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An Overview
Biological oxidations are catalyzed by intracellular enzymes. The purpose of oxidation is to obtain energy. Electron Transport: Electrons carried by reduced coenzymes (NADH or FADH2) are passed sequentially through a chain of proteins and coenzymes (so called electron transport chain)to O2 . Oxidative Phosphorylation: Coupling e- Transport (Oxidation) and ATP synthesis (Phosphorylation) . It all happens in mitochondrion or at the inner mitochondrial membrane (eukaryotic cells)
mitochondrion
the mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation
Reduction Potentials
E0=standard reduction potential. The relative tendency to accept e-s and become Crucial equation: reduced.
Half Reaction O2 O2 + + 2H+ 2H+ + + 2e2eH2O H2O2 Cytochrome a(Fe2+) Ubihydroquinone Cytochrome b(Fe2+) Succinate Butyryl-CoA Malate Lactate FADH2 Reduced glutathione Dihydrolipoic acid Isocitrate
E (V) 0.816 0.295 0.29 0.045 0.077 0.031 -0.015 -0.166 -0.19 -0.219 -0.23 -0.29 -0.32 -0.38 -0.414
Cytochrome a(Fe3+) + eUbiquinone + 2H+ + 2eCytochrome b(Fe3+) + eFumarate + 2H+ + 2eCrotonyl-CoA + 2H+ + 2eOxaloacetate + 2H+ + 2ePyruvate + 2H+ + 2eFAD + 2H+ + 2eGlutathione + 2H+ + 2eLipoic acid + 2H+ + 2eNAD+ + 2H+ + 2e2H+ + 2e H2 -ketoglutarate + 2H+ + 2e-
NADH + H+
Fumarate+2H++2e- succinate 0.031 FAD+ 2H++2e- FADH2 0 NAD++ 2H++2e- NADH+H+ -0.32
Succinate+FAD Fumarate+ FADH2 G' = - n F E' Eo' = Eo'(acceptor) - Eo'(donor) G =-2*96485*(0-0.031)= 5.98KJ/mol Succinate+ NAD+ Fumarate+ NADH+H+ G =-2*96485*(-0.32- 0.031)= 67.7KJ/mol
Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD. Thats why succinate dehydrogenase uses FAD other than NAD+ as coenzyme.
Electron Carriers
The transfer of electrons is not directly to oxygen but through coenzymes + There are 2 sites of entry NAD for electrons into the FMN electron transport chain: NAD+ or FAD FeS Both are coenzymes for FeS ubiquinone FAD dehydrogenase enzymes
Cyt b ubiquinone FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2
CONH2
XH2
H CONH2 + H+
OH CH O N
OH CH CH2 O
O P OH OH
H 3C H 3C
C C
H3C
O H3C
+
C C
H C
C C
H N
O C C C NH C H3C O H3C
+
C C
H C
C C
H N
O C C C NH C O
C H
CH2 HC HC HC H2C OH OH OH O O P O-
C H
e +H
CH2 HC HC HC OH OH OH O O P O-
C H
N CH2 HC HC HC
N H
e +H
OH OH OH O O P OO-
O-
H2C
O-
H2 C
FMN
FMNH
FMNH2
it can accept/donate 1 or 2 e. FMN has an important role in mediating e transfer between carriers that transfer 2 e (e.g., NADH) and those that transfer 1 e (e.g., Fe+++).
Role of FMN mediating between 2e & 1e carriers: For example, when NADH donates electrons to the respiratory chain, the initial electron transfers are: NADH + H+ + FMN NAD+ + FMNH2 FMNH2 + Fe+++ FMNH + Fe++ + H+
Iron-sulfur centers (Fe-S) are 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 +++ 3 , Fe ++ 1 (oxidized) + 1 e Fe +++ 2 , Fe ++ 2 (reduced)
S S Fe Fe S Fe
Fe S
Fe S S Cys
Cys
Iron-Sulfur Centers
NAD+
Ubiquinone
O CH3O
CH3 CH3
CH3O O
ubiquinone FeS
(CH2 CH
CH2)nH
coenzyme Q
2 e+ 2 H+
OH CH3O CH3 CH3 CH3O OH (CH2 CH C CH2)nH
coenzyme QH2
When bound to special sites in respiratory complexes, CoQ can accept 1 e- to form a semiquinone radical (Q-).
O CH3O CH3 CH3 CH3O O (CH2 CH C CH3O O CH3 CH3 CH3O O (CH2 CH C CH2)nH
CH2)nH
coenzyme Q
coenzyme Q
e + 2 H+
OH CH3O CH3 CH3 CH3O OH (CH2 CH C CH2)nH
coenzyme QH2
it can accept/donate 1 or 2 e-. Q can mediate etransfer between 2 e- that transfer and 1 ecarriers
Cytochromes
NAD+ FMN FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c Cyt a Cyt a3 1/2 O2
proteins that accept electrons from QH2 or FeS Ultimately transfers the electrons to oxygen
Cytochromes
Cytochromes are electron carriers containing hemes . Hemes 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.
Heme is a prosthetic group of cytochromes. Heme contains an iron atom in a porphyrin ring system.
The heme iron can undergo 1 e- transition between ferric and ferrous states: Fe3+ + e Fe2+ Copper ions besides two heme A groups (a and a3) act as electron carriers in Cyta,a3 Cu2++e- Cu+
Electron carriers
NAD+, flavins and Q carry electrons and H+ Cytochromes and non-haem iron proteins carry only electrons NAD+ FAD undergoes only a 2 e- reaction; cytochromes undergo only 1e- reactions FMN Q undergoes 1e- and 2 e- reaction
The electron transport chain in the inner mitochondrial membrane can be isolated in four proteins complexes(I, II, III, IV). A lipid soluble coenzyme (Q) and a water soluble protein (cyt c) shuttle between protein complexes Electrons transfer through the chain - from complexes I and II to complex IV
Mitochondrial Complexes
NAD+ FMN FeS FAD FeS ubiquinone Cyt b ubiquinone FeS Cyt c1 Cyt c
NADH Dehydrogenase
Succinate dehydrogenase
II
Cytochrome Oxidase
Cyt a Cyt a3 1/2 O2
III
CoQ-cyt c Reductase
IV
-0.32 -0.3
+0.03
+0. 55
Cyt a3 1/2 O2
+0. 22
+0. 25 +0.82
Amytal I rotenone
FAD FeS
II
Antimycin A
ubiquinone
CN- CO
binding tightly to the ferric form (Fe3+) of a3
FeS
Cyt c1
Cyt c
Cyt a
Cyt a3 1/2 O2
III
IV
When the chain is blocked, electron carriers will be in a reduced state before the block point and in an oxidized state after it. This can easily be monitored using difference spectra.
H Transport
+
Complex I, III, IV drive H + transport from matrix to the cytosol When e- flow through, which creates proton gradient(electrochemical potential) across the inner membrane Complex I and Complex IV : The mechanism of H+ transport is still not known. The mechanism of H+ transport in Complex III is Q cycle.
Matrix
H+ + NADH NAD+ + 2H+ 2H+ + O2 H2O
2 e Q
III cyt c IV
++
4H+ 4H+ 2H+
Intermembrane Space
4H+ are pumped per 2e passing through complex III. The H+/e ratio is less certain for the other complexes: probably 4H+/2e for complex I; 2H+/2e for complex IV.
Fe-S
cyt c1 cyt c
intermembrane space
1.Electrons are transported along the inner mitochondrial membrane, through a series of electron carriers 2.Protons (indicated by + charge) are translocated across the membrane, from the matrix to the intermembrane space 3.Oxygen is the terminal electron acceptor, combining with electrons and H+ ions to produce water 4. As NADH delivers more H+ and electrons into the ETS, the proton gradient increases, with H+ building up outside the inner mitochondrial membrane, and OH- inside the membrane.