Download - Biological oxidation i
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Dr. Subodhini Abhang
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Points to be covered Over view
Redox potential .Arrangement of components in ETC.
Coupled nature of respiration in mitochondria
Substrate Level Phosphorylation
Components of electron transport chain
P : O ratio and its calculation
Mechanism of oxidative phosphorylation
Inhibitors
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An Overview
• Biological oxidations are catalyzed by intracellular enzymes, to obtain energy.
• Electron Transport: Electrons carried by reduced coenzymes (NADH or FADH2) are passed sequentially through a chain of proteins and coenzymes ( electron transport chain)to O2 .
• Oxidative Phosphorylation: Coupling e- transport, oxidation and ATP synthesis (Phosphorylation) .
• Site of oxidative phosphorylation : inner mitochondrial membrane (Eukaryotic cells)
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Redox potential E₀
Redox potential or oxidation –reduction potential is a quantitative measure of the tendency of redox pair to loose or gain electrons.
Free energy changes can be expressed in terms of
Free energy change is proportionate to the tendency of reactants to donate or accept the electrons.
−ve redox potential
+ve redox potential
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Arrangement of components in ETC
• Arranged in the order of increasing redox potential.
From electro − ve to electro + ve
Redox pair E₀
NAD⁺/NADH -0.32 FMN/FMNH₂ -0.22Pyruvate/Lactate -0.19Cytochrome c Fe³⁺/Fe²⁺ +0.07O₂/ H₂O + 0.82
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NADH & FADH₂
C
Carbohydrates
LipidsProteins
TCA cycle
Oxidation is coupled to phosphorylation of ADP
Respiration (consumption of oxygen)proceeds only when ADP is present
•Amount of o₂ consumeddepends on amount of ADP added .
Coupled nature of respiration in mitochondria.
Energy rich
A pair of electrons
Having high transfer potential
O₂ H₂O
Donated to
Free energyliberated
Utilized for ATP generation
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Substrate Level Phosphorylation
Glyceraldehyde -3-Phosphate+ NAD ⁺+ Pi 1~3 bis phosphoglycerate + NADH
1~3bis phosphoglycerate + ADP 3 Phosphoglycerate + ATP
2) 2~ Phosphoenol pyruvate Pyruvate + ATP
3) α-ketoglutarate + NAD⁺ + CoA Succinyl ~ CoA + NADH + H⁺
Succinyl~CoA + GDP + Pi Succinate + GTP
1~
ADP
Synthesis of ATP without involving electron transport chain.
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Definition
• The process of synthesizing ATP from
ADP and Pi coupled with the electron
transport chain is known as oxidative
phosphorylation.
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Oxidative Phosphorylation
• Energy is released when electrons are transported from higher energy
NADH/FADH2 to lower energy O2 .
• This energy is used to phosphorylate ADP.
• This coupling of ATP synthesis to NADH/FADH2 oxidation is called oxidative phosphorylation.
• Oxidative phosphorylation is responsible for 90 % of total ATP synthesis in the cell.
Site …................ Mitochondria
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Mitochondria
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Impermeable
to ions and
most other
compounds
In inner
membrane
knobs
MitochondrionThe enzymes responsible for electron transport and
oxidative phosphorylation present in mitochondria.
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∙∙∙∙∙∙∙∙∙ ∙∙∙∙ ∙∙∙
Structure of Mitochondria
Β-oxi
TCA
Phosphorylating subunits
Matrix
Inner Membrane
Outer membrane
Cristae
(B)
(B)
Inner membrane
F1 subunit
Fo subunit
ATP synthase
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Electron Carriers
NAD+
FMN
FeS
ubiquinoneFAD FeS
Cyt b
FeS Cyt c1 Cyt c Cyt a Cyt a3
1/2 O2
ubiquinone
NAD+ or FAD
There are 2 sites of entry
for electrons into the
electron transport chain:
Both are coenzymes for
dehydrogenase enzymes
The transfer of electrons is not directly to oxygen but
through coenzymes
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Components of electron transport chain
• Components are arranged in order of increasing redox potential.
• From electro –ve to +ve
• NAD⁺/NADH to O₂/H₂O
• -0.32 to +0.82
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Components of electron transport chain
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Complex I
• NADH -ubiquinone oxidoreductase (NADH dehydrogenase )
• Embedded in mitochondrial membrane
• Transfers electrons from NADH to Q
• NADH transfers two electrons as a hydride ion (H: H:-) to FMN
• Electrons, one at a time , are passed through Complex I to Q via FMN and iron -sulfur proteins
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• Succinate -ubiquinone oxidoreductase(or succinate dehydrogenase complex)• Accepts electrons from succinate and catalyzes the reduction of Q to QH 2• FAD of II is reduced in a 2 -electron transfer of a hydride ion fromsuccinate• Complex II does not contribute to proton gradient , but supplieselectrons from succinate
Complex II
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Fe
Fe
S
S
S
Fe
Fe
S
S
S
SS
Cys
Cys
Cys
Cys
S
Fe
S
Fe
S
S
S
S
Cys
CysCys
Cys
Iron-Sulfur Centers
3. Iron sulfur proteins-: Exist in oxidized ( Fe³⁺) & reduced (Fe²⁺) state.
Transfers electrons FMNH₂ Q & Q b, c₁
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Iron-sulfur centers (Fe-S) have prosthetic groups containing 1-4 iron atoms
Iron-sulfur centers transfer only one electron, even if they contain two or more iron atoms.
E.g., a 4-Fe center might cycle between redox states:
Fe+++, Fe++1 (oxidized) + 1 e- Fe+++
1, Fe++ (reduced)
Iron-sulfur Centers (clusters)
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biquinone
QCoenzyme Q CoQ
Other names and abbreviations:
FAD FeS
FeS
FeS
FMN
NAD+
ubiquinone
Cyt b
ubiquinone
O
O
CH3O
CH3CH3O
(CH2 CH C CH2)nH
CH3
OH
OH
CH3O
CH3CH3O
(CH2 CH C CH2)nH
CH3
2 e- + 2 H
+
coenzyme Q
coenzyme QH2
Free CoQ can undergo a 2 e-
oxidation/reduction:
Q + 2 e- + 2 H+ QH2.
Ubiquinone or Coenzyme Q
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Coenzyme Q
• Coenzyme Q (CoQ, Q or ubiquinone) is lipid-soluble. It dissolves in the hydrocarbon core of a membrane.
• The only electron carrier not bound to a protein. It is a mobile electron carrier.
• It has ability to accept electrons in pairs and pass them one at a time through a semiquinone intermediate to complex III.
• This is called Q cycle.
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Cytochromes
NAD+
FMN
FeS
ubiquinoneFAD FeS
Cyt b
FeS Cyt c1 Cyt c Cyt a Cyt a3
1/2 O2
ubiquinone
proteins that accept
electrons from QH2 or
FeS
Ultimately transfers the
electrons to oxygen
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Cytochromes are electron carriers containing heme . Heme in the 3 classes of cytochrome (a, b,c) differ in substituents on the porphyrin ring.
Some cytochromes(b,c1,a,a3) are part of large integral membrane protein complexes.
Cytochrome c is a small, water-soluble protein.
Cytochrome c is also a mobile electron carrier.
Cytochromes
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Oxidative Phosphorylation
Oxidation and phosphorylation are coupled processes by proton gradient across the inner mitochondrial membrane.
Mechanism of oxidative phosphorylation :
1. Chemical hypothesis.
2. Chemiosmotic
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Chemical Hypothesis
• A series of phosphorylated high energy intermediates are formed and utilized for ATP synthesis.
• 1.Only substrate level phosphorylation can be explained.
• 2.Lacks experimental evidence.
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Chemiosmotic Theory
Proposed by Peter Mitchell in 1961
Most accepted.
It explains how transport of electrons through respiratory chain is utilized to produce ATP from ADP +Pi
Explains action of uncouplers.
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NAD+
FMN
FeS
ubiquinoneFAD FeS
Cyt b
FeS Cyt c1 Cyt c Cyt a Cyt a3
1/2 O2
ubiquinone
I
II
III IV
Mitochondrial Complexes
NADH Dehydrogenase
Succinate
dehydrogenase
CoQ-cyt c Reductase
Cytochrome Oxidase
NADH
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NADHα-Ketoglutarate
IsocitratePyruvate
β-HydroxybutyrateMalate
β-hydroxy acyl coA
FMN,Fe.SComplex I
ADP + Pi
ATP
QFADFe.S
Complex II
Fatty acyl coA
Glycerol -3-Phosphate
Succinate
CCcccCCyt b, Fe-S, cytC₁Complex III
ADP + Pi
ATP
Cyt. C
Heme a heme a₃Cu Cu
O₂
ADP + Pi
ATP
Complex IV
Entry of reducing equivalents and synthesis of ATP
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Complex I
Complex I: NADH-CoQ oxidoreductase
*Entry site for NADH + H+
*Contains:Fe-S cluster (non-heme protein)
flavin mononucleotide phosphate (FMN)
Coenzyme Q (free in membrane)
*Net reaction: NADH + H+ + CoQ ---> NAD+ + CoQH2*ΔG°' = -81.0 kJ/mol•complex I pumps protons outside the mitochondria•ATP produced
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Complex II
Complex II: Succinate-CoQ reductase
*Entry site for FADH2
*Contains:Fe-S cluster (non-heme protein)Coenzyme Q (free in membrane)*Net reaction: Succinate + CoQ --> Fumarate +CoQH2*ΔG°' = -13.5 kJ/mol* Conversion of succinate to fumarate is reaction of TCAcycle and is catalyzed by succinate dehydrogenase
Not a proton pumpNo ATP produced
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Complex III
Complex III: CoQH2-cytochrome c oxidoreductase
*Contains:cytochrome c (free in membrane)cytochrome bcytochrome c1Several Fe-S cluster (non-heme protein)*Net reaction: CoQH2 + 2 cyt c [Fe ³⁺] ---> CoQ + 2 cytc[Fe ²⁺ ] + 2 H+*ΔG°' = -34.2 kJ/mol•Complex III pumps protons outside the mitochondria•ATP produced.
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Complex IV
Complex IV: cytochrome oxidase
*Contains:cytochrome acytochrome a3Copper*Net reaction: 2 cyt c [Fe ²⁺]+ 1/2 O2 + 2 H+ ---> 2 cyt c[Fe ³⁺] + H2O*ΔG°' = -110.0 kJ/mol* Complex IV pumps protons outside the mitochondria* ATP produced
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III IVI
F1
FоQ
NADH+H⁺ NAD
II
Succinate Fumarate
4H⁺4H⁺2H⁺
Cyt c H⁺
H⁺
H⁺
H⁺
Uncouplers
H⁺
H⁺½O₂ + 2H⁺ H₂O
Inter membrane space +++++ +++++++ +++ +++
Inner mitochondrial membrane
Mitochondrial matrix
ADP + Pi ATP
The chemiosmotic theory 0f oxidative phosphorylation
−− −−−−− − −− −−−
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Salient features of chemiosmotic theory
Inner mitochondrial membrane is impermeable to ions particularly to protons (H
Complex I, III, and IV acts as a proton pump.Pumping of electrons results in: a) Electrical gradient : as
protons are +vely charged , inter membrane space becomes more electro +ve as compare to mitochondrial matrix or in other words mitochondrial matrix becomes electro –ve. Thus potential difference is produced. b)Chemical gradient : accumulation of H+ causes lowering of pH in inter membrane space where as mitochondrial matrix become alkaline as compare to inter membrane space . Thus chemical gradient is produced.
Hence this is called electrochemical or proton gradient.
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Salient features of chemiosmotic theory
The electrochemical potential difference across the membrane, once established as a result of proton translocation , inhibits further transport of reducing equivalents through the respiratory chain unless discharged by back translocation of protons across the membrane through ATP synthase .
This in turn depends on availability of ADP and Pi.
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P : O Ratio
Refers to number phosphate group incorporated into ATP for every atom of O₂ consumed in oxidation.
OR
Represents number of ATP synthesized per pair electron carried through ETC.
P:o = 3 Mitochondrial oxidation of NADH
NADH + H⁺ +½ O₂ + 3ADP + 3Pi 3 NAD + 3ATP + 4 H₂O
P:o = 2 Mitochondrial oxidation of FADH₂
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Calculation of the P:O ratio
molecules of ADP phosphorylated
P:O ratio = -----------------------------------------atoms of oxygen reduced
Complex I II III IV#H⁺ translocated/2e 4 0 4 2
Since 4 H⁺ are required for each ATP synthesized:
For NADH: 10 H⁺ translocated / O (2e⁻ ) P/O = (10 H ⁺/ 4 H⁺ ) = 2.5 ATP/O
For succinate substrate = 6 H⁺ / O (2e⁻ )P/O = (6 H⁺ / 4 H⁺ ) = 1.5 ATP/O
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Energetics of oxidative phosphorylation
NAD⁺/NADH 1/2O₂/H₂O—O.32 +0.82
1/2O₂ + NADH + H⁺ H₂O + NAD⁺
Potential difference 1.14V = 52 cal/mol
3 ATP = 21.9cal
Efficiency of energy conservation
21.9 x 100 52
42%
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Sites of ATP Synthesis• Site 1---Oxidation of FMNH₂ by Coenz Q
• Site2--- Oxidation of cyt.b by cyt.c₁
• Site3---cyt. Oxidase reaction (bet. a+a₃)
• When difference of redox potential betweentwo redox pairs >0.15 volts
• Or ∆ G > 7.3 Kcal
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NADH
FMN,Fe.SComplex I
ADP + Pi
ATP
QFADFe.S
Complex II
Fatty acyl coA
Glycerol -3-Phosphate
Succinate
CCcccCCyt b, Fe-S, cytC₁Complex III
ADP + Pi
ATP
Cyt. C
Heme a heme a₃Cu Cu
O₂
ADP + Pi
ATP
Complex IV
PiericidineAmobarbitalRotenone
Oligomycin
BAL(dimercaprol)Antimycin A
Sites of ATP synthesis & Inhibitors
H₂S, CO, CN
Uncouplers
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Uncouplers
Can uncouple or delink Allow oxidation without phosphorylation No ATP formation O₂ Consumption
Eg.1)2-4 dinitro phenol(DNP) - lipid soluble uncouple
2) Thermogenin- Physiological uncoupler
3) High doses of AspirinTri-fluorocarbamylcynide phenylhydrazone(FCCP) : 100 times more effective as an than dinitrophenol (DNP)
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IONOPHORES
• Ionophores—Lipid soluble compounds
permiability of lipid bilayers
to certain ions.
Eg. Valinomycin & Nigercin
Permit potassium ion to penetrate through mitochondrial membrane discharging the membrane potential..
K⁺ ions exchanges with H⁺
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Rate limiting factors
• Availabity of ADP & Substrate.
• Availabity of Substrate.
• Availibity of ADP only.
• Availibity of O₂ only.
• Capacity of respiratory chain.
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The inner mitochondrial membrane is impermeable.
Therefore ,NADH produced in cytosol cannot directly enter mitochondria.
• Two shuttle systems for transport of reducing equivalents:
• Transport : Cytosol To Mitochondria but not vice versa
(1) Glycerol phosphate shuttle : insect flight muscles
(2) Malate Malate-aspartate shuttle : predominant in liver and other mammalian tissues
Aerobic Oxidation of Cytosolic NADH
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Oxaloacetate Glutamate
Cytosolic malatedehydrogenase
NADH + H⁺
NAD⁺
MalateCytosol
Oxaloacetate Glutamate
Malate
Mitochondrial malatedehydrogenase
NADH + H⁺
NAD⁺
Electron transport chain
α-Ketoglutarate Aspartate
Aspartatetransaminase
α-Ketoglutarate Aspartate
Aspartatetransaminase
Mitochondrial Matrix
Malate – Aspartate shuttle
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Glycerophosphate shuttle