Chapter 18:

Oxidative Phosphorylation
 Cellular Respiration

8 high-energy e- s e- s reduce ATP synthesized

are removed O2 to generate a from proton gradient
from C fuels proton gradient
Mitochondria Are the Result of an Endosymbiotic Event
â semiautonomous organelles (endosymbiotic relation with host)
â contain their own DNA

The structure of this DNA is very similar to that found in prokaryotic cells.

Endosymbiotic event - engulfment of bacteria capable of oxidative phosphorylation

Possible ancestor - Rickettsia prowazekii , the cause of louse-borne typhus

Mitochondrion Nucleus

Endoplasmic
reticulum
Engulfing of
photosynthetic
Some prokaryote
cells
Engulfing Chloroplast
of oxygen-
Host cell
using
prokaryote
DNA sequence analysis suggests that
an ancestor of an existing bacterium
Mitochondrion is the source for extant mitochondria.
DNA coding region for genes with
Host cell known function is shown in red.
Oxidative phosphorylation takes place
in the mitochondria and
is the major source of ATP in aerobic organisms.

Lehninger Principles of Biochemistry fourth ed. David L. Nelson and Michael M. Cox
 Decarboxylation of pyruvate
Mitochondrion Citric acid cycle
Fatty acid oxidation
impermeable to
protons intrinsically impermeable
a large family of transporters
shuttle ATP, pyruvate, and citrate

Oxidative
-+
phosphorylation
-+
-+ Permeable to
small molecules
permeable to most small molecules
inner mitochondrial porin, or VDAC
transport phosphate, chloride, organic anions,
membrane and the adenine nucleotides
Cristae
space
Why do we have cristae?
matrix side (N)
Cristae
increase the surface area of the inner mitochondrial membrane cytosolic side (P)
providing more sites or oxidative phosphorylation
to take place than would be possible with a single fold membrane.
 The process by which ATP is formed as e –s
Oxidative Phosphorylation are transferred from NADH or FADH2 to O2.

O2 is reduced to H2O, a large amount of free E is liberated.

This free E mostly generates ATP and some heat.

NADH + ½ O2 + H+ ª H2O + NAD + DGo’ = -220.1kJ/mol

Oxidative Phosphorylation
couples oxidation of C fuels
to ATP synthesis
with a proton gradient

ADP+ Pi + H+ ª ATP + H2O DGo’ = +30.5kJ/mol

Oxidative phosphorylation (OP*) is the culmination of a
series of energy transformations, called cellular respiration

First, carbon fuels are oxidized in the citric acid cycle to yield e-s with high transfer
potential
Then, this electron-motive force is converted into a proton-motive force and,
Finally, the proton-motive force is converted into phosphoryl transfer potential

NADH or FADH2
formed in
Ø glycolysis
Ø fatty acid oxidation When these e- s are
Ø citric acid cycle transferred to reduce
are high E molecules O2 to H2O in OP*,
because they each have 2 e- s a large amount of
with high-transfer potential free E is liberated and
can be used to
generate ATP
The transfer of e- s from NADH or FADH2
to O2 through protein complexes
(respiratory chain) lead to pumping of
protons out of the mitochondrial matrix
 Oxidative Phosphorylation Depends on Electron Transfer
In oxidative phosphorylation, te ntial al
d ox po te nti
re o
u c tio n p
NADH/FADH2 E 0¢ red
the electron-transfer potential

ATP DGo¢
the phosphoryl-transfer potential

The metabolic breakdown

Lippincott’s Illustrated Reviews fifth ed. Richard Harvey and Denise Ferrier of energy yielding molecules
 The electron-transfer potential of an electron is measured as redox potential

D E0¢ â pH:7.0
D E0 â pH:0.0

Biological Redox Reactions , January 17, 2003, Bryant Miles Oxidation of NADH by FMN,
separated into two component
redox pairs. Standard reduction potentials
of some reactions.

d ox
e ple
NAD + : NADH withdraws electrons r
co u
X : X-
FMN : FMNH2 pushes electrons
Oxidant : Reductant
Lippincott’s Illustrated Reviews fifth ed. Richard Harvey and Denise Ferrier
reduction potential difference between NADH and O2
drives electron transport and favors formation of a proton gradient

NADH

1.14 Volt

O2

the more (+) the reduction potential voltage

the stronger the pull is for electrons.
 pyruvate + 2 H+ + 2 e- lactate
pyruvate + NADH + H+ NAD + + H+ + 2 e-

pyruvate + NADH + H+ lactate + NAD +

NADH + H+ lactate

NAD + pyruvate
e-

NADH is oxidized to NAD+

pyruvate is reduced to lactate
NADH and lactate are the reduced forms
NAD+ and pyruvate are the oxidized forms
pyruvate is the oxidizing agent
NADH is the reducing agent
The free-energy change of an oxidation-reduction reaction
can be calculated from the reduction potentials of the reactants

pyruvate + 2 H+ + 2 e- lactate E0¢ = - 0.19 V

pyruvate + NADH + H+ NAD + + H+ + 2 e- E0¢ = + 0.32 V

pyruvate + NADH + H+ lactate + NAD +

DE0¢ = - 0.19 V + 0.32 V = + 0.13 V

DE0¢ > 0 spontaneous g

DE0¢ < 0 spontaneous f
 The free-energy change of an oxidation-reduction reaction
can be calculated from the reduction potentials of the reactants

pyruvate + 2 H+ + 2 e- lactate E0¢ = - 0.19 V

pyruvate + NADH + H+ NAD + + H+ + 2 e- E0¢ = + 0.32 V

pyruvate + NADH + H+ lactate + NAD +

DGo¢ = ???

DE0¢ = - 0.19 V + 0.32 V = + 0.13 V

DGo¢ (overall) = - 2 x (96.48 kJ mol-1 V -1) x (+ 0.13V) = - 25.08 kJ/mol

DGo¢ can be calculated n = number of electrons transferred per rxn

from redox potential DGo¢ = - nF D E0¢ F = the Faraday constant (96.48 kJ mol-1 V -1)
by the following formula D E0¢ = redox potential (Vo)

pyruvate + 2 H+ + 2 e- lactate E0¢ = - 0.19 V

DGo¢ = - 2 x (96.48 kJ mol-1 V -1) x (- 0.19V) = + 36.7 kJ/mol
pyruvate + NADH + H+ NAD + + H+ + 2 e- E0¢ = + 0.32 V
DGo¢ = - 2 x (96.48 kJ mol-1 V -1) x (+ 0.32V) = - 61.8 kJ/mol

DGo¢ (overall) = (+ 36.7 kJ/mol ) + (- 61.8 kJ/mol) = - 25.1 kJ /mol

 So what kind of DGo’ drives oxidative phosphorylation?

1/2 O2 + 2 H+ + 2 e- H2 O E0¢ = + 0.82 V

NADH + H+ NAD + + H+ + 2 e- E0¢ = + 0.32 V
1/2 O2 + H+ + NADH H2O + NAD+
DGo¢ = - 2 x (96.48 kJ mol-1 V -1) x (+ 0.82) = - 158.2 kJ/mol
DGo¢ = - 2 x (96.48 kJ mol-1 V -1) x (+ 0.32V) = - 61.8 kJ/mol
DGo¢ (overall) = (- 158.2 kJ/mol ) + (- 61.8 kJ/mol) = - 220.1 kJ /mol

Energy released is used

Ê to make a proton gradient and then
Ê for the synthesis of ATP and
Ê for the transport of other metabolites across the mitochondrial membrane
The respiratory chain consists of 4 complexes:
3 proton pumps and a physical link to the citric acid cycle

NADH

II

III
contains
succinate
dehydrogenase
that generates

electron flow within FADH2 in the

these complexes leads IV citric acid cycle.
to transport of protons
across the IMM
they contain
OX. RED. centers:
§ quinones
§ flavins
§ iron-sulfur clusters
§ hemes
§ copper ions
Coenzyme Q - ubiquinone ubiquitous quinone
hydrophobic
diffuses rapidly within the IMM – membrane soluble
NOT a protein
accept e-s from Complex-I and Complex-II in pairs
delivers its e- s to Complex-III one at a time

du c t io n of
re
o ( Q H 2)
(Q ) t
form hold e-s
reduced binds e- and H+

Q pool there is a pool of Q and QH2 in the mitochondrial membrane

NADH-Q oxidoreductase- Complex-I Sequence of events :

prosthetic
1 NADH binding group
2 transfer of 2e- s to FMN which yields FMNH2.
Flavins -also- bind protons when they are reduced.

3 e- s are then transferred from FMNH2

to a series of iron-sulfur clusters.
prosthetic
Fe-S clusters undergo redox rxns w/o H+ binding/releasing group

4 e- s in the Fe-S clusters of Complex-1 are shuttled to Co-Q

5 The flow of 2e- s from NADH to Co-Q through

Complex-1 leads to the pumping of 4 H+ out of the
matrix of the mitochondrion.
Q is reduced to QH2
and leaves the enzyme
for the hydrophobic interior of membrane
Why is less ATP formed from FADH2 then NADH?
1 succinate DH – a part ofcomplex-2
e- s from NADH
enter from here 2 FADH2 doesn’t leave the complex,
and transfer its e- s to Fe-S clusters.
I
3 e- s in the Fe-S clusters are shuttled to Q.

II
e- s from FADH2
enter from here
III
does not pump
H+ out of the
matrix of the
mitochondrion

IV

e- s from FADH2
are generated here
The function of Q-cytochrome c oxidoreductase (Complex-III) is
to catalyze transfer of e- s from QH2 to oxidized cytochrome c (Cyt c)
to pump H+ out of the matrix of the mitochondrion
Cyt c
water-soluble protein
e- transferring protein
contains heme prosthetic group His
replace
contains a 2Fe-2S center Cys
Ø Rieske center

The flow of e- pair results in x- III

pumping 2H+ to cytoplasmic site mple
Co
Coenzyme Q - ubiquinone ubiquitous quinone
hydrophobic
diffuses rapidly within the IMM – membrane soluble
NOT a protein
accept e-s from Complex-I and Complex-II in pairs
delivers its e- s to Complex-III one at a time

du c t io n of
re
o ( Q H 2)
(Q ) t
form hold e-s
reduced binds e- and H+

Q pool there is a pool of Q and QH2 in the mitochondrial membrane

 x- IV
mple
Co
O2
4 H+ from matrix side to
reduce one O2 to two H2O :
“chemical protons”

4 additional H+ pumped to
cytosolic side :
“pumped protons”

The pumped protons double

the efficiency of free-energy
storage in the form of a
proton gradient
for this final step in the
electron transport chain
although reduction of O2 is remarkably successful,
partial reduction may generate
Ê superoxide anion
Ê hydrogen peroxide

enzymes and vitamins

help protect from ROS:
ü superoxide dismutase
ü catalase
ü antioxidant vitamins:
vit E and vit C
CHEMIOSMOSIS : The diffusion of ions across a selectively-permeable membrane.
More specifically, it relates to the generation of ATP by the movement of hydrogen
ions across a membrane during cellular respiration.

Hydrogen ions(protons) will diffuse

from an area of high proton
concentration to an area of lower
proton concentration. It has been
proposed that an electrochemical
concentration gradient of protons
across a membrane could be
harnessed to make ATP.

This process is referred to osmosis,

the diffusion of water across a
membrane, which is why it is called
chemiosmosis.

ATP synthase is the enzyme that

makes ATP by chemiosmosis. It
allows protons to pass through the
membrane using the kinetic energy
to phosphorylate ADP making ATP.
Chemiosmotic hypothesis
 electron transport chain & ATP synthesis
are separate systems - linked by a proton gradient

the enzyme complex

in the IMM that carries out
ATP synthesis
also called
• Complex V
• mit. ATPase
• F1F0ATPase
 ATP Synthase is composed of
a proton-conducting unit and a catalytic unit

Open Loose

Tight

ATP Synthase Nucleotide-Binding Sites Are Not Equivalent

 Binding-change mechanism for ATP synthase

1) ADP and Pi binding

2) ATP synthesis
3) ATP release

ATP can be synthesized and released by rotation of g subunit

 Binding-change mechanism for ATP synthase

interactions
1) ADP and Pi binding
with the g subunit
2) ATP synthesis
make the 3b subunits
3) ATP release
inequivalent

ATP can be synthesized and released by rotation of g subunit

Proton Flow Around the c Ring Powers ATP Synthesis
+ + + + Positive charge + + + + cytoplasmic side of IMM
The a subunit has 2 hydrophilic
half-channels that do not span
the membrane.
(protons cannot cross)

Each half-channel
interacts with one c subunit
- - - - - - Negative charge - - - - - - matrix side of IMM

c subunit – 2 alpha helices that span the membrane

Asp61 in the middle can take a proton.
Then this subunit c with bound proton rotates
until it is in a proton-poor environment then proton is released.

Each proton enters

the cytoplasmic half-channel,
follows a complete rotation of the c ring, Proton flow
and exits through the other depends on
half-channel into the matrix. a and c subunits of F0
 Many Shuttles Allow Movement
Across the Mitochondrial Membranes

e-s from cytoplasmic NADH

enter mitochondria via shuttles

Glycolysis generates NADH in the cytoplasm through

the oxidation of glyceraldehyde 3-phosphate
Problem:
NAD+ must be regenerated for glycolysis to continue.

Solution:
e-s from NADH, rather than NADH itself, are carried
across the otherwise impermeable IMM. HOW?
Glycerol 3-phosphate shuttle

membrane-bound
 e-s from NADH in the
Malate-aspartate shuttle cytoplasm are transferred
to oxaloacetate,
forming malate, which
traverses the IMM and
is then reoxidized by
NAD+ in the matrix to
form NADH.
Oxaloacetate is
transaminated
by glutamate to
a-ketoglutarate and
forms aspartate and then
goes back to cytoplasm.
The major function of oxidative phosphorylation is to generate ATP from ADP.

However, ATP and ADP do not diffuse freely across the IMM.

How are these highly charged molecules

moved across the inner membrane into the cytosol?
by ATP-ADP translocase: the flows of ADP and ATP are coupled.

ADP enters the mitochondrial matrix only if ATP exists. and vice versa.
 Mitochondrial Transporters for Metabolites
Have a Common Tripartite Motif

Mitochondrial transporters carry specific ions and metabolites

Similar structure of three tandem repeats of a 100 amino acid module
 The COMPLETE oxidation
of glucose yields
about 30 molecules of ATP
The chemiosmotic model: oxidation and phosphorylation become coupled
(absence of one inhibits the other)

IMM(inner mitochondrial membrane) is impermeable to H+

H+ can reenter the matrix only through proton-specific channels (Fo).
The proton-motive force that drives H+ back into the matrix provides the E for ATP synthesis,
catalyzed by the F1 complex associated with Fo.
 Electrons do not flow
The rate of OP* is determined by the ATP demand
from fuel molecules to O2
How is the rate of the electron-transport chain controlled? unless
ATP needs to be synthesized.
(ATP is required)
[ADP] — OP* rate —

respiratory control
Adding ADP increases
rate of O2 consumption by mitochondria.

ATP synthesis
controls the flow of e- s
from NADH/FADH2 to O2

The availability of NAD+ and FAD

controls the rate of
the citric acid cycle
 ability to uncouple OP* from ATP synthesis = ability to generate heat
to maintain body temperature
˜ in hibernating animals,
˜ in some newborn animals (including human beings),
˜ in mammals adapted to cold.
uncoupling protein (UCP), or
thermogenin
Temperature drop activates hormones
mediates flow of Protons from
that lead to formation of fatty acids
cytosol to matrix by
from triglycerides and FFAs
short-circuiting
Activate UCP-1.
the mitochondrial proton
battery.
Energy is released as HEAT!

Brown adipose tissue:

very rich in mitochondria
is specialized for this
Uncoupling protein (UCP-1) generates HEAT process of nonshivering
by permitting proton flow into the mitochondria thermogenesis.
without the synthesis of ATP
Many potent poisons exert their effect
by inhibiting Oxidative Phosphorylation.

Some compounds inhibiting

the Electron Transport Chain
are shown on the left.