This document summarizes oxidative phosphorylation and the structures and mechanisms of the electron transport chain complexes involved. It describes how complexes I, III, and IV of the electron transport chain use the spontaneous transfer of electrons to pump protons out of the mitochondrial matrix and into the intermembrane space, building up an electrochemical proton gradient. This proton gradient is then used by ATP synthase to generate ATP from ADP and inorganic phosphate, conserving energy from the electron transfers.
Electron Transport Chain and oxidative phosphorylation @meetpadhiyarmeetpadhiyar88
A story of electron transport to the ATP synthase complex by 4 complexes and oxidative phosphorylation.
Present at College of basic science and Humanities, Dantiwada.
The electron transport chain is comprised of a series of enzymatic reactions within the inner membrane of the mitochondria, which are cell organelles that release and store energy for all physiological needs.
As electrons are passed through the chain by a series of oxidation-reduction reactions, energy is released, creating a gradient of hydrogen ions, or protons, across the membrane. The proton gradient provides energy to make ATP, which is used in oxidative phosphorylation.
Electron Transport Chain and oxidative phosphorylation @meetpadhiyarmeetpadhiyar88
A story of electron transport to the ATP synthase complex by 4 complexes and oxidative phosphorylation.
Present at College of basic science and Humanities, Dantiwada.
The electron transport chain is comprised of a series of enzymatic reactions within the inner membrane of the mitochondria, which are cell organelles that release and store energy for all physiological needs.
As electrons are passed through the chain by a series of oxidation-reduction reactions, energy is released, creating a gradient of hydrogen ions, or protons, across the membrane. The proton gradient provides energy to make ATP, which is used in oxidative phosphorylation.
_ETC and Oxidative phosphorylation.pptxAlisha Shaikh
The electrons generated from different metabolic pathways of the cell are channeled to the electron transport chain by electron acceptors. The electrons then contributes for the synthesis of ATP.
Electron Transport Chain by Salman SaeedSalman Saeed
Electron Transport Chain lecture for Biology, Botany, Zoolog
y, and Chemistry Students by Salman Saeed lecturer Botany University College of Management and Sciences Khanewal, Pakistan.
About Author: Salman Saeed
Qualification: M.SC (Botany), M. Phil (Biotechnology) from BZU Multan.
M. Ed & B. Ed from GCU Faisalabad, Pakistan.
Electron Transport Chain and oxidative phosphorylationusmanzafar66
substrate level phosphorylation and chemiosmosis
in Eukaryotes and in prokaryotes
in plant and animal
uncoupler oxidative phosphorylation
fat and protein ATP calculation
Describe the major components of the electron transport chain. How w.pdfduttakajal70
Describe the major components of the electron transport chain. How would the following
conditions affect ATP production by the electron transport chain? (7 marks)
Abundance of NADH and O2
Cyanide added
Lack of O2
Solution
There are five major components in electron system. They are:
Complex I:
It is a large, multisubunit complex with about 40 polypeptide chains passes electron from NADH
to CoQ. It contains one molecule of FMN and 6-7 Fe-S clusters that participate in electron
transport process. During transport of each pair of electrons from NADH to coenzyme Q,
complex I pumps 4 protons across the inner mitochondrial membrane.
Complex II:
Succinate dehrogenase, an inner mitochondrial membrane bound enzyme is an integral
component of the succinate-CoQ reductase complex. It converts succinate to fumarate during
krebs cycle. The two electrons released in the conversion of succinate to fumarate are transferred
first to FAD, then to an Fe-S centre, and finally to CoQ. Thus, CoQ draws electrons into the
respiratory chain, not only from NADH but also from succinate. No protons are translocated
across the membrane by this complex.
Complex III:
Complex I and complex II donates two electrons to the complex III and regenerates oxidized
CoQ. Concomitantly, it releases two protons picked up on the cytosolic face into the
intermembrane space generating proton gradient. Within complex III, the released electrons are
transferred to an Fe-S centre and then to 2b-type cytochromes or cytochrome c. finally, the two
electrons are transferred to 2 molecules of the oxidized form of cytochrome c. 2 additional
protons are translocated from the mitochondrial matrix across the inner mitochondrial membrane
for each pair of electrons transferred.
Complex IV:
Cytochrome c transports electrons, one at a time, to the complex IV. Within this complex,
electrons are transferred, first to a pair of copper ions, then to cytochrome a, next to a complex of
another copper ion and cyt a3 and finally to O2, the ultimate electron acceptor, yielding H2O.
ATP synthase:
The use of proton motive force for synthesis is catalyzed by ATP synthase. The multi protein
ATP synthase for F0-F1 complex or complex V catalyzes ATP synthesis as protons flow back
through the inner membrane down the electrochemical proton gradient.
2. Abundance of NADH and O2:
ETC occurs more to produce more ATP.
Cyanide added:
Cyanide binds with cytochrome oxidase complex and inhibits the terminal transfer of electrons
to oxygen. Cyanide react with the oxidized form of cytochrome.
Lack of O2:
ETC will not take place in the absence of oxygen..
1 The proton motive force PMF generated by the electron t.pdfADARSHMANISH
1. The proton motive force (PMF) generated by the electron transport chain is associated with
which features?
Options:
A.It has a charge component such that it is more positive in the Mitochondrial inner membrane
space than in the matrix
B.The pH of the mitochondrial inner membrane space is higher than in the matrix
C.Protons are pumped across the inner mitochondrial membrane by Complexes I-IV.
D.It has a concentration component such that the [H+] is lower in the mitochondrial matrix than in
the inner membrane space.
E.The Phosphate transporter dissipates only the pH component of the PMF
F.Exchange of ATP and ADP through a transporter dissipates both the pH and the charge
component of the PMF.
2.Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory
chain. For example, when NADH and O2O2 are abundant, the steadystate degree of reduction of
the carriers decreases as electrons pass from the substrate to O2.O2. When electron transfer is
blocked, the carriers before the block become more reduced and those beyond the block become
more oxidized.
Match the state of oxidation for ubiquinone (Q) and cytochromes b, c , c, a, and a with each
1 3
mitochondrial condition.
A.Abundant NADH and O2, but cynide added Match the following to the correct letter: 1. All
carriers remain oxidized., 2. Early carriers undergo reduction, and later carriers
B.ABUsndant NADH , but 02 exhausted. remain oxidized , 3. Early carriers remain oxideized ,
later carriers undergo reductio
C.Abundant O2, but NADH exhausted 4.All carriers undergo reduction.
D. ABUsndant NADH and O2
3.ATP-Synthase is powered by the PMF that is generated by the transfer of electrons from NADH
or FADH2 to reduce oxygen. Which of the following are INCORRECT concerning ATP synthase
and its mechanism?
Options:
A.The catalysis of the -subunits is allosterically regulated by the -subunits.
B.The formation of mitochondrial cristae by ATP-synthase dimerization facilitates rapid exchange
of substrates between the two enzymes.
C.ATP synthase catalyzes a bimolecular reaction in which the transition state consists of a high
energy conformation of the -subunit in the presence of ATP.
D.The number of c-subunits in the F0 system varies across species resulting in differing ratios of
ATP produced per Oxygen atom reduced.
E.If the F0 and F1 were separated, F1 would promote hydrolysis of ATP.
1 The proton motive force PMF generated by the electron t.pdfamir421526
1. The proton motive force (PMF) generated by the electron transport chain is associated with
which features?
Options:
A.It has a charge component such that it is more positive in the Mitochondrial inner membrane
space than in the matrix
B.The pH of the mitochondrial inner membrane space is higher than in the matrix
C.Protons are pumped across the inner mitochondrial membrane by Complexes I-IV.
D.It has a concentration component such that the [H+] is lower in the mitochondrial matrix than in
the inner membrane space.
E.The Phosphate transporter dissipates only the pH component of the PMF
F.Exchange of ATP and ADP through a transporter dissipates both the pH and the charge
component of the PMF.
2.Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory
chain. For example, when NADH and O2O2 are abundant, the steadystate degree of reduction of
the carriers decreases as electrons pass from the substrate to O2.O2. When electron transfer is
blocked, the carriers before the block become more reduced and those beyond the block become
more oxidized.
Match the state of oxidation for ubiquinone (Q) and cytochromes b, c , c, a, and a with each
1 3
mitochondrial condition.
A.Abundant NADH and O2, but cynide added Match the following to the correct letter: 1. All
carriers remain oxidized., 2. Early carriers undergo reduction, and later carriers
B.ABUsndant NADH , but 02 exhausted. remain oxidized , 3. Early carriers remain oxideized ,
later carriers undergo reductio
C.Abundant O2, but NADH exhausted 4.All carriers undergo reduction.
D. ABUsndant NADH and O2
3.ATP-Synthase is powered by the PMF that is generated by the transfer of electrons from NADH
or FADH2 to reduce oxygen. Which of the following are INCORRECT concerning ATP synthase
and its mechanism?
Options:
A.The catalysis of the -subunits is allosterically regulated by the -subunits.
B.The formation of mitochondrial cristae by ATP-synthase dimerization facilitates rapid exchange
of substrates between the two enzymes.
C.ATP synthase catalyzes a bimolecular reaction in which the transition state consists of a high
energy conformation of the -subunit in the presence of ATP.
D.The number of c-subunits in the F0 system varies across species resulting in differing ratios of
ATP produced per Oxygen atom reduced.
E.If the F0 and F1 were separated, F1 would promote hydrolysis of ATP.
_ETC and Oxidative phosphorylation.pptxAlisha Shaikh
The electrons generated from different metabolic pathways of the cell are channeled to the electron transport chain by electron acceptors. The electrons then contributes for the synthesis of ATP.
Electron Transport Chain by Salman SaeedSalman Saeed
Electron Transport Chain lecture for Biology, Botany, Zoolog
y, and Chemistry Students by Salman Saeed lecturer Botany University College of Management and Sciences Khanewal, Pakistan.
About Author: Salman Saeed
Qualification: M.SC (Botany), M. Phil (Biotechnology) from BZU Multan.
M. Ed & B. Ed from GCU Faisalabad, Pakistan.
Electron Transport Chain and oxidative phosphorylationusmanzafar66
substrate level phosphorylation and chemiosmosis
in Eukaryotes and in prokaryotes
in plant and animal
uncoupler oxidative phosphorylation
fat and protein ATP calculation
Describe the major components of the electron transport chain. How w.pdfduttakajal70
Describe the major components of the electron transport chain. How would the following
conditions affect ATP production by the electron transport chain? (7 marks)
Abundance of NADH and O2
Cyanide added
Lack of O2
Solution
There are five major components in electron system. They are:
Complex I:
It is a large, multisubunit complex with about 40 polypeptide chains passes electron from NADH
to CoQ. It contains one molecule of FMN and 6-7 Fe-S clusters that participate in electron
transport process. During transport of each pair of electrons from NADH to coenzyme Q,
complex I pumps 4 protons across the inner mitochondrial membrane.
Complex II:
Succinate dehrogenase, an inner mitochondrial membrane bound enzyme is an integral
component of the succinate-CoQ reductase complex. It converts succinate to fumarate during
krebs cycle. The two electrons released in the conversion of succinate to fumarate are transferred
first to FAD, then to an Fe-S centre, and finally to CoQ. Thus, CoQ draws electrons into the
respiratory chain, not only from NADH but also from succinate. No protons are translocated
across the membrane by this complex.
Complex III:
Complex I and complex II donates two electrons to the complex III and regenerates oxidized
CoQ. Concomitantly, it releases two protons picked up on the cytosolic face into the
intermembrane space generating proton gradient. Within complex III, the released electrons are
transferred to an Fe-S centre and then to 2b-type cytochromes or cytochrome c. finally, the two
electrons are transferred to 2 molecules of the oxidized form of cytochrome c. 2 additional
protons are translocated from the mitochondrial matrix across the inner mitochondrial membrane
for each pair of electrons transferred.
Complex IV:
Cytochrome c transports electrons, one at a time, to the complex IV. Within this complex,
electrons are transferred, first to a pair of copper ions, then to cytochrome a, next to a complex of
another copper ion and cyt a3 and finally to O2, the ultimate electron acceptor, yielding H2O.
ATP synthase:
The use of proton motive force for synthesis is catalyzed by ATP synthase. The multi protein
ATP synthase for F0-F1 complex or complex V catalyzes ATP synthesis as protons flow back
through the inner membrane down the electrochemical proton gradient.
2. Abundance of NADH and O2:
ETC occurs more to produce more ATP.
Cyanide added:
Cyanide binds with cytochrome oxidase complex and inhibits the terminal transfer of electrons
to oxygen. Cyanide react with the oxidized form of cytochrome.
Lack of O2:
ETC will not take place in the absence of oxygen..
1 The proton motive force PMF generated by the electron t.pdfADARSHMANISH
1. The proton motive force (PMF) generated by the electron transport chain is associated with
which features?
Options:
A.It has a charge component such that it is more positive in the Mitochondrial inner membrane
space than in the matrix
B.The pH of the mitochondrial inner membrane space is higher than in the matrix
C.Protons are pumped across the inner mitochondrial membrane by Complexes I-IV.
D.It has a concentration component such that the [H+] is lower in the mitochondrial matrix than in
the inner membrane space.
E.The Phosphate transporter dissipates only the pH component of the PMF
F.Exchange of ATP and ADP through a transporter dissipates both the pH and the charge
component of the PMF.
2.Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory
chain. For example, when NADH and O2O2 are abundant, the steadystate degree of reduction of
the carriers decreases as electrons pass from the substrate to O2.O2. When electron transfer is
blocked, the carriers before the block become more reduced and those beyond the block become
more oxidized.
Match the state of oxidation for ubiquinone (Q) and cytochromes b, c , c, a, and a with each
1 3
mitochondrial condition.
A.Abundant NADH and O2, but cynide added Match the following to the correct letter: 1. All
carriers remain oxidized., 2. Early carriers undergo reduction, and later carriers
B.ABUsndant NADH , but 02 exhausted. remain oxidized , 3. Early carriers remain oxideized ,
later carriers undergo reductio
C.Abundant O2, but NADH exhausted 4.All carriers undergo reduction.
D. ABUsndant NADH and O2
3.ATP-Synthase is powered by the PMF that is generated by the transfer of electrons from NADH
or FADH2 to reduce oxygen. Which of the following are INCORRECT concerning ATP synthase
and its mechanism?
Options:
A.The catalysis of the -subunits is allosterically regulated by the -subunits.
B.The formation of mitochondrial cristae by ATP-synthase dimerization facilitates rapid exchange
of substrates between the two enzymes.
C.ATP synthase catalyzes a bimolecular reaction in which the transition state consists of a high
energy conformation of the -subunit in the presence of ATP.
D.The number of c-subunits in the F0 system varies across species resulting in differing ratios of
ATP produced per Oxygen atom reduced.
E.If the F0 and F1 were separated, F1 would promote hydrolysis of ATP.
1 The proton motive force PMF generated by the electron t.pdfamir421526
1. The proton motive force (PMF) generated by the electron transport chain is associated with
which features?
Options:
A.It has a charge component such that it is more positive in the Mitochondrial inner membrane
space than in the matrix
B.The pH of the mitochondrial inner membrane space is higher than in the matrix
C.Protons are pumped across the inner mitochondrial membrane by Complexes I-IV.
D.It has a concentration component such that the [H+] is lower in the mitochondrial matrix than in
the inner membrane space.
E.The Phosphate transporter dissipates only the pH component of the PMF
F.Exchange of ATP and ADP through a transporter dissipates both the pH and the charge
component of the PMF.
2.Mitochondrial conditions determine the degree of reduction of each carrier in the respiratory
chain. For example, when NADH and O2O2 are abundant, the steadystate degree of reduction of
the carriers decreases as electrons pass from the substrate to O2.O2. When electron transfer is
blocked, the carriers before the block become more reduced and those beyond the block become
more oxidized.
Match the state of oxidation for ubiquinone (Q) and cytochromes b, c , c, a, and a with each
1 3
mitochondrial condition.
A.Abundant NADH and O2, but cynide added Match the following to the correct letter: 1. All
carriers remain oxidized., 2. Early carriers undergo reduction, and later carriers
B.ABUsndant NADH , but 02 exhausted. remain oxidized , 3. Early carriers remain oxideized ,
later carriers undergo reductio
C.Abundant O2, but NADH exhausted 4.All carriers undergo reduction.
D. ABUsndant NADH and O2
3.ATP-Synthase is powered by the PMF that is generated by the transfer of electrons from NADH
or FADH2 to reduce oxygen. Which of the following are INCORRECT concerning ATP synthase
and its mechanism?
Options:
A.The catalysis of the -subunits is allosterically regulated by the -subunits.
B.The formation of mitochondrial cristae by ATP-synthase dimerization facilitates rapid exchange
of substrates between the two enzymes.
C.ATP synthase catalyzes a bimolecular reaction in which the transition state consists of a high
energy conformation of the -subunit in the presence of ATP.
D.The number of c-subunits in the F0 system varies across species resulting in differing ratios of
ATP produced per Oxygen atom reduced.
E.If the F0 and F1 were separated, F1 would promote hydrolysis of ATP.
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2. Respiration-linked H+ pumping out of the matrix conserves
some of the free energy of spontaneous e- transfers as
potential energy of an electrochemical H+ gradient.
matrix
inner
membrane
outer
membrane
inter-
membrane
space
mitochondrion
cristae
Conventional view of
mitochondrial
structure is at right.
Respiratory chain is
in cristae of the inner
membrane.
Spontaneous electron
transfer through
respiratory chain complexes I, III & IV is coupled to
H+ ejection from the matrix to the intermembrane space.
Because the outer membrane contains large channels,
these protons may equilibrate with the cytosol.
3. 3-D reconstructions based on electron micrographs of
isolated mitochondria taken with a large depth of field, at
different tilt angles have indicated that the infoldings of
the inner mitochondrial membrane are variable in shape
and are connected to the periphery and to each other by
narrow tubular regions.
matrix
inner
membrane
outer
membrane
inter-
membrane
space
mitochondrion
cristae
4. between the lumen of cristae & the intermembrane space.
There is evidence also that protons pumped out of the
matrix spread along the anionic membrane surface and
only slowly equilibrate with the surrounding bulk phase,
maximizing the effective H+ gradient.
Electron micrograph by Dr. C.
Mannella of a Neurospora
mitochondrion in a frozen sample
in the absence of fixatives or
stains that might alter appearance
of internal structures.
Wadsworth Center website.
Tubular cristae connect to the
inner membrane via narrow
passageways that may limit
the rate of H+ equilibration
5. A total of 10 H+ are ejected from the mitochondrial matrix
per 2 e- transferred from NADH to oxygen via the
respiratory chain.
The H+/e- ratio for each respiratory chain complex will be
discussed separately.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
Spontaneous
electron flow
through each
of complexes
I, III, & IV is
coupled to H+
ejection from
the matrix.
6. Complex I (NADH Dehydrogenase) transports
4H+ out of the mitochondrial matrix per 2e-
transferred from NADH to CoQ.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
7. Lack of high-resolution structural information for the
membrane domain of complex I has hindered elucidation
of the mechanism of H+ transport.
Direct coupling of transmembrane H+ flux & e- transfer is
unlikely, because the electron-tranferring prosthetic groups,
FMN & Fe-S, are all in the peripheral domain of complex I.
Thus is assumed that protein conformational changes are
involved in H+ transport, as with an ion pump.
inner mitochondrial
membrane
matrix
NAD+
NADH
Complex I
FMN peripheral
domain
membrane domain
FMN
A B
FMN
Peripheral domain of a bacterial Complex I
membrane
domain
PDB 2FUG
N2
8. Complex III (bc1 complex):
H+ transport in complex III involves coenzyme Q (CoQ).
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
9. The “Q cycle” depends on mobility of coenzyme Q within
the lipid bilayer.
There is evidence for one-electron transfers, with an
intermediate semiquinone radical.
O
O
CH3O
CH3
CH3O
(CH2 CH C CH2)nH
CH3
OH
OH
CH3O
CH3
CH3O
(CH2 CH C CH2)nH
CH3
e-
+ 2 H+
coenzyme Q
coenzyme QH2
O-
O
CH3O
CH3
CH3O
(CH2 CH C CH2)nH
CH3
e-
coenzyme Q •-
10. Electrons enter complex III via coenzyme QH2,
which binds at a site on the positive side of the inner
mitochondrial membrane, adjacent to the intermembrane
space.
One version
of Q Cycle:
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
11. The loss of one electron from QH2 would generate a
semiquinone radical, shown here as Q·-, though the
semiquinone might initially retain a proton as QH·.
QH2 gives up 1e-
to the Rieske
iron-sulfur center,
Fe-S.
Fe-S is reoxidized
by transfer of the
e- to cyt c1, which
passes it out of the
complex to cyt c.
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
12. The fully oxidized CoQ, generated as the 2nd e- is passed
to the b cytochromes, may then dissociate from its binding
site adjacent to the intermembrane space.
Accompanying the two-electron oxidation of bound QH2,
2H+ are released to the intermembrane space.
A 2nd e- is
transferred from
the semiquinone to
cyt bL (heme bL)
which passes it via
cyt bH across the
membrane to
another CoQ
bound at a site on
the matrix side.
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
13. In an alternative mechanism that has been proposed, the
2 e- transfers, from QH2 to Fe-S & cyt bL, may be
essentially simultaneous, eliminating the semiquinone
intermediate.
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
14. It takes 2 cycles for CoQ bound at the site hear the matrix
to be reduced to QH2, as it accepts 2e- from the b hemes,
and 2H+ are extracted from the matrix compartment.
In 2 cycles, 2QH2 enter the pathway & one is regenerated.
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
15. QH2 + 2H+
(matrix) + 2 cyt c (Fe3+)
Q + 4H+
(outside) + 2 cyt c (Fe2+)
Per 2e- transferred through the complex to cyt c, 4H+ are
released to the intermembrane space.
Animation
Overall reaction
catalyzed by
complex III,
including net
inputs & outputs
of the Q cycle :
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
16. While 4H+ appear outside per net 2e- transferred in 2
cycles, only 2H+ are taken up on the matrix side.
In complex IV, there is a similarly uncompensated proton
uptake from the matrix side (4H+ per O2 or 2 per 2e-).
-
2H+
Q Q -
QH2 QH2
cyt bH
cyt bL
Q Q·
Fe-S cyt c1
2H+
matrix
Complex III
e-
intermembrane space
.
cyt c
e-
17. Thus there are 2H+ per 2e- that are effectively transported
by a combination of complexes III & IV.
They are listed with complex III in diagrams depicting
H+/e- stoichiometry.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
18. Complex III:
Half of the homodimeric
structure is shown.
Approximate location of
the membrane bilayer is
indicated.
Not shown are the CoQ
binding sites near heme
bH and near heme bL.
The b hemes are
positioned to provide a
pathway for electrons
across the membrane.
heme bL
heme c1
Fe-S
PDB
1BE3
Complex III
(bc1 Complex)
membrane
heme bH
19. The domain with
attached Rieske Fe-S has
a flexible link to the rest
of the complex.
(Fe-S protein in green.)
Fe-S changes position
during e- transfer.
After Fe-S extracts an e-
from QH2, it moves
closer to heme c1, to
which it transfers the e-.
View an animation.
heme bL
heme c1
Fe-S
PDB
1BE3
Complex III
(bc1 Complex)
membrane
heme bH
20. After the 1st e- transfer
from QH2 to Fe-S, the
CoQ semiquinone is
postulated to shift position
within the Q-binding site,
moving closer to its e-
acceptor, heme bL.
This would help to
prevent transfer of the
2nd electron from the
semiquinone to Fe-S.
heme bL
heme c1
Fe-S
PDB
1BE3
Complex III
(bc1 Complex)
membrane
heme bH
21. Complex III is an
obligate homo-dimer.
Fe-S in one half of the
dimer may interact with
bound CoQ & heme c1
in the other half of the
dimer.
Arrows point at:
• Fe-S in the half of
complex colored
white/grey
• heme c1 in the half of
complex with proteins
colored blue or green.
PDB-1BGY Complex III
homo-dimer
Fe-S
heme c1
22. Electrons are donated to complex IV, one at a time, by
cytochrome c, which binds from the intermembrane space.
Each e- passes via CuA & heme a to the binuclear center,
buried within the complex, that catalyzes O2 reduction:
4e- + 4H+ + O2 → 2H2O.
Protons utilized in this reaction are taken up from the
matrix compartment.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
Complex IV
(Cytochrome
Oxidase):
23. H+ pumping by complex IV:
In addition to protons utilized in reduction of O2, there
is electron transfer-linked transport of 2H+ per 2e-
(4H+ per 4e-) from the matrix to the intermembrane
space.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
24. Structural & mutational studies indicate that protons pass
through complex IV via chains of groups subject to
protonation/deprotonation, called "proton wires."
These consist mainly of chains of buried water molecules,
along with amino acid side-chains, & propionate side-
chains of hemes.
Separate H+-conducting pathways link each side of the
membrane to the buried binuclear center where O2
reduction takes place.
These include 2 proton pathways, designated "D" & "K"
(named after constituent Asp & Lys residues) extending
from the mitochondrial matrix to near the binuclear center
deep within complex IV.
Images in web pages of: IBI, & Crofts.
25. A switch mechanism controlled by the reaction cycle is
proposed to effect transfer of a proton from one half-
wire (half-channel) to the other.
There cannot be an open pathway for H+ completely
through the membrane, or oxidative phosphorylation
would be uncoupled. (Pumped protons would leak back.)
Switching may involve conformational changes, and
oxidation/reduction-linked changes in pKa of groups
associated with the catalytic metal centers.
Detailed mechanisms have been proposed.
26. Ejection of a total of 20H+ from the matrix per 4e-
transferred from 2 NADH to O2 (10H+ per ½O2).
Not shown is OH- that would accumulate in the matrix
as protons, generated by dissociation of water
(H2O H+ + OH-), are pumped out.
Also not depicted is the effect of buffering.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
Simplified
animation
depicting:
27. ATP synthase, embedded in cristae of the inner
mitochondrial membrane, includes:
F1 catalytic subunit, made of 5 polypeptides
with stoichiometry a3b3gde.
Fo complex of integral membrane proteins that
mediates proton transport.
ADP + Pi ATP
F1
Fo
3 H+
matrix
intermembrane
space
28. F1Fo couples ATP synthesis to H+ transport into the
mitochondrial matrix. Transport of least 3 H+ per ATP is
required, as estimated from comparison of:
DG for ATP synthesis under cellular conditions (free
energy required)
DG for transfer of each H+ into the matrix, given the
electrochemical H+ gradient (energy available per H+).
ADP + Pi ATP
F1
Fo
3 H+
matrix
intermembrane
space
29. The Chemiosmotic Theory of oxidative phosphorylation,
for which Peter Mitchell received the Nobel prize:
Coupling of ATP synthesis to respiration is indirect,
via a H+ electrochemical gradient.
Matrix
H+
+ NADH NAD+
+2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c 3H+
F1
Fo
ADP + Pi ATP
30. Chemiosmotic theory - respiration:
Spontaneous e- transfer through complexes I, III, & IV is
coupled to non-spontaneous H+ ejection from the matrix.
H+ ejection creates a membrane potential (DY, negative
in matrix) and a pH gradient (DpH, alkaline in matrix).
Matrix
H+
+ NADH NAD+
+2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c 3H+
F1
Fo
ADP + Pi ATP
31. Chemiosmotic theory - F1Fo ATP synthase:
Non-spontaneous ATP synthesis is coupled to spontaneous
H+ transport into the matrix. The pH & electrical gradients
created by respiration are the driving force for H+ uptake.
H+ return to the matrix via Fo "uses up" pH & electrical
gradients.
Matrix
H+
+ NADH NAD+
+2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c 3H+
F1
Fo
ADP + Pi ATP
32. Transport of ATP, ADP, & Pi
ATP produced in the mitochondrial matrix must exit to
the cytosol to be used by transport pumps, kinases, etc.
ADP & Pi arising from ATP hydrolysis in the cytosol
must reenter the matrix to be converted again to ATP.
Two carrier proteins in the inner mitochondrial
membrane are required.
The outer membrane is considered not a permeability
barrier. Large outer membrane VDAC channels are
assumed to allow passage of adenine nucleotides and Pi.
33. Adenine nucleotide translocase (ADP/ATP carrier) is an
antiporter that catalyzes exchange of ADP for ATP
across the inner mitochondrial membrane.
At cell pH, ATP has 4 (-) charges, ADP 3 (-) charges.
ADP3-/ATP4- exchange is driven by, and uses up,
membrane potential (one charge per ATP).
ADP + Pi ATP matrix
lower [H+
]
_ _
3 H+
ATP4-
ADP3-
H2PO4
-
H+
higher [H+
]
ADP + Pi cytosol
energy
requiring
reactions
ATP4-
+ +
34. Phosphate re-enters the matrix with H+ by an electroneutral
symport mechanism. Pi entry is driven by, & uses up, the
pH gradient (equivalent to one mol H+ per mol ATP).
Thus the equivalent of one mol H+ enters the matrix with
ADP/ATP exchange & Pi uptake. Assuming 3H+ transported
by F1Fo, 4H+ total enter the matrix per ATP synthesized.
ADP + Pi ATP matrix
lower [H+
]
_ _
3 H+
ATP4-
ADP3-
H2PO4
-
H+
higher [H+
]
ADP + Pi cytosol
energy
requiring
reactions
ATP4-
+ +
Animation
35. Questions: Based on the assumed number of H+ pumped
out per site shown above, and assuming 4H+ are
transferred back to the matrix per ATP synthesized:
What would be the predicted P/O ratio, the # of ATP
synthesized per 2e- transferred from NADH to ½O2?
What would be the predicted P/O ratio, if the e- source is
succinate rather than NADH?
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
36. 2.5 ~P bonds synthesized during oxidation of NADH
produced via Pyruvate Dehydrogenase & Krebs Cycle
(10 H+ pumped; 4 H+ used up per ATP).
1.5 ~P bonds synthesized per NADH produced in the
cytosol in Glycolysis (electron transfer via FAD to CoQ).
1.5 ~P bonds synthesized during oxidation of QH2
produced in Krebs Cycle (Succinate Dehydrogenase –
electrons transferred via FAD & Fe-S to coenzyme Q).
For, summing up
synthesis of ~P
bonds via ox
phos, assume:
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c
37. Above is represented an O2 electrode recording while
mitochondria respire in the presence of Pi and an e- donor
(succinate or a substrate of a reaction to generate NADH).
The dependence of respiration rate on availability of ADP,
the ATP Synthase substrate, is called respiratory control.
[O2]
time
ADP added
ADP all
converted
to ATP
a
b
c
An oxygen electrode
may be used to record
[O2] in a closed vessel.
Electron transfer, e.g.,
NADH O2, is
monitored by the rate
of O2 disappearance.
38. Respiratory control ratio is the ratio of slopes after and
before ADP addition (b/a).
P/O ratio is the moles of ADP divided by the moles of O
consumed (based on c) while phosphorylating the ADP.
[O2]
time
ADP added
ADP all
converted
to ATP
a
b
c
39. Chemiosmotic explanation of respiratory control:
Electron transfer is obligatorily coupled to H+ ejection
from the matrix. Whether this coupled reaction is
spontaneous depends on pH and electrical gradients.
Reaction DG
e- transfer (NADHO2) negative value*
H+ ejection from matrix positive; depends on H+
gradient**
e- transfer with H+ ejection algebraic sum of above
*DGo' = -nFDEo' = -218 kJ/mol for 2e- NADHO2.
**For ejection of 1 H+ from the matrix:
DG = RT ln ([H+]cytosol/[H+]matrix) + FDY
DG = 2.3 RT (pHmatrix - pHcytosol) + FDY
40. With no ADP, H+ cannot flow through Fo. DpH & DY are
maximal. As respiration/H+ pumping proceed, DG for H+
ejection increases, approaching that for e- transfer.
When the coupled reaction is non-spontaneous,
respiration stops. This is referred to as a static head.
In fact there is usually a low rate of respiration in the
absence of ADP, attributed to H+ leaks.
Matrix
H+
+ NADH NAD+
+2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c 3H+
F1
Fo
ADP + Pi ATP
41. When ADP is added, H+ enters the matrix via Fo, as ATP
is synthesized. This reduces DpH & DY.
DG of H+ ejection decreases.
The coupled reaction of electron transfer with H+ ejection
becomes spontaneous.
Respiration resumes or is stimulated.
Matrix
H+
+ NADH NAD+
+2H+
2H+
+ ½ O2 H2O
2e-
– –
I Q III IV
+ +
4H+
4H+
2H+
Intermembrane Space
cyt c 3H+
F1
Fo
ADP + Pi ATP
42. Uncoupling reagents (uncouplers) are lipid-soluble
weak acids. E.g., H+ can dissociate from the OH group
of the uncoupler dinitrophenol.
Uncouplers dissolve in the membrane and function as
carriers for H+.
OH
NO2
NO2
2,4-dinitrophenol
43. Uncouplers block oxidative phosphorylation by
dissipating the H+ electrochemical gradient.
Protons pumped out leak back into the mitochondrial
matrix, preventing development of DpH or DY.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
I Q III IV
4H+
4H+
2H+
H+
Intermembrane Space
cyt c
uncoupler
44. With uncoupler present, there is no DpH or DY.
DG for H+ ejection is zero
DG for e- transfer coupled to H+ ejection is maximal
(spontaneous).
Respiration proceeds in the presence of an uncoupler,
whether or not ADP is present.
Matrix
H+
+ NADH NAD+
+ 2H+
2H+
+ ½ O2 H2O
2e-
I Q III IV
4H+
4H+
2H+
H+
Intermembrane Space
cyt c
uncoupler
45. DG for H+ flux is zero in the absence of a H+ gradient.
Hydrolysis of ATP is spontaneous.
The ATP Synthase reaction runs backward in presence
of an uncoupler.
ADP + Pi ATP
F1
Fo
3 H+
ATPase with H+
gradient dissipated
matrix
intermembrane
space
46. Uncoupling Protein
An uncoupling protein (thermogenin) is produced in
brown adipose tissue of newborn mammals and
hibernating mammals.
This protein of the inner mitochondrial membrane
functions as a H+carrier.
The uncoupling protein blocks development of a H+
electrochemical gradient, thereby stimulating
respiration. DG of respiration is dissipated as heat.
This "non-shivering thermogenesis" is costly in terms
of respiratory energy unavailable for ATP synthesis,
but provides valuable warming of the organism.