harvests chemical energy by oxidizing glucose

to pyruvate

The energy input and

output of glycolysis.
Concentrate on the
totals, not the
details!

Glycolysis movie

6 CH OPO 2
2
3
5
O

H
4

OH

H
OH
3

H
2

H
1

OH

OH

glucose-6-phosphate
Glycolysis takes place in the cytosol of cells.
Glucose enters the Glycolysis pathway by
conversion to glucose-6-phosphate.
Initially there is energy input corresponding to
cleavage of two ~P bonds of ATP.

6 CH2OH
5

H
4

OH

H
OH

H
2

OH

glucose

6 CH OPO 2
2
3
5
O

ATP ADP
H
H
1

OH

Mg2+

OH

Hexokinase

H
OH
3

H
2

H
1

OH

OH

glucose-6-phosphate

1. Hexokinase catalyzes:
Glucose + ATP glucose-6-P + ADP
The reaction involves nucleophilic attack of
the C6 hydroxyl O of glucose on P of the
terminal phosphate of ATP.
ATP binds to the enzyme as a complex with
Mg++.

NH2

ATP

adenosine triphosphate
O

O
O

P
O

O
O

P
O

CH2

adenine

OH

H
OH

ribose

Mg++ interacts with negatively charged

phosphate oxygen atoms, providing charge
compensation & promoting a favorable
conformation of ATP at the active site of the
Hexokinase enzyme.

6 CH2OH
5

H
4

OH

H
OH

H
2

OH

glucose

6 CH OPO 2
2
3
5
O

ATP ADP
H
H
1

OH

Mg2+

OH

H
OH
3

H
2

H
1

OH

Hexokinase H
OH
glucose-6-phosphate

The reaction catalyzed by Hexokinase is

highly spontaneous.
A phosphoanhydride bond of ATP (~P) is
cleaved.
The phosphate ester formed in glucose-6phosphate has a lower G of hydrolysis.

6 CH2OH

Induced fit:
Glucose
binding to
Hexokinase
stabilizes a
conformatio
n
in
which:

H
4

OH

H
OH

H
2

OH

ATP ADP
H
H
1

OH

Mg

2+

OH

Hexokinase

glucose

the C6 hydroxyl of the

bound glucose is close to
the terminal phosphate of
ATP, promoting catalysis.

6 CH OPO 2
2
3
5
O

H
OH
3

H
2

H
1

OH

OH

glucose-6-phosphate

glucose

Hexokinase

water is excluded from the active site.

This prevents the enzyme from catalyzing ATP hydrolysis,
rather than transfer of phosphate to glucose.

glucose

Hexokinase

It is a common motif for an enzyme active

site to be located at an interface between
protein domains that are connected by a
flexible hinge region.
The structural flexibility allows access to
the active site, while permitting precise
positioning of active site residues, and in
some cases exclusion of water, as substrate
binding promotes a particular conformation.

6 CH OPO 2
2
3
5
O

H
4

OH

H
OH
3

H
2

OH

H
1

OH

6 CH OPO 2
2
3

1 CH2OH

H
4

OH

HO

3 OH

Phosphoglucose Isomerase
glucose-6-phosphate
fructose-6-phosphate
2. Phosphoglucose Isomerase catalyzes:
glucose-6-P (aldose) fructose-6-P (ketose)
The mechanism involves acid/base catalysis, with
ring opening, isomerization via an enediolate
intermediate, and then ring closure. A similar
reaction catalyzed by Triosephosphate Isomerase will
be presented in detail.

Phosphofructokinase
6 CH OPO 2
2
3

1CH2OH

H
4

OH

ATP ADP

HO

3 OH

fructose-6-phosphate

6 CH OPO 2
2
3

Mg2+

1CH2OPO32

H
4

OH

HO

3 OH

fructose-1,6-bisphosphate

3. Phosphofructokinase catalyzes:
fructose-6-P + ATP fructose-1,6-bisP
+ ADP
This highly spontaneous reaction has a
mechanism similar to that of Hexokinase.
The Phosphofructokinase reaction is the ratelimiting step of Glycolysis.
The enzyme is highly regulated, as will be

1CH2OPO3
2C

HO 3C
H 4C

Aldolase

2
CH
OPO
2
3
3

OH

2C

OH

1CH2OH

2
CH
OPO
2
3
6

dihydroxyacetone
phosphate

fructose-1,6bisphosphate

O
1C

H 2C OH
2
3 CH2OPO3

glyceraldehyde-3phosphate

Triosephosphate Isomerase

4.
Aldolase
catalyzes:
fructose-1,6bisphosphate
dihydroxyacetone-P +
glyceraldehyde-3-P
The reaction is an aldol cleavage, the reverse
of an aldol condensation.

A lysine residue at the active site functions

in catalysis.
The keto group of fructose-1,6-bisphosphate
reacts with the -amino group of the active
site lysine, to form a protonated Schiff base
intermediate.

1CH2OPO3
2C

HO 3C
H 4C

Aldolase

2
CH
OPO
2
3
3

OH

2C

OH

1CH2OH

2
CH
OPO
2
3
6

dihydroxyacetone
phosphate

fructose-1,6bisphosphate

O
1C

H 2C OH
2
3 CH2OPO3

glyceraldehyde-3phosphate

Triosephosphate Isomerase

5. Triose Phosphate Isomerase (TIM)

catalyzes:
dihydroxyacetone-P glyceraldehyde3-P
Glycolysis continues from glyceraldehyde-3-P.
TIM's K favors dihydroxyacetone-P. Removal of

Triosephosphate Isomerase
H
H

OH

H H

CH2OPO32

dihydroxyacetone
phosphate

OH
C
C

H H

OH

CH2OPO32

enediol
intermediate

O
C

OH

CH2OPO32

glyceraldehyde3-phosphate

The ketose/aldose conversion involves

acid/base catalysis, and is thought to proceed
via an enediol intermediate, as with
Phosphoglucose Isomerase.
Active site Glu and His residues are thought to
extract and donate protons during catalysis.

OH
O

HC

CH2OPO32

CH2OPO32

proposed
enediolate
intermediate

phosphoglycolate
transition state
analog

2-Phosphoglycolate is a transition state

analog that binds tightly at the active site of
Triose Phosphate Isomerase (TIM).
This inhibitor of catalysis by TIM is similar in
structure to the proposed enediolate
intermediate.
TIM is judged a "perfect enzyme." Reaction
rate is limited only by the rate that substrate

Triosephosphate
Isomerase structure is
an barrel, or TIM
barrel.
In an barrel there are
8 parallel strands surrounded by 8
-helices.
Short loops connect
alternating -strands &
-helices.

TIM

TIM barrels serve as

scaffolds for active site
residues in a diverse
array of enzymes.
Residues of the active
site are always at the
same end of the barrel,
TIM
on C-terminal ends of strands & loops
There
is debate
whether
connecting
these
to - the many different
enzymes
helices. with TIM barrel structures are
evolutionarily related.
In spite of the structural similarities there is
tremendous diversity in catalytic

OH
O

HC

TIM

CH2OPO32

CH2OPO32

proposed
enediolate
intermediate

phosphoglycolate
transition state
analog

Explore the structure of the Triosephosphate

Isomerase (TIM) homodimer, with the
transition state inhibitor
2phosphoglycolate bound to one of the TIM
monomers.
Note the structure of the TIM barrel, and the

Glyceraldehyde-3-phosphate
Dehydrogenase
H

NAD+

1C

+ Pi

OH

2
CH
OPO
2
3
3

glyceraldehyde3-phosphate

OPO32
+ H+ O
NADH
1C
H

OH

2
CH
OPO
2
3
3

1,3-bisphosphoglycerate

6. Glyceraldehyde-3-phosphate
Dehydrogenase catalyzes:
glyceraldehyde-3-P + NAD+ + Pi
1,3-bisphosphoglycerate
+

Glyceraldehyde-3-phosphate
Dehydrogenase
H

O
1C

OH

OPO32
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH

2
CH
OPO
2
3
3

glyceraldehyde3-phosphate

2
CH
OPO
2
3
3

1,3-bisphosphoglycerate

Exergonic oxidation of the aldehyde in

glyceraldehyde- 3-phosphate, to a carboxylic
acid, drives formation of an acyl phosphate,
a "high energy" bond (~P).
This is the only step in Glycolysis in which
NAD+ is reduced to NADH.

1C

H 2 C OH
2
3 CH2OPO3

glyceraldehyde-3phosphate

A cysteine thiol at the active site of

Glyceraldehyde-3-phosphate Dehydrogenase
has a role in catalysis.
The aldehyde of glyceraldehyde-3-phosphate
reacts with the cysteine thiol to form a
thiohemiacetal intermediate.

Enz-Cys

Oxidation to a
carboxylic
acid (in a ~
thioester)
occurs, as
NAD+ is
reduced to
NADH.

Enz-Cys

OH

HC

CH

SH

OH

OH

CH

CH

CH2OPO32

glyceraldehyde-3phosphate
CH2OPO32

thiohemiacetal
intermediate

NAD +
NADH

Enz-Cys

OH

CH

CH2OPO32

acyl-thioester
intermediate

Pi

Enz-Cys

SH

O3PO

OH

CH

CH2OPO32

1,3-bisphosphoglycerate

The high energy acyl thioester is

attacked by Pi to yield the acyl phosphate
(~P) product.

O
C

+
N

NH2

2e + H

NH2

NAD+

NADH

Recall that NAD+ accepts 2 e plus one H+ (a

hydride) in going to its reduced form.

Phosphoglycerate Kinase
O

OPO32 ADP ATP O

O
1C

H 2C OH
2
3 CH2OPO3

1,3-bisphosphoglycerate

Mg

2+

H 2C OH
2
3 CH2OPO3

3-phosphoglycerate

7. Phosphoglycerate Kinase catalyzes:

1,3-bisphosphoglycerate + ADP
3-

phosphoglycerate + ATP
This phosphate transfer is reversible (low G),
since
one ~P bond is cleaved & another
synthesized.

Phosphoglycerate Mutase
O

O
C

O
C

H 2C OH
2
3 CH2OPO3

H 2C OPO32
3 CH2OH

3-phosphoglycerate

2-phosphoglycerate

8. Phosphoglycerate Mutase catalyzes:

3-phosphoglycerate 2phosphoglycerate
Phosphate is shifted from the OH on
C3 to the OH on C2.

Phosphoglycerate Mutase
O

O
C

O
C

H 2C OH
2
CH
OPO
2
3
3

H 2C OPO32
3 CH2OH

3-phosphoglycerate

2-phosphoglycerate

An active site histidine

side-chain participates in Pi
transfer, by donating &
accepting phosphate.
The process involves a
2,3-bisphosphate
View
an animation of the
intermediate.
Phosphoglycerate Mutase

O
C

H 2C OPO32
2
3 CH2OPO3

2,3-bisphosphoglycerate

Enolase

O
C

H 2 C OPO32
3 CH2OH

O
C
C

OH

O
1

OPO32

CH2OH

2C

OPO32

3 CH2

2-phosphoglycerate enolate intermediate phosphoenolpyruvate

9. Enolase catalyzes:
2-phosphoglycerate
phosphoenolpyruvate + H2O

This dehydration reaction is Mg++-dependent.

2 Mg++ ions interact with oxygen atoms of the
substrate carboxyl group at the active site.
The Mg++ ions help to stabilize the enolate
anion intermediate that forms when a Lys

Pyruvate Kinase
O

O
C
1
C
2

ADP ATP

O
C

OPO32

3 CH2

phosphoenolpyruvate

3 CH3

pyruvate

10. Pyruvate Kinase catalyzes:

phosphoenolpyruvate + ADP
pyruvate + ATP

Pyruvate Kinase
O

O
C
1
C
2

ADP ATP

OPO32

3 CH2

phosphoenolpyruvate

O
C

O
1

OH

3 CH2

enolpyruvate

3 CH3

pyruvate

This phosphate transfer from PEP to ADP is spontaneous.

PEP has a larger G of phosphate hydrolysis than ATP.
Removal of Pi from PEP yields an unstable enol, which
spontaneously converts to the keto form of pyruvate.
Required inorganic cations K+ and Mg++ bind to anionic
residues at the active site of Pyruvate Kinase.

glucose
ATP

Glycolysis

Hexokinase

ADP
glucose-6-phosphate

Phosphoglucose Isomerase
fructose-6-phosphate
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase
glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued

glyceraldehyde-3-phosphate
NAD+ + Pi
Glyceraldehyde-3-phosphate
Dehydrogenase
NADH + H+

Glycolysi
s
continue
d.
Recall
that
there are
2 GAP
per
glucose.

1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate

Balance sheet for ~P bonds of ATP:

2 ATP expended
4 ATP produced (2 from each of two 3C fragments from
glucose)
Net production of 2 ~P bonds of ATP per glucose.

Glycolysis - total pathway, omitting H+:

glucose + 2 NAD+ + 2 ADP + 2 Pi
2 pyruvate + 2
NADH + 2 ATP
In aerobic organisms:
pyruvate produced in Glycolysis is oxidized to CO2 via Krebs
Cycle
NADH produced in Glycolysis & Krebs Cycle is reoxidized via
the respiratory chain, with production of much additional
ATP.

Glyceraldehyde-3-phosphate
Dehydrogenase
H

Fermentation
:

O
1C

OH

OPO32
+ H+ O
NAD+ NADH
1C
+ Pi
H C OH

2
CH
OPO
2
3
3

2
CH
OPO
2
3
3

Anaerobic
glyceraldehyde1,3-bisphospho3-phosphate
glycerate
organisms
lack a
They
must reoxidize NADH produced in
respiratory
Glycolysis
through some other reaction,
chain.
because NAD+ is needed for the
Glyceraldehyde-3-phosphate Dehydrogenase
reaction.
Usually NADH is reoxidized as pyruvate is
converted to a more reduced compound.

Lactate Dehydrogenase
O

O
C
C

NADH + H+ NAD+

O
C
HC

OH

CH3

CH3

pyruvate

lactate

E.g., Lactate Dehydrogenase catalyzes

reduction of
the keto in pyruvate to a
hydroxyl, yielding lactate, as
NADH is
oxidized to NAD+.
Lactate, in addition to being an end-product
of fermentation, serves as a mobile form of
nutrient energy, & possibly as a signal
molecule in mammalian organisms.

Lactate Dehydrogenase
O

O
C
C

NADH + H+ NAD+

O
C
HC

OH

CH3

CH3

pyruvate

lactate

Skeletal muscles ferment glucose to lactate

during exercise, when the exertion is brief and
intense.
Lactate released to the blood may be taken
up by other tissues, or by skeletal muscle after
exercise, and converted via Lactate
Dehydrogenase back to pyruvate, which may
be oxidized in Krebs Cycle or (in liver)

Lactate Dehydrogenase
O

O
C
C

NADH + H+ NAD+

O
C
HC

OH

CH3

CH3

pyruvate

lactate

Lactate serves as a fuel source for cardiac

muscle as well as brain neurons.
Astrocytes, which surround and protect
neurons in the brain, ferment glucose to
lactate and release it.
Lactate taken up by adjacent neurons is
converted to pyruvate that is oxidized via

Pyruvate
Decarboxylase

Alcohol
Dehydrogenase

CO2

NADH + H+ NAD+

O
C
C

CH3

pyruvate

O
C

CH3

acetaldehyde

OH

CH3

ethanol

Some anaerobic organisms metabolize

pyruvate to ethanol, which is excreted
as a waste product.
NADH is converted to NAD+ in the
reaction catalyzed by Alcohol

Glycolysis, omitting H+:

glucose + 2 NAD+ + 2 ADP + 2 Pi
2 pyruvate + 2
NADH + 2 ATP
Fermentation, from glucose to lactate:
glucose + 2 ADP + 2 Pi 2 lactate +
2 ATP
Anaerobic catabolism of glucose yields
only 2 high energy bonds of ATP.

Glycolysis Enzyme/Reaction

Go'
G
kJ/mol kJ/mol

Hexokinase
Phosphoglucose Isomerase
Phosphofructokinase
Aldolase
Triosephosphate Isomerase
Glyceraldehyde-3-P Dehydrogenase
& Phosphoglycerate Kinase

-20.9 -27.2
+2.2
-1.4
-17.2 -25.9
+22.8
-5.9
+7.9 negative
-16.7
-1.1

Phosphoglycerate Mutase
Enolase
Pyruvate Kinase

+4.7
-3.2
-23.0

-0.6
-2.4
-13.9

*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3 rd

Edition, John Wiley & Sons, New York, p. 613.

Flux through the Glycolysis pathway is

regulated by control of 3 enzymes that
catalyze spontaneous reactions:
Hexokinase, Phosphofructokinase &
Pyruvate Kinase.
Local control of metabolism involves regulatory effects of
varied concentrations of pathway substrates or
intermediates, to benefit the cell.
Global control is for the benefit of the whole organism, &
often involves hormone-activated signal cascades.
Liver cells have major roles in metabolism, including
maintaining blood levels various of nutrients such as glucose.
Thus global control especially involves liver.
Some aspects of global control by hormone-activated signal
cascades will be discussed later.

6 CH2OH
5

H
4

OH

H
OH

H
2

OH

glucose

6 CH OPO 2
2
3
5
O

ATP ADP
H
H
1

OH

Mg2+

OH

H
OH
3

H
2

H
1

OH

Hexokinase H
OH
glucose-6-phosphate

Hexokinase is inhibited by product

glucose-6-phosphate:
by competition at the active site
by allosteric interaction at a separate enzyme site.

Cells trap glucose by phosphorylating it,

preventing exit on glucose carriers.
Product inhibition of Hexokinase ensures that
cells will not continue to accumulate glucose
from the blood, if [glucose-6-phosphate] within

6 CH2OH
5

H
4

H
OH

6 CH OPO 2
2
3
5
O

ATP ADP
H
H
1

H
OH

H
1

Glucokina
Mg2+
OH
OH
OH
OH
se is a
2
3
2
3
Hexokinase H
variant of
OH
H
OH
glucose
glucose-6-phosphate
Hexokinase
found in
Glucokinase has a high KM for glucose.
liver.
It is active only at high [glucose].
One effect of insulin, a hormone produced
when blood glucose is high, is activation in
liver of transcription of the gene that
encodes the Glucokinase enzyme.
Glucokinase is not subject to product
inhibition by glucose-6-phosphate. Liver
H

 Glucokinase is subject to inhibition by

glucokinase regulatory protein (GKRP).
The ratio of Glucokinase to GKRP in liver
changes in different metabolic states,
providing a mechanism for modulating
glucose phosphorylation.

Glycogen

Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway

Glucokinase,
with high KM
Glucose-1-P
for glucose,
allows liver
to
store
Pyruvate
glucose
Glucose metabolism in liver.
as glycogen in
the fed
Glucose-6-phosphatase catalyzes hydrolytic
state
release of Pi from glucose-6-P. Thus glucose is
when blood
released is
from
the liver
to the blood as
[glucose]
high.
needed to maintain blood [glucose].
The enzymes Glucokinase & Glucose-6phosphatase, both found in liver but not in
most other body cells, allow the liver to control

Pyruvate Kinase
O

Pyruvate Kinase,
ADP ATP
C
C
1
1
the last step
2
C
OPO
C O
3
Glycolysis, is
2
2
controlled in liver
3 CH2
3 CH3
phosphoenolpyruvate
pyruvate
partly by
modulation of the
amount
of
High
[glucose]
within liver cells causes a
enzyme.
transcription
factor carbohydrate responsive
element binding protein (ChREBP) to be
transferred into the nucleus, where it activates
transcription of the gene for Pyruvate Kinase.
O

This facilitates converting excess glucose to

pyruvate, which is metabolized to acetylCoA, the main precursor for synthesis of fatty

Phosphofructokinase
6 CH OPO 2
2
3

1CH2OH

H
4

OH

ATP ADP

HO

3 OH

fructose-6-phosphate

6 CH OPO 2
2
3

Mg2+

1CH2OPO32

H
4

OH

HO

3 OH

fructose-1,6-bisphosphate

Phosphofructokinase is usually the ratelimiting step of the Glycolysis pathway.

Phosphofructokinase is allosterically
inhibited by ATP.
At low concentration, the substrate ATP binds only at the
active site.
At high concentration, ATP binds also at a low-affinity
regulatory site, promoting the tense conformation.

60

low [ATP]

PFK Activity

50
40
30

high [ATP]

20
10
0
0

0.5
1
1.5
[Fructose-6-phosphate] mM

The tense conformation of PFK, at high [ATP],

has lower affinity for the other substrate,
fructose-6-P. Sigmoidal dependence of reaction
rate on [fructose-6-P] is seen.
AMP, present at significant levels only when

Glycogen

Glucose-1-P

Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway

Pyruvate
Glucose metabolism in liver.

Inhibition of the Glycolysis enzyme

Phosphofructokinase when [ATP] is high
prevents breakdown of glucose in a pathway
whose main role is to make ATP.
It is more useful to the cell to store glucose as
glycogen when ATP is plentiful.

Stage 2: The Krebs Cycle

The Krebs cycle finishes the breakdown of pyruvic acid molecules to
carbon dioxide, releasing more energy in the process. The enzymes
for the Krebs cycle are dissolved in the fluid matrix within a
mitchondrion's inner membrane.

Krebs Cycle:
Where does this
occur?
Identify the products.

Krebs Cycle movie

Citric Acid cycle or Tricarboxylic Acid cycle or Krebs

Cycle
Overview and brief history
Pyruvate Dehydrogenase Complex (PDC) and its control
Reactions of TCA cycle or CAC
Amphibolic nature of TCA cycle
Regulation of TCA cycle
Reactions of Glycolysis are localized in Cytosol, and do not
require any oxygen.
whereas pyruvate dehydrogenase and TCA cycle reactions
take place in mitochondria where oxygen is utilized to
generate ATP by oxydative phosphorylation.
Consumption of oxygen (respiration) depends on the rate of
PDC and TCA reactions.

In Cytosol

In
Mitochondria

Reactions of Citric Acid Cycle

1. Citrate synthase: Formation of Citroyl CoA intermediate.
2. Binding of Oxaloacetate to the enzyme results in conformational change
which facilitates the binding of the next substrate, the acetyl Coenzyme A.
There is a further conformational change which leads to formation of
products. This mechanism of reaction is referred as induced fit model.

2. Aconitase: This enzyme catalyses the isomerization

reaction by removing and then adding back the water ( H
and OH ) to cis-aconitate in at different positions.
Isocitrate is consumed rapidly by the next step thus
deriving the reaction in forward direction.

3. Isocitrate dehydrogenase: There are two isoforms of this

enzyme, one uses NAD+ and other uses NADP+ as electron
acceptor.

4. -Ketoglutarate dehydrogenase: This is a complex of

different enzymatic activities similar to the pyruvate
dyhdogenase complex. It has the same mechanism of
reaction with E1, E2 and E3 enzyme units. NAD+ is an
electron acceptor.

5. Succinyl CoA synthatse: Sccinyl CoA, like Acetyl CoA

has a thioester bond with very negative free energy of
hydrolysis. In this reaction, the hydrolysis of the
thioester bond leads to the formation of phosphoester
bond with inorganic phosphate. This phosphate is
transferred to Histidine residue of the enzyme and this
high energy, unstable phosphate is finally transferred to
GDP resulting in the generation of GTP.

6. Succinate Dehydrogenase: Oxidation of succinate to

fumarate. This is the only citric acid cycle enzyme that is
tightly bound to the inner mitochondrial membrane. It is an
FAD dependent enzyme.
Malonate has similar structure to Succinate, and it
competitively inhibits SDH.

7. Fumarase: Hydration of Fumarate to malate: It is a highly

stereospecific enzyme. Cis-Maleate (the cis form of
fumarate is not recognized by this enzyme.

8. L-Malate dehydrogenase: Oxidation of malate to

oxaloacetate: It is an NAD+dependent enzyme. Reaction is
pulled in forward direction by the next reaction (citrate
synthase reaction) as the oxaloacetate is depleted at a very
fast rate.

Conservation of energy of oxidation in the CAC: The two

carbon acetyl group generated in PDC reaction enter the CAC, and
two molecules of CO2 are released in on cycle. Thus there is
complete oxidation of two carbons during one cycle. Although
the two carbons which enter the cycle become the part of
oxaloacetate, and are released as CO2 only in the third round of
the cycle. The energy released due to this oxidation is conserved
in the reduction of 3 NAD+, 1 FAD molecule and synthesis of one
GTP molecule which is converted to ATP.

Regulation of CAC:
Rate controlling enzymes:
Citrate synthatase
Isocitrate dehydrogenase
-keoglutaratedehydrogenase
Regulation of activity by:
Substrate availability
Product inhibition
Allosteric inhibition or
activation by other
intermediates

Stage 3: Electron Transport Chain and ATP Synthase Action

The final stage occurs in the inner membranes of mitochondria. This
stage has two parts: an electron transport chain and ATP production
by ATP synthase

ATP Formation Through

Mitochondrial Electron-Transport
(Chapter 17)

Components of the Electron-Transport Chain

Oxidative Phosphorylation
Recycling of Cytoplasmic NADH

Electron transport, also known as aerobic

respiration, is the last stage of aerobic
metabolism.
After glycolysis and TCA cycle, 10 NADH
and 2 FADH2 are generated from the
oxydation of one glucose molecule.

dehydrogenase

AH2 + NAD+
NADH + H+
dehydrogenase
Or
BH2 + FAD

A+

B +

When there is sufficient O2 supply, NADH and

FADH2 enter electron transport chain to becom
reoxidized

Large amount of energy is recovered, when

electrons are passed from NADH and FADH2
to O2.

This is accomplished by a series of carrier prot

n the inner mitochondrial membrane .

Mitochondrial Electron Transport

Folding in the mitochondria inner membrane provides
a large surface area.
Electron-transport chain components are arranged in
packages called respiratory assemblies.

lectron Transport and Oxidative

hosphorylation:

n ETS, electrons are transferred from NADH

nd FADH2 to O2 step by step.

n the mean time, energy released from electro

ow is coupled to ATP synthesis.

Here, phosphorylation of ADP is coupled with

he oxidation of NADH or FADH2.

ADP + Pi

NADH + H+ + 1/2 O2
FADH2 + 1/2 O2

ATP

+
NAD
+ H2O
ATP synthase

ADP + Pi

ATP

FAD + H2O

ATP synthase

Theenergyreleasedduringtheelectronflowis
coupledtoATPsynthesis.

Composition of the Electron Transport

Chain
Four large protein complexes.

Complex I - NADH-Coenzyme Q reductase

Complex II - Succinate-Coenzyme Q
reductase
Complex III - Cytochrome c reductase
Complex IV - Cytochrome c oxidase
Many of the components are proteins with prosthetic
groups to move electrons.

Complex
I

Complex
IV

Complex
III
cyt
c1

CoQ

cyt b
FADH2

Fe
S

cyt
c

(Cu)
cyt
a/a3

Complex II
NADH

matrix

O
2

 Important characteristic of the electrontransport chain:

Electron carriers are arranged in order of increasing
electron affinity, from low to high.
This results in the spontaneous flow of
electrons from carrier to carrier.

Eo, standard reduction potentials.

The more positive the Eo value a molecule has,
the
better it serves as an electron acceptor.
O2 has the highest Eo (0.82V), - highest affinity
for electrons and is located in the end of the
chain.

Eo for NAD is 0.32, lowest in the system, its

the
poorest electron carrier, located in the
beginning.

-0.4
-0.2

NADH
(-0.32)
NAD+

Path of
Electrons
Complex I

0.0
0.2

succinate
(FADH2)
(0.18)
fumarate

Q
Complex II

Complex III
cyt c

(0.25)

0.4

Complex IV
0.6
0.8
1.0

Table 17.2 !

1/2 O2 + 2 H+

(0.82)

HO
2

Components of the
electron transport chain
Complex I
Electrons pass from
NADH FMN Fe-S cluster ubiquinone

(flavin mononucleotide)

(coenzyme Q

Fe-S cluster: iron cycles between 3+

and 2+
states.

2 H+

FMN

Complex I

FMNH2

2 electrons

Fe-S

QH2

2 electrons

2 H+
NADH

H+

NAD+

 CoQ ubiquinone
Highlighted region serves as an anchor to inner
mitochondrial membrane.

O
H3CO

CH3
CH3

H3CO

(CH2
O

CH

CH2 )10

Reduction of CoQ
Oxidized form
Ubiquinone (CoQ)

Reduced form
Ubiquinol (CoQH2)

OH

H3CO

CH3

H3CO

CH3

H3CO

H3CO

2e 2H+

OH

 Complex II
Entry point for FADH2.
Succinate dehydrogenase

(from the citric acid

cycle) directs transfer of electrons from

succinate to CoQ via FADH2.
(from -oxidation of
fatty acids) also transfers electrons to CoQ
via FADH2.

Acyl-CoA dehydrogenase

 All electrons from FADH2 and NADH must pass through

CoQ.

innermembrane
space
I
Fe-S
FMN

II

Fe-S
FAD

NADH

NAD+

CoQ

FAD

Succinate
Fatty acyl
CoA

matrix

 Complex III (cytochromes b, c1 and c).

Electron transfer from ubiquinol to

cytochrome c.

cytochrome c

heme prosthetic group

ytochromes are electron-transfer proteins tha

ontain a heme prosthetic group.

he iron atom in heme also cycles through

educed form (Fe2+) and the oxidized form (Fe3+

ed muscles are rich in mitochondria, which

ontains electron transport system and
ytochromes.

 Complex IV

Combination of cytochromes a and a3,

10 protein subunits, 2 types of
prosthetic groups: 2 heme and 2 Cu.
Electrons are delivered from
cytochromes a and a3 to O2.

Several chemicals can inhibit the pathway at

different locations.

Cyanide and CO can block e transport betwee

a/a3 and O2.

Flow of electrons
-0.4
NADH
-0.2

Path of
Electrons

NAD+
Complex I

0.0
0.2

succinate
(FADH2)
fumarate

Complex II

Q
Complex III
cyt c

0.4
Complex IV
0.6
1/2 O2 + 2 H+
0.8

HO
2

Energy
is not released at once, but in increment
1.0
amounts at each step.

Energy Yield
The amount of energy can be
calculated in
terms of Go .
Go = - nF Eo
n = electron number,
F = faraday constant = 96.5kJ/volt
.
mole
Eo = Eoacceptor - Eodonor

Energy Yield
NADH + H+ + 1/2 O2

NAD+ + H2O

Go = - 220 kJ/mol
FADH2 + 1/2 O2

FAD + H2O

Go = - 152 kJ/mol
Note: ADP + Pi
kJ/mol

ATP

Go = + 31

Energy yield from one FADH2 is less than one

NADH.

Oxidative phosphorylation
The electron-transport chain moves
electrons from NADH and FADH2 to O2.
In the mean time, ADP is phosphorylated to
ATP.
The two processes are dependent on each
other.
ATP cannot be synthesized unless there is
energy from electron transport (Go= +31
kj/mol).
Electrons do not flow to O2, unless there is
need for ATP.

3 ATP are generated when two electrons

are transported from NADH to O2.

The oxidation of FADH2 only produces 2 ATP.

Coupling of electron-transport
with ATP synthesis
Chemiosmotic coupling mechanism

Electron-transport causes unidirectional

movement of H+ into the innermembrane space.
The results in a H+ gradient being produced.
The gradient then drives the synthesis of ATP.

Outer mitochondrial membrane

H+

H+

H+

H+
H

H+
H+

H+

H+
H+

H+

Inner mitochondrial membrane

H+ H+

H+
Electron
Electron
Transport
Transport
Chain
Chain

ATP
ATP
synthase
synthase
complex
complex

ADP + Pi

H+

ATP

Components of ATP synthase

These are knob-like projections into the matrix side of
the inner membrane.
Two units
F1 contains the catalytic site for ATP synthesis.
F0 serves as a transmembrane channel for H+ flow.
F1-F0 complex serves as the molecular apparatus for
coupling H+ movement to ATP synthase.

Components of ATP synthase

H+
Cytosole
H+

H+

H+
H+

H+

H+

H+
H+

H+

F0

Matrix

F
1

ADP + Pi

ATP

ATP is transported from the matrix of mitochondria

to cytosole by ATP-ADP translocase.

ATP and ADP cannot diffuse through the mitochondri

membrane freely.

The exit of ATP is coupled with the entry of AD

nto mitochondria.

Regulation of oxidative
phosphorylation
Electrons do not flow unless ADP is present for
phosphorylation
Increased ADP levels cause an increase in the activity of
various enzymes including:
glycogen phosphorylase
phosphofructokinase
citrate synthase

Uncoupling of electron-transport
and oxidative phosphorylation
In some special cases, the coupling of the
two processes can be disrupted.
Large amounts of O2 are consumed but no
ATP is produced.
Used by newborn animals and hibernating
mammals.
Occurs in brown fat- which contain
thermogenin (uncoupling protein).
Thermogenin allows the release of energy as
heat instead of ATP.

Energy production from

glucose
Glycolysis

2 ATP

2 NADH

2 ATP
3 ATP/NADH

Citric Acid Cycle

2 GTP
1 ATP/GTP

6 NADH
3 ATP/NADH
2 FADH2
2 ATP/FADH2

38 ATP

(in heart)
* 4 ATP in muscle and brain.
36 ATP / glucose

6 ATP*
2 ATP
18 ATP
4 ATP

Mitochondria
Glycolysis
Glucose
Glucose

22Pyruvate
Pyruvate

22NADH
NADH

22Acetyl
AcetylCoA
CoA

22NADH
NADH

66NADH+
NADH+
22FADH
FADH2
2

22GTP
GTP

Oxidative
Oxidative
phosphorylation
phosphorylation

22 ATP
ATP

32-34
32-34 ATP
ATP

22 ATP
ATP

Recycling of cytoplasmic NADH(?)

Different methods are used to recycle
NADH. This accounts for the different
energy productions from glucose.
Glycerol-3-phosphate shuttle

Used by skeletal muscles and the

brain
Malate-aspartate shuttle

Used by the heart and liver

Glucose-3-phosphate shuttle
NAD+

NAD+

cytoplasmic
glycerol-3-phosphate
dehydrogenase

cytosolic
glycerol-3-phosphate
dehydrogenase

Glycerol-3-phosphate

NADH + H+

NADH + H+

Dihydroxyacetone
phosphate
mitochondrial
glycerol-3-phosphate
dehydrogenase

Cytoplasm

FAD

FADH2
II

Mitochondrial matrix

III

Malate-aspartate shuttle
Matrix
L-aspartate

L-aspartate

-ketoglutarate

Glycolysis

cytoplasmic
aspartate
aminotransferase

L-glutamate
oxaloacetate

cytoplasmic malate
dehydrogenase

NADH+ H+

L-malate
NAD+

-ketoglutarate
mitochondrial
aspartate
aminotransferase

L-glutamate
mitochondrial
malate
dehydrogenase

L-malate

oxaloacetate

NAD+

3 ATP
NADH
+ H+

The inner mitochondrial membrane couples electron

transport to ATP synthesis.
The Pathway of Electron
Transport
This energy change is
used to pump
hydrogen to the inner
membrane space
creating a gradient
which can power cell
processes.
.

Chemiosmosis couples the electron transport chain to

ATP synthesis

Chemiosmosis: The Energy-Coupling Mechanism

ATP synthase protein
complex functions as a mill,
powered by the flow of
hydrogen ions.
This complex resides in
mitochondrial and
chloroplast membranes of
eukaryotes and in the
plasma membranes of
prokaryotes..
The gradient of hydrogen
ions pushes the ATP
synthesis.

Animation of ATP synthesis in Mitochondria

Copyright 1997. Thomas

M. Terry, The University
of Connecticut

Cellular respiration generates many ATP molecules for each sugar

molecule it oxidizes
During respiration, most energy flows in this sequence:
Glucose NADH electron transport chain protonmotive
force ATP

Harvesting Energy without Oxygen

Fermentation in Human Muscle Cells
When your lungs and bloodstream can't supply oxygen fast enough to
meet your muscles' need for ATP. Your muscle cells use fermentation,
to make ATP without using oxygen.

How does the energy production of Lactic Acid fermentation

compare to aerobic respiration?

ctic Acid fermentation occurs in animal cells deficient in oxyg

Lactic Acid Fermentation movie

Fermentation in Microorganisms
Yeast (a microscopic fungus) is capable of both cellular respiration
and fermentation.

Fermentation in yeast produces ethyl alcohol. The carbon dioxide that

is released during fermentation creates bubbles and pockets that
make bread rise. The alcohol evaporates during baking.

Fermentation enables some cells to produce ATP without

the help of oxygen. Alcoholic fermentation occurs in
yeast.

Alcoholic Fermentation movie

Pyruvate as a key juncture in catabolism. Glycolysis is

common to fermentation and respiration.

How does the net gain of

ATP compare in aerobic
vs. fermentation?

The catabolism of various

food molecules.
Carbohydrates, fats, and
proteins can all be used
as fuel for cellular
respiration.

Summar
y
Glucose
ATP

Fig.169