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Biological Oxidation
生 物 氧 化
Jiao Li
Department of biochemistry and
molecular biology
021-65986142
[email protected]
exercise
* How food provide energy?
glycogen
glucose
triglyceride
protein
Fatty acid+glycerol
Amino acid
Acetyl-CoA
TAC
CO2
2H
ADP+Pi ATP
Respiratory
chain
Reducing equavalents( H or e-)
H2O
Learning Objectives
After completion of this section, you should be able to :
1. List the components of four protein complexes Ⅰ,Ⅱ,Ⅲ and
Ⅳ that involved in electron transport.
2. Describe the electron transport along the protein complexes
of respiratory chain.
3. Understand the mechanism of ATP synthesis by oxidative
phosphorylation.
4. Illustrate the site of action of common poisons that block
electron transport and oxidative phosphorylation.
Outline of the section
1. Large four protein complexes Ⅰ,Ⅱ,Ⅲ and
Ⅳ embedded in the inner mitochondrial membrane.
2. Electron transport via the respiratory chain.
3. ATP synthesis by oxidative phosphorylation.
4. common poisons that block electron transport and oxidative
phosphorylation.
5. Substrates shuttle into mitochondial respiratory chain.
Section 1
Large four protein complexes Ⅰ,Ⅱ,Ⅲ and
Ⅳ embedded in the inner mitochondrial
membrane.
glycogen
glucose
triglyceride
protein
Fatty acid+glycerol
Amino acid
Acetyl-CoA
mitochondrion
TAC
CO2
2H
ADP+Pi ATP
Respiratory
chain
H2O
Mitochondrion
The inner membrane is protein rich
(4:1 protein:lipid). not permeable
and contains within it the
electron transporter, ATP
synthase, membrane transport
proteins.
The outer membrane is readily
permeable to molecules with
molecular weights below 10,000 Da.
.
ADP+Pi ATP
2H
Respiratory
chain
O2
H2O
Transfer of reducing equavalents to oxygen occurs
in inner mitochondrial membrane
1. Respiratory chain
Definition
H+ and e- are passed through a set of
electron transporters embedded in inner mt
membrane to O2. Respiratory chain comprises
enzymatic series electron transporters (electron
donors and acceptors), also called electron
transfer chain（ETC）.
Composition
Electron transporters（2H  2H+ + 2e）
4 protein complexes + 2 free electron transporter（CoQ and Cyt c ）
Human ETC
protein complex
mw
polypeptides
cofactor
ComplexⅠ
FMN, Fe-S
（NADH-Q reductase）
880
44
ComplexⅡ
FAD, Fe-S, Cytb
（succinate-Q reductase）
140
4
ComplexⅢ
S, CytbL/H,
(Q-Cytc reductase）
280
≥11
ComplexⅣ
Rieske FeCytc1
200
14
CuA、Cyta、
Location of each complex of respiratory chain
Cytochrome c
cytosol
Inner
membrane
matrix
CoQ and Cytochrome c are mobile component.
2. ComplexⅠ （NADH-CoQ reductase）
 composition: flavoprotein（FMN）、iron-sulphur
protein（Fe-S）
 function: electron tansfer from NADH to
CoQ(ubiquinone)
NADH
NADH + H+
NAD+
FMN; Fe-S
FMN
FMNH2
CoQ
Fe2+S
Fe3+S
CoQ
CoQH2
Structure of NAD+ and NADP+
Nicotinamide
R=H: NAD+;
R=H2PO3: NADP+
Conversion between NAD+（NADP+） and NADH+H+
（NADPH+H+）
or
or
FMN（flavin mononucleotide）
isoallo
xazine
Fe-S prosthetic group in iron-sulphur
protein carries the reaction:
Fe2+  Fe3++e-
Ⓢ indicates inorganic
sulphur
Fe-S
S
Inorganic sulphur
S
S-cys
Ubiquinone (Coenzyme Q, CoQ, Q）contains
a long isoprenoid units tail（human:CoQ10) and
transfer H+ and e- by conversion as follows.
Quinone
(oxidized
form)
Semiquinone
(free radical
form)
Quinone
(reduced form)
NADH+H+
NAD+
FMN
Reduced Fe2+-S
Q
FMNH2
Oxidized Fe3+-S
QH2
Function of complexⅠ
matrix
cytosol
3. Complex Ⅱ: Succinate-CoQ Reductase
 composition: flavoprotein（FAD）、iron-sulphur
protein（Fe-S）、cytochrome b
 function: electron tansfer from succinate to
CoQ(ubiquinone)
succinate→
Complex Ⅱ
FAD; Fe-S; b; Fe-S
→
CoQ
isoalloxazine
AMP
FAD（flavin adenine dinulceotide）
Cytochrome
A hemeprotein contains heme group and
is primarily responsible for transferring
electrons.
It is divided into several types via their
absorbance of wavelength, such as
cytochrome a, b, c, etc.
protein
b
a
c
Function of complex Ⅱ
cytosol
matrix
succinate
Succinate
Fumarate
FAD
FADH2
Fe2+S
Fe3+S
CoQ
CoQH2
4. Complex Ⅲ: CoQ-Cytochrome c Reductase
 composition: cytochrome bL/bH, iron-sulphur
protein（Rieske Fe-S ), cytochrome c1
 function: electron tansfer from CoQ(ubiquinone) to
cytochrome c
• CoQ passes electrons to cyt c (and pumps H+)
in a unique redox cycle known as the Q cycle.
Complex Ⅲ
QH2→
b; Fe-S; c1
→Cyt c
One e- is transferred
to cyt c
Second e- is back to
Q
Q cycle in complex Ⅲ catalyzed electron transfer
5. Complex Ⅳ: Cytochrome c Oxidase
 composition: CuA, cytochrome a/a3, CuB
 function: electron tansfer from cytochrome c to O2.
Cyt a3 /CuB binuclear center transfers e- to O2。
Complex Ⅳ
Reduced Cyt c → CuA→a→a3→CuB → O2
• Oxygen is thus the terminal acceptor of electrons
in the electron transport pathway -
the end!
cytosol
matrix
Function of Complex Ⅳ
Section 2
Electron transport via the
respiratory chain
1. Direction of electron flow in ETC
is from experiment
Experiments included:
① determination of standard redox potential
② separation and recombination of each
component.
③ specific inhibitors
④ oxygen is added to reduced ETC at a slow rate.
Standard reduction potentials of
the major respiratory electron
carriers.
• Electrons generally fall in energy through the
chain - from complexes I and II to complex IV
2. Reducing equavalence enter ETC
by two routes
1. NADH redox respiratory chain
NADH →complexⅠ→Q →complex Ⅲ→Cyt c
→complex Ⅳ→O2
2. Succinate redox respiratory chain
Succinate →complexⅡ →Q →complex Ⅲ→Cyt
c →complexⅣ→O2
NADH respiratory chain
FADH2 respiratory chain
complex Ⅱ
complex Ⅰ
complex Ⅲ
complex Ⅳ
Electron transport occurs in inner mitochondrial
membrane via a series of protein complexes.
※ complex Ⅰ,Ⅲ, Ⅳ are also a pump to pump out H+ to
intermembrane space.
Section 3
ATP synthesis by oxidative
phosphorylation
1. Oxidative phosporylation provides
most energy captured during catablism
* Definition
Electron transfer from reducing equavalents
to oxygen via ETC couples the synthesis of ATP
from ADP, which is oxidative phosphorylation,
also called coupled phosphorylation.
Substrate
level
phosphorylation
another way to synthesize ATP.
is
2. Coupling sites of oxidative
phosphorylation in ETC
Experiments:
(1) P:O ratio
(2) Free energy change- ΔG°′
(1) P:O ratio：For per half mol O2, the number of
inorganic phosphate consumed is equal to the
number of ATP generated.
线粒体离体实验测得的一些底物的P/O比值
P:O ratio
determined from mitochondrion in vitro
substrate
底 物
β-hydroxybutyric
β-羟丁酸
acid
Respiratory
呼吸链的组成chain
NAD+→复合体Ⅰ→CoQ→复合体Ⅲ
complexⅠ
complexⅢ
P:O
ratio
P/O比值
2.4～2.8
Number of
ATP
可能生成的
ATP数
3
complexⅣ
→Cyt c→复合体Ⅳ→O
2
琥珀酸
Succinate
complexⅢ
复合体Ⅱ→CoQ→复合体Ⅲ
complexⅡ
1.7
2
complexⅣ
→Cyt c→复合体Ⅳ→O
2
抗坏血酸
Ascorbic acid
Cytochrome
c
细胞色素c
complexⅣ
Cyt c→复合体Ⅳ→O
2
(Fe2+)
complexⅣ
复合体Ⅳ→O
2
0.88
1
0.61-0.68
1
(2) Redox potential in respiratory chain
呼吸链中各种氧化还原对的标准氧化还原电位
氧化还原对
System
complex Ⅰ
-70.4kJ/mol
complex Ⅲ
-36.7kJ/mol
complex Ⅳ
-83kJ/mol
NAD+/NADH+H+
FMN/ FMNH2
FAD/ FADH2
Cyt b Fe3+/Fe2+
Q10/Q10H2
Cyt c1 Fe3+/ Fe2+
Cyt c Fe3+/Fe2+
Cyt a Fe3+ / Fe2+
Cyt a3 Fe3+ / Fe2+
1/2 O2/ H2O
ΔG°′＝ － nFΔ E°′
Eº' (V)
-0.32
-0.30
ATP → ADP + P i
ΔG°′＝ −30.5kJ/mol
-0.06
0.08
0.04（或0.10）
0.07
0.22
0.25
0.29
0.55
0.82
ΔG°′: free energy change
Δ E°′: potential change
n: number of electrons
F : constant（96.5 kJ /mol · V ）
Coupling sites：ComplexⅠ, Ⅲ, Ⅳ
Succinate
Complex Ⅱ
Coupling sites of oxidative
phosphorylation
ATP
ATP
ComplexⅠ
Complex Ⅲ
ATP
ComplexⅣ
How ATP is produced ?
3. Chemiosmotic theory account for
oxidative phosphorylation.
ComplexⅠ, Ⅲ, Ⅳ act as a proton pump. H+ in
electron flow is pumped out to cytosol
to form
electrochemical gradient across inner membrane.
Back flow of H+ drives generation of ATP from ADP
and Pi.
ComplexⅠ, Ⅲ, Ⅳ act as a proton pump
Cytosol 4H+
+
Cyt c
4H+
+ + + + +
2H+
+
4H+
+ +
Q
-
Ⅰ
F
Ⅱ
-
-
Ⅲ
NAD+
Succinate
0
Ⅳ
- - -
Fumaric acid
NADH+H+
+
1/2O2+2H+
- -
-
H2O
F1
Matrix
ADP+Pi
ATP
H+
4. ATP synthase (ComplexⅤ)
Cytosol
Inner
membrane
Matrix
ATP synthase model
4. ATP synthase
(complexⅤ)
b
F1:hydrophilic,
（ α3β3γδε subunits ）
F0:hydrophobic,
（a1b2c9～12 subunits）
Fo not F0 since O
stands
for
oligomycin!
c
a
Structure:
F0
δ
γ ε
F6
Catalytic
subunit
β
OSCP
Catalytic
subunit
β
α
ATP synthase
F1
Mechanism of ATP production
by ATP synthase.
Pro-tons passing through the disk of “C”
units cause it and γ-subunitRed:
to rotate.
OADP and P i
are taken up sequentially byOrange:
the β-subunitsLto form
ATP, which is expelled as Pink:
the rotating
T γ-subunit
squeezes each β-subunit in turn. Thus, three ATP
molecules are generated per revolution.
H+ flow
5. Factors affecting oxidative phosphorylation
ADP （major regulation）
Rate of respiratory in Mt is mainly controlled by
availability of ADP
ADP
O2 consumption
Resting state
6. ATP
(1) High energy bond and high energy phosphate
compound
High energy phosphate bond
≥21kJ/mol free energy is release during
hydrolysis of some phosphate ester bond or
phosphate anhydride bond, P.
 High energy phosphate
Standard free energy of hydrolysis
of some organophosphates of biochemical
importance
γ
β
~
~
α
(2) Synthesis and use of ATP
ATP
creatine
Creatine
phosphate
oxidative phosphorylation
~P
substrate phosphorylation
ADP
ATP is the center of energy
generation and use
~P
mechanic(contraction)
osmotic(active transport)
chemical(anabolism)
electrical(bioelectricity)
heat(body temperature)
Section 4
Common poisons that block electron
transport and oxidative phosphorylation
1. Many poisons inhibit respiratory chain
(1) Electron transport inhibitor
Piericidin A
Amobarbital
Rotenone
(2) Oxidative phosphorylation inhibitor
Oligomycin
(3) Oxidative phosphorylation uncoupler
2,4-dinitrophenol
Thermogenin (physical uncoupler)
Site of action of some poisons
2. Mechanism of action of uncoupler
H
NO2
NO2
DNP： 2,4-dinitrophenol
O2N
OH
O 2N
O
H+
Cytosol
Cyt c
uncoupler
Q
Ⅰ
Ⅱ
F
Ⅲ
Ⅳ
Matrix
0
F1
ADP+Pi ATP
H+
Thermogenin (physical uncoupler) provide heat for body.
Heat
3. Mechanism of action of oligomycin
cytosol
Oligomycin
blocks H+ flow
through c
channel of ATP
synthase
matrix
oligomycin
ATP synthase
Section 5
Substrates shuttle into mitochondrial
respiratory chain
1. Transporter in inner
mitochondrial membrane
Impermeability of inner
membrane requires
transporter to help
transport substrate and
substance.
2. Shuttle systems
 Cytoplasmic NADH enter into
mitochondrial matrix through two shuttles.
Shuttle 1: α-glycerophosphate shuttle
Shuttle 2: malate-asparate shuttle
(1) α-glycerophosphate shuttle （brain and white muscle）
CH2OH
CH2OH
ETC
NADH+H+
α-glycerolphosphate
dehydrogenase
NAD+
C=O
C=O
CH2O- Pi
CH2O- Pi
Dihydroxyacetone
phosphate
αglycerolphosphate
dehydrogenase
CH2OH
CH2OH
CHOH
CHOH
CH2O- Pi
CH2O- Pi
α-glycerolphosphate
FADH2
FAD
inner
outer
Intermembrane
membrane
membrane
space
matrix
(2) Malate-asparate shuttle （universal）
(3) Adenine nucleotide transporter
ADP3- ATP4-
3H+
H2PO4- H+
胞液侧
F
0
基质侧 腺苷酸
转运蛋白
F1
ATP4-
磷酸
转运蛋白
H2PO4- H+
ADP3H+
ATP and ADP are antiport in inner membrane
Clinical case
MELAS ( Mitochondrial encephalopathy, lactic acidosis, and stroke )
1. Genetic disease due to mutations in genes of mitochondrial
DNA( eg. NADH-Q reductase)
2. Brain and muscle are
mainly affected.
Summary
1. Four protein complexes Ⅰ,Ⅱ,Ⅲ and
Ⅳ are mainly responsible for transferring electron in ETC.
2. ATP is synthesized by oxidative phosphorylation driven by
H+ gradient .
4. Some poisons inhibit biologic oxidation by blocking
electron transport and oxidative phosphorylation.
Study question
1. Which of following subunit is mainly responsible to catalyze the
synthesis of ATP in ATP synthase?
A. α
B. β
C. γ
D. ε
E. c
2. What is the consequence of thermalgenin effect.
A. Producing more heat
B. ETC is inhibited
C. Producing more ATP
D. Oxidative phosphorylation is increased