respirasomes functional combination of two or more electron-transfer complexes respirasome of...
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Respirasomes
Functional combination of two or more electron-transfer complexes
Respirasome of complex III & IV
Cardiolipin (abundant in inner mito membrane) critical to formation of respirasomes
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Conservation of the e- Transfer Energy in a Proton Gradient
Standard free energy change of e- from NADH to O2
NADH + H+ + ½ O2 NAD+ + H2O G’o = -nFE’o = -220 kJ/mol
Oxidation of succinate G’o = -150 kJ/mol
Using the energy to pump protons out of the matrix 4H+ (complex I), 4H+ (complex III), 2H+ (complex IV)
; NADH + 11 HN+ + 1/2O2 NAD+ + 10Hp
+ + H2O
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Conservation of the e- Transfer Energy in a Proton Gradient
Electrochemcial energy proton-motive force Energy stored in proton gradient Two components
Chemical potential energy : separation of chemical H+ Electrical potential energy : separation of charge
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Proton-Motive Force
Free energy change for the creation of electrochemical gradient by ion pump
G = RTln(C2 / C1) + ZF C : concentrations of an ion, C2 > C1
Z : absolute value of electrical charge (1 for a proton) transmembrane difference in electrical potential
ln(C2 / C1) = 2.3 (log [H+]P - log [H+]N)
= 2.3 (pHN – pHP) = 2.3 pH
G = 2.3 RTpH +F Active mitochondria
; 0.15 ~ 0.2 V, pH = 0.74 G = 19 kJ/mol (of H+)
~ 200 kJ/mol (NADH)
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Reactive oxygen species (ROS)
ROS generation of oxidative phosphorylation
- Radical •Q- intermediate generated during complex I QH2
QH2 complex III
Pass an electron to O2
O2 + e- •O2- (superoxide)
- Detoxification systems Superoxide dismutase (SOD) Glutathione peroxidase (GPx)
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Oxidation of NADH in plant mitochondria
Analogous to mito ATP synthesis mechanism of animal Plant specific alternative mechanism
Regeneration of NAD+ from unneeded NADH
Direct e- transfer from ubiquinone to O2 (bypassing complex III and complex IV)
Energy from e- transfer heat generation
CN- alternative QH2 oxidase
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Oxidative Phosphorylation19.2 ATP Synthesis
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Chemiosmotic Model
“ The electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane – the proton-motive force – drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with ATP synthase”
Chemical potential∆ pH
Electrical potential∆ ψ
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Coupling of Electron Transfer & ATP Synthesis
Experiment to demonstrate ‘coupling’
ADP + Pi
succinate(1) Substrate oxidation(2) O2 consumption(3) ATP synthesis
Isolated mitochondria
Experiment 1
(1) (ADP + Pi) addition no respiration & ATP synthesis
(2) Succinate addition respiration & ATP synthesis
(3) CN- addition inhibiting respiration & ATP synthesis
Coupling of electron transfer & ATP synthesis
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Coupling of Electron Transfer & ATP Synthesis
Experiment 2
(1) Succinate addition no respiration & ATP synthesis(2) (ADP + Pi) addition respiration & ATP synthesis(3) Oligomycin or venturicidin addition inhibiting respiration & ATP synthesis(4) DNP addition continuing respiration without ATP synthesis (uncoupling)
Further demonstration of coupling of electron transfer & ATP synthesis
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Chemical uncouplers 2,4-dinitrophenol (DNP) &
carbonylcyanide-ρ-trifluoromethoxyphenylhydrazone (FCCP) Weak acids with hydrophobic properties
Release protons in the matrix dissipation of proton gradient
Ionophores Valinomycin (peptide ionophore binding K+) Transport of inorganic ions through
membranes dissipation of electrical gradient
Coupling of Electron Transfer & ATP Synthesis
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Evidence for the Role of a Proton Gradient in ATP Synthesis
Artificial generation of electrochemical gradient
Leads to ATP synthesis without oxidizable substrate
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Mechanism of ATP formation
1. Karl Lohman (1929)2. Fritz Lipmann (1953)3. Efraim Racker (1960)4. Peter Mitchell (1961)5. Masasuke Yoshida (1997) 6. Paul D. Boyer
How is the pmf transmitted to the ATP synthesis?
FoF1-ATPase
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ATP Synthase
Mitochondrial ATP synthase (complex V) F-type ATP synthase Similar to ATP synthase of chloroplast
and bacteria Two functional domains F1 : peripheral membrane protein
ATP synthesis Isolated F1 : ATP hydrolysis
(originally called F1 ATPase) Fo (o : oligomycin-sensitive)
Membrane integrated Proton pore
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ATP Synthase : F1
33(9 subunits) Knoblike structure with alternating and arrangement subunits; catalytic sites for ATP synthesis subunit
Central shaft Association with one of the three subunits (-empty)
Induction of conformational difference in subunits difference in ADP/ATP binding sites of subunits-ATP, -ADP, -empty
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ATP Synthase : Fo
ab2c10-12 (3 subunits) C subunit
Small (Mr 8,000), hydrophobic two transmembrane helices Two concentric circles
Inner circle : N-terminal helices Outer circle (55 Å in diameter) : C-terminal helices
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Mechanism of ATP synthesis in F1
18O-exchange experiment with purified F1
Incubation of purified F1 with ATP in 18O-labelled water
Analysis of 18O incorporation into Pi 3 or 4 isotopes in Pi
Repetitive reaction of both ATP hydrolysis and ATP synthesis
; ADP + Pi ATP + H2O, G’o ≈ 0 reversible reaction!
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Mechanism of ATP synthesis in F1
Kinetic study for confirmation of G’o ≈ 0 Enz-ATP Enz-(ADP + Pi)
K’eq = k-1/k1 = 24 s-1/10 s-1 = 2.4 G’o ≈ 0 differ from ATP (free in solution) hydrolysis
G’o = -30.5 kJ/mol (K’eq=105) FoF1 has high affinity to ATP (Kd < 10-12 M) than ADP (Kd ≈ 10-5 M) 40 kJ/mol
difference in binding energy Equilibrium toward ATP synthesis
Release of ATP from the enzyme surface ; major energy barrier (not ATP formation) Proton gradient makes it possible