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Lecture
8
Bioenergetics and
Oxidative Phosphorylation
Outline
 Introduction
 Free energy
 ATP as an energy carrier
 Electron transport chain
 Oxidative phosphorylation
1. Introduction
• Energy
– A central theme of Biochemistry
• Organisms employ this energy to:
–
–
–
–
–
Grow
Protect Themselves
Repair Themselves
Compete with other Organisms
Make new Organisms (I.e., babies)
• Energy cannot be produced by a cell. It can
only be borrowed from someplace else. We
are Energy Parasites!
• The ultimate source of energy for life on
Earth is the SUN.
2. Free energy, G
• The amount of energy available
to do work during a chemical
process.
• Free energy change (DG)
– Predict the direction of a reaction
– If DG is negative (DG<0), the
reaction is spontaneously, the
process is exergonic and energy is
released.
– If DG is positive (DG>0), the
reaction is nonspontaneously, the
process is endergonic and energy
is absorbed.
– If DG=0,the reactants are in
equilibrium.
Coupling Reactions
•Exergonic reactions can supply
energy for endergonic reactions
3. ATP: an energy carrier
 DGo of ATP
 DGo of hydrolysis of ATP is
approximately –7.3 kcal/mol
(-30.5 KJ/mol) for each of
the two terminal phosphate
groups.
• Because of this large,
negative DGo, ATP is called
a high-energy phosphate
compound.
•ATP + H2O
•ADP + H2O
•AMP + H2O
ADP + Pi
ΔG0’=-30.5 KJ/mol
AMP + Pi
ΔG0’=-30.5 KJ/mol
Adenosine+ Pi ΔG0’=-14.2 KJ/mol
High-energy phosphate
• High-energy phospthae bonds
energy release in hydrolysis of these bonds
is over 21KJ/mol
–high energy phosphate bonds are denoted
by the character '~ P ‘
–
• Compounds which contain these bonds
called high-energy phosphates.
High-energy Compounds
• ATP、ADP、UTP、CTP、PPi；
• Phosphate with energy higher than that of
ATP. DGo: greater than –10 kcal/mol
(41.8KJ/mol).
– Enol phosphate ：PEP
– Mix-anhydride ：glycerate 1,3-bisphosphate
– Guanidine phosphate：phosphocreatine
• Thioester bond：Acetyl-CoA、Acyl-CoA, etc.
High-energy Compounds
Phosphenol
pyruvate (PEP)
O
O
glycerate
1,3R C O¡« P O
bisphosphate
O
CH2
O
HOOC C O¡« P O
O
HO
P
Acetyl-CoA
O
O
O
P
O
¡«
OH2C CH C
O
61.9 kJ/mol
NH
NH
O
phosphocreatine
P
O
R C NH ¡«
O
61.9 kJ/mol
HOOC CH2
O
N C NH ¡« P O
O
CH3
CH3 C¡« SCoA
O
43.9 kJ/mol
31.4 kJ/mol
Function of ATP
• Chemical work: as an energy carrier,
supplying the needed energy for primary
step of metabolism of synthesis or
degradation.
• Mechanical work: supplying the energy
needed for the activities of organism.
• Produce NTP.
• Transform the high-energy phosphate to
creatine to store the energy in
phosphocreatine.
Function of ATP
ATP Energy Storage in Creatine Phosphate
ATP
ADP
creatine kinase
•Creatine phosphate is an important energy
store in skeletal muscle and in the brain.
•ATP as an Intermediate Phosphate Transfer
How is ATP produced
• Two methods
(1) Substrate level phosphorylation
– the production of ATP from ADP by a direct transfer
of a high-energy phosphate group from a
phosphorylated intermediate metabolic compound.
COO
～P
CH2OH
CH2O-P
ADP
Mg2+
1,3-bisphosphoglycerate
ATP
COOH
CH2OH
CH2O-P
3-phosphoglycerate
How is ATP produced
(2) Oxidative phosphorylation
– the process in which ATP is formed as a
result of the transfer of electrons from
NADH or FADH2to O2 by a series of
electron carriers.
4. Electron transfer chain (ETC, or
Respiratory chain)
• Definition: a specialized set of electron
carriers, can accept or donate electrons,
finally electrons combine with O2 and protons
to form H2O.
• Associated with cell breath, also called
respiratory chain.
• Electron carriers located in mitochondria
according to a order.
• Significance of Electron transfer
chain
– Electron transport and oxidative
phosphorylation re-oxidize NADH
and FADH2 and trap the energy
released as ATP.
• Site:
– Located in the inner membrane of
mitochondria.
Mitochondria
•The
components of
the respiratory
chain located in
the inner
membrane of
mitochondria.
Respiratory chain
Components of respiratory chain
•
6 Components
–
–
–
–
–
–
complex I: NADH dehydrogenase
complex II: Succinate dehydrogenase
Mobile carriers: Ubiquinone (CoQ)
complex III: Cytochrome bc1 complex
Mobile carriers: Cytochrome c (Cyt c)
complex IV: Cytochrome c oxidase
NADH → Complex I → Q → Complex III → cyt c → Complex IV → O2
Complex II
Co-enzyme
(Prosthetic group)
Complexes
Name of the enzyme
Complexes Ⅰ
NADH dehydrogenase
FMN，Fe-S
Complexes Ⅱ
Succinate dehydrogenase
FAD， Fe-S
Complexes III
Cyt bc1 complex
Heme b, Heme c1, Fe-S
Complexes Ⅳ
Cyt C oxidase
Heme a, Heme a3 ,Cu
Two respiratory chain
•NADH oxidation respiratory chain
•Succinic acid oxidation respiratory chain
Succinic acid oxidation
respiratory chain
NADH oxidation
respiratory chain
(1) ComplexⅠ— NADH dehydrogenase
ComplexⅠ
• Structure: 45
Intermembrane
peptide chains,
space
850 kD.
• Prosthetic
group:
Matrix
– FMN, ironsulphur (Fe-S)
Iron-sulfur protein
Fe-S clusters
Fe2S2,
Fe2+
Fe3+ + e
Fe4S4
(2) Coenzyme Q (CoQ, Ubiquinone) ： CoQ10
• Ubiquinone: ubiquitous in living
system.
• CoQ contain a polyisoprene side
chain.
 Liposolubility，make it move in
mitochondrial inner membrane easily.
4H+
FMN
Fe-S
Q
NADH+H+
• Function of
complexes I
NAD+
FMN
Fe2+
Fe-S
NAD+
QH2
matrix
2e-
NADH+H+
Intermembrane
space
FMNH2
Fe3+
– bind and oxidize
NADH
– transfer
electrons to CoQ
Q
– release 4H+ to
QH2 interspace of
inner and outer
membrane.
Electron transport from complexes I to CoQ
(3) ComplexⅡ- Succinate dehydrogenase
Intermembrane
space
Matrix
succinic acid
succinic acid →FAD→Fe-S→Q
Structure: succinate
dehydrogenase,
consists at list 4
peptides.
Prosthetic group:
FAD, Fe-S
Function
Transfer electron
from succinic acid
to CoQ, do not
release H+ to the
interspace.
(4) Complex Ⅲ: Cytochrome bc1 complex
Structure:
consists of 11
peptide chains
different, existing
as a dimer.
Prosthetic group:
heme b,heme
c1, Fe-S
Cytochrome, Cyt
• Structure
– colourant protein containing iron
porphyrin (heme group).
• Typing
– Cyta: Cyta, a3
– Cytb: Cytb562 、Cytb566
– Cytc: Cytc, c1
Cytochrome
Cyt a
Cyt b
Fe3+ + e
Cyt c
Fe2+
Complex Ⅲ: Cyt bc1 complex
Function
Catalyze
electron transfer
from Q to cyt c.
QH2
bFe-Sc1
Cytc
every two
electrons’s
transfering lead 4
H+ pumped to the
intermembrance
space.
(5) Cytochrome c (Cyt c)
• Cyt c is a small heme protein
found loosely associated with the
inner membrane of the
mitochondrion.
• It is a highly soluble protein,
with a solubility of about 100g/L.
• It transfers electrons between
Complexes III and IV.
Fe3+ + e
Fe2+
(6) Complex Ⅳ: Cyt c oxidase
The only electron
carrier can directly react
with molecular oxygen.
Prosthetic group:
heme a, heme a3
CuA, CuB
Function:
transfer electrons from
Cu2+ + e  Cu+
Cyt c
cytochrome c to O2,
converting O2 to H20.
every two electrons’s
transfering lead 2 H+
pumped to the
intermembrance space.
CuA a  a3  CuB
O2
Intermembrane
space
2H+
4H+
+
4H+
+ + + + +
Cyt c
+
+ +
+
Q
-
Ⅰ
Ⅱ
-
-
F
Ⅲ
-
Ⅳ
- -
fumarate
NADH+H+
NAD+
Succinate
0
1/2O2+2H+
- -
-
H2 O
F1
Matrix
ADP+Pi
ATP
H+
目录
Summary: Two respiratory chain
Succinic acid
NADH oxidation
respiratory chain
NADH
FAD
(Fe-S)
FMN
(Fe-S)
complexesⅠ
CoQ
Succinic acid oxidation
respiratory chain
complexesⅡ
Cyt b→Cyt c1
Complexes Ⅲ
Cyt c
Cyt aa3
Complexes Ⅳ
1/2O2
5.
Oxidative Phosphorylation
•Defenition: The process of synthesizing
ATP from ADP and Pi coupled with the
electron transport chain(ETC) is know as
oxidative phosphorylation.
•It is main form of ATP producing in body.
2.5 ADP + 2.5 Pi
2.5 ATP
(1) P/O Ratio
• The relationship between ATP synthesis and
O2 consumption.
the number of moles of ATP made
P/O ratio = ----------------------------------------------(½) the number of moles of O2 consumed
• When NADH is substrate, P/O = 2-3
• When succinate is used, P/O = 1-2
• When ascorbic acid is used, P/O = 0.88
succinate
The sites of coupled oxidative
phosphorylation
(2) 4 H+ transported for each ATP
synthesized (important!)
Complex I
• H+ translocated/2e- :
2
• For NADH:
•
IV
4
10 H+ translocated / (2e-)
yield 2.5 ATPs when NADH is oxidized.
• For succinate:
•
III
4
6 H+ translocated / (2e-)
yield 1.5 ATPs when FADH2 is oxidized.
(3). Mechanisms of coupling electron
transport and oxidative
phosphorylation
•This coupling mechanism had ever been
a mystery for many years!
A. Chemical coupling
B. Conformational coupling
C. The chemiosmotic theory
The chemiosmotic theory
•Proposed by Peter Mitchell in the 1960’s
(Nobel Prize 1978)
• Chemiosmotic theory:
A proton concentration gradient serves
as the energy reservoir for driving ATP
formation.
Chemiosmotic coupling
• Electrons from NADH and FADH2 are transported
down a respiratory chain in the mitochondrial inner
membrane.
• The energy released is used to pump protons (H+)
across the inner membrane
• Creates an electrochemical proton gradient
• Flow of H+ back is used to drive the conversion of
ADP+Pi to ATP
+
+ __ + +
_
_
+ _ _ + __ +
_
+
_
+ _+ _ + _ _ +
_ _
_ +
+
+ _ + ___ _ +_ +
_
+ + + __
_
_
_ _ +
+
+ _ + _ _ +
+
+ _
+
+
+ + +
(4)
Mechanism of ATP synthesis
• ATP synthase also called Complex V, or
F0F1-ATPase
• FoF1 ATPase uses the proton gradient
energy for the synthesis of ATP
F1/F0 ATP Synthase
----“knob-and-stalk” structure
F1 (knob): catalytic
subunit, made of 5
polypeptides: a3b3gde
Fo (stalk): complex of
integral membrane
proteins that mediates
proton transport, made
of a1b2c9-12
Proton diffusion through
the protein drives ATP
synthesis
F1
Matrix
Intermembrane
space
F0
Pi+
ATP
ADP
e¯ e¯
NADH
+H+
HH++
H+ H+
+
H
H+
+
+
H
H H+H+
目录
Complex
Ⅰ
NADH
Fe-S
Amytal
Rotenone
Q
Complex
Ⅲ
b × c1
Antimycin A
C
Complex
Ⅳ
aa3
O2
CO, CN N3-
6. Drugs that inhibit the ETC
(Important!)
NAD+
FMN
I
Rotenone helps natives of the
Amazon rain forest catch fish!
Amytal
rotenone
FeS
FeS
FAD
II
ubiquinone
Cyt b
Inhibit Complex IV
Antimycin A
CN-, N3-
ubiquinone
Cyt c1
FeS
III
Cyt c
Cyt a
Cyt a3
IV
CO
1/2 O2
Oligomycin
H+
ADP
H+
H+
H+
Ⅳ
+
H H+
Q
Ⅲ
H+ H+
C
Ⅰ
Oligomycin: Inhibitors of ATP Synthase
H+ H+ H+
H+
H+
H+
• Fo is sensitive
to oligomycin, an
antibiotic that
binds in the
channel and blocks
H+ passage,
thereby inhibiting
ATP synthesis
Intermembrane
space
oligomycin
Matrix
Uncouplers
• Uncouplers disrupt the tight
coupling between electron
transport and the ATP synthase.
– They act by dissipating the proton
gradient across the inner
mitochondrial membrane created by
the electron transport system, so
affect ATP synthesis.
H+
ADP
Pi
H+
H+
Ⅳ
H+ H+
H+
H++
•Features: hydrophobic and a dissociable proton.
–Acquire protons on the cytosolic surface of the
membrane
–Carry them to the matrix side
–Thereby destroying the proton gradient that couples
electron transport and the ATP synthase.
•Electron transport continues, but ATP synthesis
does not occur. The energy released in electron
transport is dissipated as heat.
+
H H+
Q
Ⅲ
H+ H+
C
Ⅰ
2,4-Dinitrophenol( DNP): an
uncoupler
Points
• Free energy change (DG)
– negative (DG<0), spontaneously
– positive (DG>0), nonspontaneously
• High-energy Compounds
• Function of ATP
• How is ATP produced
– Substrate level phosphorylation
– Oxidative phosphorylation
• Electron transfer chain (or Respiratory
chain)
– Definition, Significance, Site, Components
– Two respiratory chain
• NADH oxidation respiratory chain
• Succinic acid oxidation respiratory chain
Points (continued)
• Oxidative Phosphorylation
–
–
–
–
P/O Ratio
2.5 ATP per NADH; 1.5 ATP per FADH2
The chemiosmotic theory
ATP synthase (or F0F1-ATPase)
• Drugs that inhibit the ETC or
oxidative phosphorylation
– Amytal, rotenone, antimycin A, CN-, CO,
N3– Oligomycin
– Uncouplers: DNP