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ELECTRON TRANSPORT CHAIN AND OXIDATIVE PHOSPHORYLATION.ppt
1. THEME # 7: CELL
RESPIRATION
LECTURE No. 13
ELECTRON TRANSPORT
CHAIN AND OXIDATIVE
PHOSPHORILATION
2. 1. Electron transport chain (ETC) and oxidative
phosphorylation. Concept and cell location.
2. Organization of ETC. Complexes of ETC.
3. Proton pumps associated to the electronic
transport.
4. Chemiosmotic theory.
5. ATP synthase. Molecular structure and
mechanism of action.
6. Inhibitors of ETC and oxidative
phosphorylation.
7. Uncouplers.
SUMMARY:
3. BIBLIOGRAPHY
k. Murray, et al. Harper´s
Biochemistry. 27 th. Ed. Mc Graw
Hill. 2006.
Lehninger. Principle of Biochemistry,
by David L. Nelson and Michael M.
Cox.5th Ed. 2008.
Stryer. Biochemistry, by Jeremy M.
Berg, John L. Tymoczko and Lubert
Stryer. 7th Ed. 2007
4. WAITING ROOM # 1
To your consultation room, was brought by her
friends, a young lady intoxicated with cyanide.
She was working in a chemistry laboratory in
the University.
After physical examination, you diagnosed her
dead.
One of her friends was terrify, and came to
talk with you. She was crying disgracefully and
suddenly she ask you why cyanide killed her
so rapidly.
On the base of your knowledge about cellular
respiration, how can you explain the toxicity of
cyanide?
5. A very important man came to your house to
visit you. Mohamed, your Mathematics lecturer
in your pre-university school. For you, he is a
symbol of a good lecturer, an excellent person
and a wise man. You love him very much.
He is now 40 year-old and he was diagnosed
as a hyperthyroid. He is very worry because
his heat intolerance with heavy sweating,
heart palpitations, and tremulousness. Over the
past 4 months, he has lost weight in spite of a
good appetite.
WAITING ROOM # 2
6. He explained you that he was trying to find
information in internet to help him to
understand his symptomatology.
He knew that his heat intolerance is related
with a problem with his central metabolism,
but he did not understood the explanation in
internet.
He remembered you as one of his best
students and came to your house requesting
your help.
On the base of your knowledge about central
metabolism, how can you explain him , the
cause of his heat intolerance?
WAITING ROOM # 2
7.
8. The discovery in
1948 by Eugene
Kennedy and Albert
Lehninger that
mitochondria are the
site of oxidative
phosphorylation in
eukaryotes marked
the beginning of the
modern phase of
studies in biological
energy transductions.
Albert L. Lehninger,
1917–1986
9.
10. 1) Oxidation of fuels (fat, carbohydrate,
and protein).
2) Conversion of energy from fuel
oxidation into the high-energy phosphate
bonds of ATP.
3) Utilization of ATP phosphate bond
energy to drive energy-requiring
processes.
THESE ENERGY TRANSFORMATIONS
CAN BE DIVIDED INTO THREE
PRINCIPAL PHASES:
13. In cells, the chemical bond energy of fuels is
transformed into the physiologic responses
necessary for life.
To generate ATP through cellular respiration,
fuels are degraded by oxidative reactions that
transfer most of their chemical bond energy to
NAD+ and FAD to generate the reduced form of
these coenzymes, NADH and FADH2.
When NADH and FADH2 are oxidized by O2 in
the electron transport chain, the energy is used
to regenerate ATP in the process of oxidative
phosphorylation.
THE ATP-ADP CYCLE
15. CONCEPT ABOUT ELECTRON
TRANSPORT CHAIN
Electron transport chain is a serie
of oxidation-reduction reactions,
throught which the electrons from
NADH and FADH2 are gradually
transported to the final acceptor:
molecular oxygen (O2), which
become oxidized forming H2O.
16. When NADH and FADH2 are oxidized by O2 in
the electron transport chain, some energy
released is used to regenerate ATP in the
process of oxidative phosphorylation and some
is released as heat.
We need to breathe principally because our
cells require O2 to generate adequate amounts
of ATP from the oxidation of fuels to CO2 .
Cellular respiration uses over
90% of the O2 inhaled for us.
VERY IMPORTANT TO KNOW:
17. Oxidation-reduction reactions always
involve a pair of chemicals: an electron
donor, which is oxidized, and an electron
acceptor, which is reduced.
During our metabolism, our fuels donate
electrons, and are oxidized, while, NAD+
and FAD accept electrons, and are
reduced.
OXIDATION-REDUCTION REACTIONS
18. In oxidation reactions, NAD+ accepts two
electrons as an hydride ion to form NADH,
and a proton (H +) is released into the
medium .It is generally used for metabolic
reactions involving oxidation of alcohols and
aldehydes.
In contrast FAD accepts two electrons as
hydrogen atoms, which are donated singly
from separate atoms (e.g., formation of a
double bond or a disulfide).
OXIDATION-REDUCTION REACTIONS
22. The reduction potential is an
electrochemical concept. It is also called
the redox potential or oxidation–reduction
potential and is represented by the letter:
E‘0
In oxidative phosphorylation, the
electron-transfer potential of NADH or
FADH2 is converted into the phosphoryl-
transfer potential of ATP.
THE REDUCTION POTENTIAL
23. A negative reduction potential means that
the oxidized form of a substance has lower
affinity for electrons . A positive reduction
potential means that the oxidized form of a
substance has higher affinity for electrons.
Thus, a strong reducing agent (such as
NADH) is poised to donate electrons and has
a negative reduction potential, whereas a
strong oxidizing agent (such as O2 ) is ready
to accept electrons and has a positive
reduction potential.
MEANING OF THE REDUCTION POTENTIAL
24. In the electron transport chain, or respiratory
chain, the electrons are transferred from NADH
and FADH2 to a chain of electron carriers.
The electrons flow from the more electronegative
components to the more electropositive
components.
All the components of electron transport chain
(ETC) are located in the inner membrane of
mitochondria.
ORGANIZATION OF ELECTRON
TRANSPORT CHAIN
25. ORGANIZATION OF ELECTRON
TRANSPORT CHAIN
There are four distinct multi-protein
complexes:
Complex-I (NADH - CoQ reductase).
Complex II ( Succinate - CoQ reductase).
Complex III (CoQH2 – Cytochrome C
reductase).
Complex IV (Cytochrome C oxidase).
These four complexes are connected by two
mobile carriers:
CoQ
Cytochrome C.
28. Each cytochrome is a protein that contains a bound
heme group similar in structure to the heme group
present in hemoglobin and myoglobin.
Because of differences in the protein component
of the cytochromes and small differences in the
heme structure, each heme has a different
reduction potential.
The following slide shows the general structure of
the three different type of cytochromes found
during the electron transport chain reactions.
CYTOCHROMES
29. Iron protoporphyrin IX is found in b-type
cytochromes and in hemoglobin and
myoglobin. Heme c is covalently bound to
the protein of cytochrome c through
thioether bonds to two Cys residues. Heme
a, found in the a-type cytochromes, has a
long isoprenoid tail attached to one of the
five-membered rings.
PROSTHETIC GROUPS OF
CYTOCHROMES
30. Complexes I, II and III have
also iron sulfur proteins (Fe-S).
In iron-sulfur proteins, first
discovered by Helmut Beinert,
the iron is present not in heme
but in association with inorganic
sulfur atoms or with the sulfur
atoms of Cys residues in the
protein, or both.
IRON-SULFUR PROTEINS
All iron-sulfur proteins participate in one-electron
transfers in which one iron atom of the iron-sulfur
cluster is oxidized or reduced.
Helmut Beinert
31.
32. Ubiquinone (also called coenzyme Q) is a lipid-
soluble benzoquinone with a long isoprenoid side
chain.
Because ubiquinone is both small and
hydrophobic, it is freely diffusible within the
lipid bilayer of the inner mitochondrial
membrane and can shuttle reducing equivalents
between other, less mobile electron carriers in
the membrane.
And because it carries both electrons and
protons, it plays a central role in coupling
electron flow to proton movement.
COENZYME Q
34. CoQ is the unique component of the electron
transport chain that is not protein bound.
The large hydrophobic side chain of 10
isoprenoid units (50 carbons) confers lipid
solubility, and CoQ is able to diffuse through
the lipids of the inner mitochondrial membrane.
When the oxidized Quinone form accepts a
single electron, it forms a free radical (a
compound with a single electron in an orbital).
COENZYME Q AND GENERATION OF TOXIC
OXYGEN FREE RADICALS
The transfer of single electrons makes it the
major site for generation of toxic oxygen
free radicals in the body.
35. FORMATION OF FREE RADICALS DURING
OXIDATION AND REDUCTION REACTIONS
OF CoQ
36.
37. It is also called NADH-CoQ
reductase, NADH
dehydrogenase complex or
NADH:ubiquinone oxido-
reductase complex. It is tightly
bound to the inner mitochondrial
membrane.
ETC Complex-I
38. It contains a flavoprotein consisting of FMN as
prosthetic group and an iron-sulphur protein
(Fe-S).
NADH is the donor of electrons, FMN accepts
them and gets reduced to FMNH2 Two electrons
and one hydrogen ion are transferred from
NADH to the flavin prosthetic group of the
enzyme.
NADH + H+ + FMN → FMNH2 + NAD+
The electrons from FMNH2 are then
transferred to Fe-S center and subsequently
transferred to coenzyme Q (ubiquinone or CoQ).
ETC Complex-I
39.
40. The electrons from FADH2 enter the ETC at the
level of coenzyme Q. This step does not liberate
enough energy to act as proton pump. In other
words, substrates oxidized by FAD-linked
enzymes bypass complex-I.
The three major enzyme systems that transfer
their electrons directly to ubiquinone from the
FAD prosthetic group are:
COMPLEX II OR SUCCINATE-CoQ-REDUCTASE
Succinate dehydrogenase,
Fatty acyl CoA
dehydrogenase
Mitochondrial glycerol
phosphate dehydrogenase.
42. This is a cluster of iron-sulfur center proteins, cytochrome
b and cytochrome C1, both containing heme prosthetic
group.
REACTION SEQUENCE OF COMPLEX III
( CoQH2 – Cytochrome C reductase)
43. It contains different proteins, including cytochrome a and
cytochrome a3. The Complex IV is tightly bound to the
mitochondrial membrane.
REACTION SEQUENCE OF COMPLEX IV
(Cytochrome C oxidase)
44.
45.
46.
47. Oxidative phosphorylation is the process
by which, the energy released by the
oxidation reactions of the electron
transport chain is used to synthesize
adenosine triphosphate (ATP) by the
action of complex V (ATP synthase, or
also called F0F1ATPase).
CONCEPT OF
OXIDATIVE PHOSPHORYLATION (OP)
48. Oxidative phosphorylation is the
culmination of energy-yielding
metabolism in aerobic organisms.
All oxidative steps in the degradation
of carbohydrates, fats and amino
acids, converge at this final stage of
cellular respiration, in which the
energy of oxidation drives the
synthesis of ATP.
OXIDATIVE PHOSPHORYLATION
49.
50.
51. Peter Mitchell was
awarded with the
Nobel Price in
Chemistry in 1978 for
his contribution to
the underestanding of
biological energy
transfer during ATP
synthesis, through
the formulation of
the Chemiosmotic
theory.
52. POSTULATES OF CHEMIOSMOTIC
THEORY
The inner mitochondrial membrane is
impermeable for hydrogen protons
(H+)
The complexes I , III and IV of the
electron transport chain , at the
same time that they transfer
electrons to the final aceptor, O2 ,
they pump H+ from the mitochondrial
matrix to the intermembrane space.
53. As consequence of this pumping function is
formed an electrochemical gradient in both sides
of the inner mitocondrial membrane.
The intermembrane side of the inner mitocondrial
membrane remain positivelly charged, while, the
mitocondrial matrix side is becoming negativelly
charged. Also the pH will be lower in the
intermembrane space (because high H+
concentration ) in relation with the pH inside
mitochondrila matrix (because lower H+
concentration ).This elcectrochemical gradient is
also called, proton motive force.
POSTULATES OF CHEMIOSMOTIC
THEORY
55. The proton motive force the re-entry of the
Hydrogen protons to the mitochondrial matrix,
using the H+ proton channel of F0 subunit of
complex V or ATP synthase.
ATP is then synthesized by the catalytic
subunit of ATP synthase complex (F1 subunit ),
from ADP and Pi using the energy released
during the disipation of the electrochemical
gradient between both sides of the inner
mitocondrial membrane.
POSTULATES OF CHEMIOSMOTIC
THEORY
56. POSTULATES OF CHEMIOSMOTIC
THEORY
Electron transport chain and
oxidative phosphorylation are
coupled processes, by the formation
of the electrochemical gradient
between both sides of the inner
mitochondrail membrane.
57. ETC and OP are coupled by the formation of an
electrochemical gradient between both sides of the inner
mitochondrial membrane(CHEMIOSMOTIC MODEL)
58. The H+ pumping activity of complexes I, III and IV of
ETC, generates an electrochemical gradient. Complex V
(ATP synthase) catalyze ATP synthesis using the energy
released during disipation of the gradient.
59. ATP synthase (F0F1 ATPase),, is a multisubunit
enzyme containing a transmembrane portion (F0) and
a stalk and headpiece (F1 ) that project into the
matrix .
The 12 c subunits of the F0 subunit form a rotor
that is attached to a central asymmetric shaft
composed of the Ɛ and ɣ subunits. The headpiece
(F1 subunit) is composed of three αβ subunit pairs.
Each β subunit contains a catalytic site for ATP
synthesis. The headpiece is held stationary by a δ
subunit attached to a long b subunits connected to
subunit a in the membrane.
ATP SYNTHASE STRUCTURE
60.
61. F 1 , the first
factor recognized
as essential for
oxidative
phosphorylation,
was identified and
purified by Efraim
Racker and his
colleagues in the
early 1960s.
62. The
crystallographic
determination of
the F 1 structure
by John E. Walker
and colleagues
revealed structural
details very helpful
in explaining the
catalytic
mechanism
of the enzyme.
63. The influx of protons through the proton channel
turns the rotor. The proton channel is formed by
the c subunits on one side and the a subunit on
the other side.
Although continuous, it has two offset portions,
one portion directly open to the intermembrane
space and one portion directly open to the
matrix. In the current model, each c subunit
contains a glutamyl carboxyl group that extends
into the proton channel. As this carboxyl group
accepts a proton from the intermembrane space,
the c subunit rotates into the hydrophobic lipid
membrane.
MECHANISM OF ACTION OF ATP SYNTHASE
64. The rotation exposes a different proton-
containing c subunit to the portion of the channel
directly open to the matrix side.
Because the matrix has a lower proton
concentration, the glutamyl carboxylic acid group
releases a proton into the matrix portion of the
channel.
Rotation is completed by an attraction between
the negatively charged glutamyl residue in the c
subunit and a positively charged arginyl group
on the a subunit.
MECHANISM OF ACTION OF ATP SYNTHASE
65. Each proton enters
the cytoplasmic
half-channel,
follows a complete
rotation of the c
ring, and exits
through the
other half-channel
into the matrix.
PROTON PATH THROUGH THE MEMBRANE
67. According to the binding change mechanism, as the
asymmetric shaft rotates to a new position, it
forms different binding associations with the αβ
subunits.
The new position of the shaft alters the
conformation of one β subunit so that it releases a
molecule of ATP and another subunit
spontaneously catalyzes synthesis of ATP from
inorganic phosphate, one proton, and ADP.
Thus, energy from the electrochemical gradient is
used to change the conformation of the ATP
synthase subunits so that the newly synthesized
ATP is released.
MECHANISM OF ACTION OF ATP SYNTHASE
70. THERMOGENESIS
Thermogenesis refers to energy
expended for the purpose of
generating heat in addition to that
expended for ATP production.
To maintain our body at 37ºC,
despite changes in environmental
temperature, it is necessary to
regulate fuel oxidation and its
efficiency (as well as heat
dissipation).
71. In shivering thermogenesis, we respond to
sudden cold with asynchronous muscle
contractions (shivers) that increase ATP
utilization and, therefore, fuel oxidation
and the release of energy as heat.
In nonshivering thermogenesis (adaptive
thermogenesis), the efficiency of
converting energy from fuel oxidation into
ATP is decreased. More fuel needs to be
oxidized to maintain constant ATP levels
and, thus, more heat is generated.
THERMOGENESIS
72. Most of the newly synthesized ATP that is
released into the mitochondrial matrix must be
transported out of the mitochondria, where it is
used for energy-requiring processes such as
active ion transport, muscle contraction, or
biosynthetic reactions.
Likewise, ADP, phosphate, pyruvate, and other
metabolites must be transported into the
matrix. This requires transport mechanism of
compounds through mitochondrial membranes.
TRANSPORT THROUGH INNER AND OUTER
MITOCHONDRIAL MEMBRANES
75. SHUTTLE SYSTEMS INDIRECTLY
CONVEY CYTOSOLIC NADH INTO
MITOCHONDRIA FOR OXIDATION
The NADH dehydrogenase of the inner
mitochondrial membrane of animal cells can
accept electrons only from NADH in the matrix.
Given that the inner membrane is not permeable
to NADH, how can the NADH generated by
glycolysis in the cytosol be reoxidized to NAD+ by
O2 via the respiratory chain?
Special shuttle systems carry reducing
equivalents from cytosolic NADH into
mitochondria by an indirect route.
76. NADH and FADH2 from the Krebs cycle and β-
oxidation reactions in mitochondrial matrix are
directly reoxidized by complexes I and II
respectively.
77.
78. Malate-aspartate shuttle is the most active NADH
shuttle, which functions in liver, kidney, and heart
mitochondria.
The reducing equivalents of cytosolic NADH are first
transferred to cytosolic oxaloacetate to yield malate,
catalyzed by cytosolic malate dehydrogenase. The malate
thus formed passes through the inner membrane via the
malate–α-ketoglutarate transporter.
Within the matrix the reducing equivalents are passed to
NAD+ by the action of matrix malate dehydrogenase,
forming NADH; this NADH can pass electrons directly to
the respiratory chain. About 2.5 molecules of ATP are
generated as this pair of electrons passes to O2.
Cytosolic oxaloacetate must be regenerated by
transamination reactions and the activity of membrane
transporters to start another cycle of the shuttle.
MALATE-ASPARTATE SHUTTLE
79.
80. Skeletal muscle and brain use a
different NADH
shuttle, the glycerol 3-phosphate.
It differs from the malate-aspartate
shuttle in that it delivers the reducing
equivalents from NADH to ubiquinone
and thus into Complex II, not Complex I,
providing only enough energy to
synthesize 1.5 ATP molecules per pair of
electrons.
GLYCEROL 3-PHOSPHATE
SHUTTLE
85. EFFECT OF ELECTRON CHAIN
INHIBITORS
After bind to some component of any complex,
electron chain inhibitors:
Stop substrate oxidation.
Stop the electron transport.
Stop H+ pumping.
No electrochemical gradient is formed.
Stop Oxygen consumption.
Stop H2O production.
Stop ATP synthesis.
87. Bind to F1 or Fo fractions of ATP
synthase.
Stop ATP synthesis
Proton motive force gets a
maximum level
As consequence, electron transport
chain stop working.
EFFECT OF OXIDATION
PHOSPHORYLATION
INHIBITORS
88.
89. ETCand OP are coupled by the formation of an
electrochemical gradient between both sides of the inner
mitochondrial membrane(CHEMIOSMOTIC MODEL)
90. The electrochemical gradient is formed beause
complexes I, III and IV, pump hydrogen
protons from mitocondrial matrix to the
intermembrane space. These H+ can not enter
back, because, the inner mitocondrial
membrane is not permeable for them .
REMEMBERING:
What would happen if hydrogen protons
pumped by ETC complexes can freely cross or,
are transported back to the mitochondrial
matrix?
TO THINK:
91.
92. When protons leak back into the matrix without
going through the ATP synthase pore, they
dissipate the electrochemical gradient across the
membrane without generating ATP.
This phenomenon is called “uncoupling” oxidative
phosphorylation.
It occurs with chemical transporters, known as
uncouplers, and it occurs physiologically with
uncoupling proteins that form proton
conductance channels through the membrane.
UNCOUPLING ATP SYNTHESIS FROM
ELECTRON TRANSPORT
93. Chemical uncouplers, also known as proton ionophores, are
lipid-soluble compounds that rapidly transport protons
from the cytosolic to the matrix side across the inner
mitochondrial membrane.
Because the proton concentration is higher in the
intermembrane space than in the matrix, uncouplers pick
up protons from the intermembrane space. Their lipid
solubility enables them to diffuse through the inner
mitochondrial membrane while carrying protons and release
these protons on the matrix side.
The rapid influx of protons dissipates the electrochemical
potential gradient; therefore, the mitochondria are unable
to synthesize ATP.
CHEMICAL UNCOUPLERS OF
OXIDATIVE PHOSPHORYLATION
94.
95. UNCOUPLING PROTEINS AND
THERMOGENESIS
Uncoupling proteins (UCPs) form channels through
the inner mitochondrial membrane that are able to
conduct protons from the intermembrane space to
the matrix, thereby short-circuiting ATP synthase.
UCP1 (thermogenin) is associated with heat
production in brown adipose tissue. The major
function of brown adipose tissue is nonshivering
thermogenesis, whereas the major function of
white adipose tissue is the storage of
triacylglycerols in white lipid droplets. The brown
color arises from the large number of
mitochondria that participate.
97. BROWN FAT IS VERY IMPORTANT IN
HIBERNATING ANIMALS
98.
99. Uncouple Electron Transport
and Oxidative Phosphorylation.
Destroy Proton motive force.
Stop ATP synthesis.
Activate ETC reactions.
Increase the lost of energy as heat.
EFFECT OF UNCOUPLERS
100.
101. WAITING ROOM # 1
To your consultation room, was brought by her
friends, a young lady intoxicated with cyanide.
She was working in a chemistry laboratory in
the University.
After physical examination you diagnosed her
dead.
One of her friends was terrify, and came to
talk with you. She was crying disgracefully and
suddenly she ask you why cyanide killed her
so rapidly.
On the base of your knowledge about cellular
respiration, how can you explain the toxicity of
cyanide?
102. A very important man came to your house to
visit you. Mohamed, your Mathematics lecturer
in your pre-university school. For you, he is a
symbol of a good lecturer, an excellent person
and a wise man. You love him very much.
He is now 40 year-old and he was diagnostic
as a hyperthyroid. He is very worry because
his heat intolerance with heavy sweating,
heart palpitations, and tremulousness. Over the
past 4 months, he has lost weight in spite of a
good appetite.
WAITING ROOM # 2
103. He explained you that he was trying to find
information in internet to help him to
understand his symptomatology.
He knew that his heat intolerance is related
with a problem with his central metabolism,
but he did not understand the explanation in
internet.
He remembered you, as one of his best
students and came to your house requesting
your help.
On the base of your knowledge about central
metabolism, how can you explain him , the
cause of his heat intolerance?
WAITING ROOM # 2
104. Thyroid hormones increase the synthesis and
as consequence the level of UCP2 and UCP3 in
the inner mitochondrial membrane.
UCP2 and UCP3 (termogenines) are channels
that propitiate the reenter of hydrogen
protons to mitochondrial matrix.
Electrochemical gradient is destroy. ATP
production decreases and the ETC is
enhanced.
As a consequence of the increased rate of the
electron transport chain, most released energy
is lost as heat, this is because
hyperthyroidism results in increased heat
intolerance.
WAITING ROOM # 2
