Electron Transport Chain

 ELECTRON TRANSPORT CHAIN: CHAIN: -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 mitochondrionor at the inner mitochondrial membrane(eukaryotic cells).

 Mitochondria: •outer membrane relatively permeable •inner membrane permeable only to those things with specific transporters Impermeable to NADH and FADH2  – Impermeable Permeable to pyruvate  – Permeable •Compartmentalization  –Kreb's and β-oxidation in matrix Glycolysis in cytosol  – Glycolysis the mitochondrion contained the enzymes responsible for electron transport and oxidative phosphorylation 

REDOX POTENTIALS: Redox potential is a capacity to hold electrons within itself.Molecules with lower standard redox potential have higher capacity to donate electrons.Higher standard redox potential have capacity to accept electrons from molecules with lower standard redox potential.  Nernst equation:    

=Standard redox potential

=

Standard free energy change

n= number of electrons transferred

REDOX POTENTIALS: Redox potential is a capacity to hold electrons within itself.Molecules with lower standard redox potential have higher capacity to donate electrons.Higher standard redox potential have capacity to accept electrons from molecules with lower standard redox potential.  Nernst equation:    

=Standard redox potential

=

Standard free energy change

n= number of electrons transferred F= faraday constant (96485 J/volt/mole) Electrons move from higher to lower standard free energy change. -Removal of H across a C-C bond is not sufficiently exergonic to reduce NAD+,but it does yield enough energy to reduce FAD. -That’s why succinate dehydrogenase uses FAD other than NAD+as coenzyme. Most energy from Redox: •electrons during metabolic reactions sent to NAD and FAD  – Glycolysis •In cytosol

•produces 2 NADH  – Pyruvate dehydrogenase reaction •In mitochondrial matrix •2 NADH / glucose  – Krebs •In mitochondrial matrix •6 NADH and 2 FADH2/ glucose

 ELECTRON CARRIERS:

-The transfer of electrons is not directly to oxygen but through coenzymes. -There are 2 sites of entry for electrons into the electron transport chain: NAD+ or FAD Both are coenzymes for dehydrogenase enzymes. Nicotinamide coenzymes: NAD+ Always a 2-electron reaction transferring 2 e- and 2 H+ The flavin coenzymes / flavoproteins : -flavin adenine dinucleotide (FAD) always a 2-electron reaction transferring 2 e- and 2 H+ -it can accept/donate 1 or 2 e-.FMN has an important role in mediating etransfer between carriers that transfer 2 e-(e.g., NADH) and those that transfer 1 e-(e.g., Fe+++). 

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Iron-sulfur Centers (clusters)

-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. Ubiquinone 

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 can accept/donate 1 or 2 e-.Q can mediate e-transfer between 2 e-that transfer and 1 e-carriers Cytochromes -proteins that accept electrons from QH2 or FeS -Ultimately transfers the electrons to oxygen. -Cytochromesare 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. 

THE ELECTRON TRANSPORT CHAIN:

Support for this order of events: 1. Energetically favorable. electrons pass from lower to higher standard reduction potentials 2. Spectra: the absorption spectrum for the reduced carrier differs from that of its oxidized form. carriers closer to oxygen are more oxidized. 3. Specific inhibitors. Those before the blocked step should be reduced and those after be oxidized.4. Assay of individual complexes.NADH can reduce complex I but not the other complexes

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COMPLEX 1:

•Has NADH binding site  – NADH reductase activity •NADH -→NAD+  – NADH ---> FMN--->FeS---> ubiquinone  – ubiquinone ---> ubiquinone H2  – 4 H+pumped/NADH

NADH + H++ Q →NAD++ QH2

COMPLEX 2: succinate ---FAD — ubiquinone  – Contains coenzyme Q

 – FADH2binding site •FAD reductase activity •FADH2--→FAD Reason for no proton pump by complex 2: 1)It is not a transmembrane protein and hence cannot pump proton from matrix to intermembrane space. 2) E produced by electron transfer in complex 2 is so small that it cannot pump proton from matrix to intermembrane space,because it produces very small G. COMPLEX 3: ubiquinone - →ubiquinone ox

•while cyt C gets reduced •Also contains cytochromes b  – proton pump 4H+ •Adds to gradient 8 H+/ NADH 4 H+/ FADH2 COMPLEX 4: -reduction of oxygen -cytochrome oxidase -cyt a+a3 red---> oxidized state -oxygen ---> water  – 2 H++ 2 e-+ ½ O2--

 – transfers e-one at a time to oxygen -Pumps 2H+out  – Total of 10 H+/ NADH  – Total of 6 H+/ FADH2

 INHIBITORS OF ETS: Complex 1= Rotenone,Amytol Complex 2= Malonate,Carboxyn Complex 3= Antimycin,Mixothiozole,Dimercaparol Complex 4= Cyanite, Azide, Carbon monoxide.(These three inhibitor are known as warburg inhibitor. UNCOUPLER: A) Synthetic uncoupler: 2,4-dinitrophenol,dicumarol Uncoupler has two properties: -It should be hydrophobic which help its movement in mitochondrial membrane -It should have ionizable group which can carry a proton from intermembrane space to matrix. Uncoupler has 2 stages, one is protonated and other is deprotonated.Proton is taken from intermembrane space and release into matrix by these uncoupler which finally function just reverse to the pump which decrease the electrochemical gradient and ATP synthesis. B) Natural uncoupler: Ionophores are antibiotics that generate pore in membrane to kill the bacteria. Valonomycin decrease membrane potential component of proton motive force without direct effect on pH gradient . Nigericin disrupt the chemical gradient without changing membrane potential. 

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SHUTTLE SYSTEM:

•NADH made in cytosol •Can’t get into matrix of mitochondrion •2 mechanisms  – In muscle and brain •Glycerol phosphate shuttle  – In liver and heart •Malate / aspartate shuttle GLYCEROL PHOSPHATE SHUTTLE:

•In muscle and brain •Each NADH converted to FADH2inside mitochondrion  – FADH2enters later in the electron transport chain  – Produces 1.5 ATP Total ATP per glucose in muscle and brain •Gycerol phosphate shuttle

 – 2 NADH per glucose - →2 FADH2  – 2 FADH2X 1.5 ATP / FADH2……….3.0 ATP  –2 ATP in glycoysis……………………2.0 ATP  – From pyruvate and Krebs •12.5 ATP X 2 per glucose ……………..25.0 ATP Total = 30.0 ATP/ glucose MALATE ASPARTATE SHUTTLE:

Malate –Aspartate Shuttlein cytosol •In liver and heart

•NADH oxidized while reducing oxaloacetate to malate  –Malate dehydrogenase

•Malate crosses membrane

Malate –Aspartate Shuttlein matrix •Malate reoxidized to oxaloacetate  –Malate dehydrogenase  –NAD+reduced to NADH

•NADH via electron transport yields 2.5 ATP

Total ATP per glucose in liver and heart •Malate –Aspartate Shuttle  –2 NADH per glucose -2 NADH

 –2 NADH X 2.5 ATP /  NADH…………5.0 ATP  –2 ATP from glycolysis………………..2.0 ATP  –From pyruvate and Krebs •12.5 ATP X 2 per glucose ……………..25.0 ATP Total = 32.0 ATP/ glucose

Summary •Total ATP / glucose  –Muscle and brain30.0 ATP •Uses glycerol phosphate shuttle

 –Heart and liver32.0 ATP •Uses malate aspartate shuttle