Oxidative phosphorylation
Biochemistry, 4th edition, RH Garrett & CM Grisham,
Brooks/Cole (Cengage); Boston, MA: 2010
pp 592-629
Instructor: Kirill Popov
1. The mitochondrion
2. Electron transport
3. Oxidative phosphorylation
4. Heat, oxidative stress, etc.
Biochemical anatomy of a mitochondrion
Outer membrane
freely permeable to
small molecules and ions
Inner membrane
Cristae
ATP synthase
Impermeable to most
small molecules and ions
Including H+
Contains:
• Respiratory electron carriers
• ADP/ATP translocase
• ATP synthase
• Other membrane transporters
Matrix
Ribosomes
Porin channels
Contains:
• Pyruvate dehydrogenase complex
• Citric acid cycle enzymes
• Fatty acid β-oxidation enzymes
• Amino acid oxidation enzymes
• DNA, ribosomes
• Other enzymes and metabolites
O
CH3O
CH3
CH3
CH3O
(CH2
O
H+ + e−
CH2
C CH2)10
H
Ubiquinone (Q)
(fully oxidized)
O•
CH3O
CH3
CH3O
R
Semiquinone radical
(•QH)
OH
H+ + e−
OH
CH3O
CH3
CH3O
R
OH
Ubiquinol (QH2)
(fully reduced)
Prosthetic groups of cytochromes
S Cys
H3C
CH CH2
H3C
Cys
H2C CH
CH3
N
N
Fe
H3C
S
CH3CH
N
Fe
-
CH2CH2COO
CH3
N
N
N
H3C
CHCH3
N
-
N
H3C
CH2CH2COO
-
CH2CH2COO
-
CH2CH2COO
H3C
Heme C
(in c-type cytochromes)
Iron protoporphyrin IX
(in b-type cytochromes)
H3C
CH CH2
OH
CH2 CH
H3C
CH3
CH3
CH3
Heme A
(in a-type cytochromes)
CH3
N
N
Fe
N
H3C
CHO
N
-
CH2CH2COO
-
CH2CH2COO
Adsorption spectra of cytochrome c
Relative light absorption (%)
100
γ
Oxidized
cyt c
Reduced
cyt c
50
α
β
0
300
400
500
Wavelength (nm)
600
Iron-sulfur centers
S
Cys
Fe
S
Cys
Cys
S
S
S
Cys
Fe
Fe
Cys
S
Protein
S
S
Cys
Cys
Cys
S
Cys
Fe
S
Cys
S
S
S
Fe
S
S
Cys
S
Fe
Fe
S
S
Cys
Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers
Redox reaction (half-reaction)
E'° (V)
2H+ + 2e− → H2
-0.414
NAD+ + H+ + 2e− → NADH
-0.320
NADP+ + H+ + 2e− → NADPH
-0.324
NADH dehydrogenase (FMN) + 2H+ + 2e− → NADH dehydrogenase (FMNH2)
-0.30
Ubiquinone + 2H+ + 2e− → ubiquinol
0.045
Cytochrome b (Fe3+) + e− → cytochrome b (Fe2+)
0.077
Cytochrome c1 (Fe3+) + 2e− → cytochrome c1 (Fe2+)
0.22
Cytochrome c (Fe3+) + 2e− → cytochrome c (Fe2+)
0.254
Cytochrome a (Fe3+) + 2e− → cytochrome a (Fe2+)
0.29
Cytochrome a3 (Fe3+) + 2e− → cytochrome a3 (Fe2+)
0.35
1/2O2 + 2H+ + 2e− → H2O
0.8166
Separation of functional complexes of the respiratory chain
The Protein Components of the Mitochondrial Electron-Transfer Chain
Enzyme complex/protein
Mass (kDa)
Number of subunits*
Prosthetic group(s)
I NADH dehydrogenase
850
43 (14)
FMN, Fe-S
II Succinate dehydrogenase
140
4
FAD, Fe-S
III Ubiquinone:cytochrome c
oxidoreductase
250
11
Hemes, Fe-S
Cytochrome c#
13
1
Heme
IV Cytochrome oxidase
160
13 (3-4)
Hemes, CuA, CuB
*Numbers of subunits in the bacterial equivalents in parentheses.
#Cytochrome c is not part of an enzyme complex; it moves between Complexes III and IV as a freely soluble protein.
Pathways in the mitochondrial electron transport
Fatty acyl-CoA dehydrogenase
Complex I
Flavoprotein 1
Flavoprotein 3
NADH dehydrogenase,
FMN,
Fe-S centers
Electron-transf erring
f lavoprotein, FAD,
Fe-S centers,
Complex III
NADH coenzyme Q
oxidoreductase
UQ/UQH2
pool
Complex II
Flavoprotein 2
Succinate dehydrogenase,
FAD,
Fe-S centers,
b-type heme
Succinate-coenzyme Q
oxidoreductase
1/2 O2
Flavoprotein 4
Glycerolphosphate
dehydrogenase,
FAD,
Fe-S centers,
Cytochrome bc 1 complex,
2 b-type hemes,
Rieske Fe-S center
c-type heme (cyt c 1),
Coenzyme Q-cytochrome c
oxidoreductase
H2O
Complex IV
Cytochrome c
Cytochrome aa3 complex,
2 a-type hemes,
Cu ions
Cytochrome c oxidase
+400
+600
a
CuA
c
c1
Rieske Fe/S
UQ10
(Fe/S)S1
(Fe/S)S3
0
bH
FAD
bL
(Fe/S)N2
Complex II
a3
+200
(Fe/S)N3
(Fe/S)N1
(Fe/S)N4
FMN
NAD+/NADH
-400
Fum/Succ
E (mV)
Electrons move downhill
Complex I
-200
Complex III
Complex IV
NADH:ubiquinon oxidoreductase (Complex I)
4H+
Intermembrane
space (P side)
Complex I
Q
N-2
Matrix
arm
2H+
Fe-S
Matrix
(N side)
FMN
QH2
2e−
Series
of Fe-S
centers
2e−
NAD+
NADH + H+
Membrane
arm
Succinate dehydrogenase (Complex II)
Intermembrane
space (P side)
b-type heme
Ubiquinone
Q
QH2
2e−
Matrix (N side)
2H+
Fe-S
FAD
Substrate
binding
site
Series
of Fe-S
centers
2e−
Succinate
Fumarate
Malate
Succinyl-CoA
Krebs cycle
Oxaloacetate
α-Ketoglutarate
Isocitrate
Citrate
Acetyl-CoA
Cytochrome bc1 complex (Complex III)
Cytochrome c
Rieske ironsulfur protein
Cytochrome c1
Intermembrane
space (P side)
Qp
c1
bL
Heme
QN
2Fe-2S
center
bH
Cytochrome b
Matrix (N side)
The Q cycle
Oxidation of first QH 2
Oxidation of second QH 2
Cyt c
Cyt c
2H+
Cyt c1
2H+
Cyt c1
Fe-S
Fe-S
bL
bH
Intermembrane
space (P side)
QH2
Q
Q
Q•
bL
QH2
bH
Q•
Q
QH2
2H+
Matrix (N side)
QH 2 + Cyt c1 (oxidized) →
Q•− + 2H P+ + Cyt c1 (reduced)
QH 2 + Q•− + Cyt c1 (oxidized) →
QH2 + 2H P+ + Q + Cyt c1 (reduced)
Net equation:
QH2 + 2 Cyt c1 (oxidized) + 2H N+ → Q + 2 Cyt c1 (reduced) + 4H P+
Path of electrons through comlex IV
Intermembrane
space
(P side)
2H+
4Cyt c
4e-
CuA
a
a3
CuB
O2
Fe-Cu center
III
II
I
4H+
2H+
2H2O
(substrate) (pumped)
Reaction sequence for the reduction of O 2 by the
cytochrome c oxidase
1st e−
2+
1+
Fe
Cu
O2
2+
3+
Fe
Cu
1+
2+
2nd e−
Fe
Cu
O
O
next cycle
3+
2+
Fe
Cu
4th e−
3rd e−
Fe
2H2O
2+
4+
3+
2+
Fe
O− Cu
O−
Cu
O
2H+
O
H
H+
H
H+
The flow of electrons and protons through the respiratory
chain (proton-motive force and chemiosmotic model)
4H+
4H+
Intermembrane
Space (P side)
2H+
Cyt c
+++
Cyt c
+ + + + + +
+ + + + +
Fo
Q
- - -
H+ - -
Matrix (N side)
-
-
I
IV
III
F1
II
1/2O2 + 2H+
ADP +Pi
ATP
NADH +
Chemical
potential
ΔpH
(inside
alkaline)
H+
NAD +
Succinate Fumarate
ATP
synthesis
driven by
proton-motive
force
Electrical
potential
Δψ
(inside
negative)
H2O
P side
N side
[H+]P = C2
[H+]N = C1
H+
OH−
H+
OH−
H+
OH−
H+
H+
OH−
H+
H+
H+
OH−
Proton
pump
ΔG = RT ln (C2/C1) + ZFΔψ
= 2.3RT ΔpH + FΔψ
OH−
OH−
Catalytic mechanism of F1
In the presence of a proton gradient:
ADP +
P
ATP
is released
In the absence of a proton gradient:
H+
ADP +
P
H2O
H2
ATP
Enzyme
bound
18O
O
H+
ADP +
-
18O
P
O-
OH
Mitochondrial ATP synthase complex
Rotation of Fo and γ
Actin filament
Avidin
a
C10
ε
b
γ
ADP + Pi
α
His
residues
β
α
His residues
Ni complex
ATP
Rotation of Fo and γ
A model of the FoF1 complex, a rotating molecular motor
δ
α
β
ADP + Pi
F1
b2
H+
γ
ε
Fo
ATP
β
N side
C10
a
H+
P side
Binding-change model for ATP synthase
α
β
ATP
β ADP
α
+ Pi
α
3 HP+
β
3 HN +
ATP
3 HN+
3 H P+
ATP
α
β
ATP
α
β
ADP
+ Pi
β
α
ATP
α
ADP
+ Pi
β
β
3 H P+
α
α
3 HN+
ATP
β
The P/O ratio is an index of the efficiency of coupling
P/O ratio: number of molecules of Pi incorporated (=ATP synthesized) per
atom of oxygen consumed (or pair of electrons being carried through the
chain).
Measurements: oxygen consumption during complete phosphorylation
of a fixed amount of ADP after addition of either an NAD+-linked substrate
or FAD-linked substrate.
P/O  2.5 for NADH
P/O  1.5 for FADH2
Adenine nucleotide and phosphate translocases
Intermembrane
space
Adenine
nucleotide
translocase
(antiporter)
ATP
synthase
Phosphate
translocase
(symporter)
ATP4ADP3-
Matrix
ATP4ADP3-
H+
H2PO4−
H+
H+
H2PO4−
H+
Glycerol 3-phosphate shuttle
Glycolysis
NAD+
cytosolic
glyceol 3-phosphate NADH + H+
dehydrogenase
CH2OH
Glycerol 3phosphate
C O
Dihydroxyacetone
CH2
phosphate
CH2OH
O
P
mitochondrial
glyceol 3-phosphate
dehydrogenase
CHOH
CH2
O
P
FAD
FADH2
Q
Matrix
III
Malate-aspartate shuttle
Intermembrane
space
NAD +
H+ +
Malate
malate
dehydrogenase
NADH
Oxaloacetate
Matrix
Malate
α-ketoglutarate
transporter
NAD +
Malate
malate
dehydrogenase
NADH + H+
Oxaloacetate
Glutamate
Glutamate
aspartate
aminotransferase
aspartate
aminotransferase
α-Ketoglutarate
Aspartate
Glutamate-aspartate
transporter
α-Ketoglutarate
Aspartate
ATP Yield from Complete Oxidation of Glucose
Process
Direct product
Final ATP
Glycolysis
2 NADH (cytosolic)
2 ATP
3 or 5*
2
Pyruvate oxidation (two per
glucose)
2 NADH (mitochondrial matrix)
5
Acetyl-CoA oxidation in citric acid
cycle (two per glucose)
6 NADH (mitochondrial matrix)
2 FADH2
2 GTP
15
3
2
Total yield per glucose
30 or 32
*The number depends on which shuttle system transfers reducing equivalents into the mitochondrion.
Heat generation by uncoupled mitochondria
Intermembrane
space
Matrix
IV
Cyt c
III
II
H+
H+
I
ADP +Pi
ATP
H+
Fo
Uncoupling
protein
(thermogenin)
F1
Heat
ROS formation in mitochondria and mitochondrial defenses
Nicotinamide
nucleotide
transhydrogenase
Inner
mitochondrial
membrane
Cyt c
Q
IV
III
I
O2
•O −
2
NAD+
NADH
NAD+
•OH
NADP+
GSSG
glutathione
reductase
NADPH
2 GSH
inactive
oxidative
stress
Enz
S
S
SH
SH
active
2 GSH
protein thiol
reduction
GSSG
superoxide
dismutase
H2 O2
glutathione
peroxidase
H2O
Hypoxia-inducible factor (HIF-1)
Hypoxia (low pO2)
increased
level
of HIF-1
HIF-1
HIF-1 increases transcription
of other enzymes and proteins
(green arrows)
Glucose
transporter
Glucose
ATP
Glycolytic
enzymes
ATP production
by glycolysis
increases
lactate
dehydrogenase
Lactate
Pyruvate
PDH
kinase
pyruvate
dehydrogenase
(PDH)
Protease
Degrades
COX4-1
subunit
COX4-2
subunit
Replaces
COX4-1
Complex IV properties
Are adapted to low pO2
Acetyl-CoA
Citric
acid
cycle
O2
NADH, respiratory chain Complex IV
FADH 2
Electron flow from
NADH
and FADH2 to
Respiratory chain
decreases
O2
•O −, •OH
2
ROS production is
reduced
H2O
Role of cytochrome c in apoptosis
DNA
damage
Developmental
signal
Stress
ROS
Cytochrome c
Permeability transition
pore complex opens
Cytochrome c
moves to cytosol
Apaf-1 (apoptosis
protease activating factor-1)
ATP
Procaspase-9 monomers
(inactive)
Binding of cytochrome c
and ATP induces Apaf-1 to
form an apoptosome
Apoptosome
Apoptosome causes dimerization
of procaspase-9, creating active
caspase-9 dimers
Procaspase-3
Procaspase-7
Caspase-9 dimers
(active)
Caspase-9 catalyzes proteolytic activation of caspase-3
and caspase-7
Caspase-3
These caspases lead
to the death and resorption of the cell
Cell death
Caspase-7
1.
In mitochondria, hydride ions removed from substrates by NAD-linked dehydrogenases
donate electrons to the respiratory (electron-transfer) chain, which transfers electrons
to molecular O2 reducing it to H2O
2.
The energy of electron flow is conserved by the concomitant pumping of protons
across the membrane, producing an electrochemical gradient, the proton-motive force
3.
Proton gradient provides the energy (in the form of the proton-motive force) for ATP
synthesis from ADP and Pi by ATP synthase (FoF1 complex) in the inner membrane
4.
ATP synthase carries out “rotational catalysis,” in which the flow of protons through Fo
causes each of three nucleotide-binding sites in F1 to cycle from (ADP + Pi)-bound to
ATP-bound to empty conformations
5.
Energy conserved in proton gradient can drive solute transport uphill across a
membrane
6.
In brown fat, electron transfer is uncoupled from ATP synthesis and the energy of fatty
acid oxidation is dissipated as heat
7.
Reactive oxygen species produced in mitochondria are inactivated by a set of protective
enzymes that prevent oxidative stress
8.
Mitochondrial cytochrome c, released into the cytosol can cause activation of caspases
and apoptosis