1. Electron Transport Chain and Oxidative Phosphorylation Dr. Sooad Al-Daihan Biochemistry department

2. Introduction The NADH and FADH 2 formed in glycolysis, fatty acid oxidation, and the citric acid cycle are energy-rich molecules. because each contains a pair of electrons having a high transfer potential. The NADH and FADH 2 formed in glycolysis, fatty acid oxidation, and the citric acid cycle are energy-rich molecules. because each contains a pair of electrons having a high transfer potential. When these electrons are used to reduce molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP. When these electrons are used to reduce molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers. Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of electron carriers.

3. Continue.. The inner mitochondrial membrane contains 5 separate enzyme complexes, called compelexes I, II, III, IV and V. Each complex accepts or donates electrons to mobile carrier, such as coenzyme Q and cytochrome c. The electrons ultimately combine with oxygen and protons to form water.

4. The Components of the ETC The components of the electron transport chain are organized into 4 complexes. Each complex contains several different electron carriers. 1. Complex I also known as the NADH-coenzyme Q reductase or NADH dehydrogenase. 2. Complex II also known as succinate-coenzyme Q reductase or succinate dehydrogenase. 3. Complex III also known as coenzyme Q reductase. 4. Complex IV also known as cytochrome oxidase. Each of these complexes are large multisubunit complexes embedded in the inner mitochondrial membrane.

5. Complex I: Also called NADH-Coenzyme Q reductase because this large protein complex transfers 2 electrons from NADH to coenzyme Q. Complex I was known as NADH dehydrogenase. Complex I (850,000 kD) contains a FMN prosthetic group which is absolutely required for activity and seven or more Fe-S clusters. This complex binds NADH, transfers two electrons in the form of a hydride to FMN to produce NAD + and FMNH 2. The subsequent steps involve the transfer of electrons one at a time to a series of iron-sulfer complexes.

6. Continue.. The importance of FMN The importance of FMN. First First it functions as a 2 electron acceptor in the hydride transfer from NADH. Second Second it functions as a 1 electron donor to the series of iron sulfur clusters. The process of transferring electrons from NADH to CoQ by complex I results in the net transport of protons from the matrix side of the inner mitochondrial membrane to the inter membrane space where the H + ions accumulate generating a proton motive force. The stiochiometry is 4 H + transported per 2 electrons.

7. Complex II It is none other than succinate dehydrogenase, the only enzyme of the citric acid cycle that is an integral membrane protein, so its the only membrane-bound enzyme in the citric acid cycle This complex is composed of four subunits. Two of which are iron-sulfur proteins and the other two subunits together bind FAD through a covalent link to a histidine residue.

8. Continue.. In the first step of this complex, succinate is bound and a hydride is transferred to FAD to generate FADH 2 and fumarate. FADH 2 then transfers its electrons one at a time to the Fe-S centers. Thus once again FAD functions as 2 electron acceptor and a 1 electron donor. The final step of this complex is the transfer of 2 electrons one at a time to coenzyme Q to produce CoQH 2.

9. Complex III Q-cycle. This complex is also known as coenzyme Q- cytochrome c reductase because it passes the electrons form CoQH 2 to cyt c through a very unique electron transport pathway called the Q-cycle. In complex III we find two b-type cytochromes and one c-type cytochrome and iron sulfur proteins.

10. Complex IV Complex IV is also known as cytochrome c oxidase because it accepts the electrons from cytochrome c and directs them towards the four electron reduction of O 2 to form 2 molecules of H 2 O. Cytochrome c oxidase contains 2 heme centres, cytochrome a and cytochrome a3 and two copper proteins. The reduction of oxygen involves the transfer of four electrons. Four protons are abstracted from the matrix and two protons are released into the intermembrane space

11. Summary ComplexFunctional Groups FunctionPoisons Complex I- NADH-CoQ reductase FMN, Fe-S protein -Oxidizes NADH to NAD + -Transfers electrons to CoQ Retenone Complex II- Succinate dehydrogenase FAD, Fe-S protein -Oxidizes succinate to fumarate with reduction of FAD to FADH 2 -Electron transfer to CoQ Complex III- CoQ-cytochrome c reductase Cytochrome b, cytochrome c, Fe-S protein -Transfers electrons between CoQ and cytochrome C Antimycin A Complex IV- Cytochrom c oxidase Cytochrome a, Cytochrome a3, Cu Protein -Oxidizes cytochrome C -Reduces ½ O 2 to H 2 O Carbon monoxide, Cyanide

12. Overall Reaction NADH + 11H + matrix + ½ O 2  NAD + + 10 H + intermem +H2O Protons Pumped out to the intermembrane space : Complex I 4H + Complex III 4H + Complex IV 2H +

13. ATP synthetase: ATPase (Complex V) This enzyme complex synthesizes ATP, utilizing the energy of the proton gradient (proton motive force) generated by the electron transport chain. The Chemiosmotic theory proposes that after proton have transferred to the cytosolic side of inner mitochondrial membrane, they can re-enter the matrix by passing through the proton channel in the ATPase (F 0 ), resulting in the synthesis of ATP in (F 1 ) subunit.

15. Properties of ATP synthetase Multisubunit transmembrane protein Molecular mass ~450 kDa Functional units ◦ F0: water ‐ insoluble transmembrane protein (up to 8 different subunits) ◦ F1: water ‐ soluble peripheral membrane protein (5 subunits),contains the catalytic site for ATP synthesis Flow of 3 protons through ATP synthase leads to phosphorylation of 1 ADP

17. Electron transport inhibitors These compounds prevent the passage of electrons by binding to chain components, blocking the oxidation/reduction reaction Inhibition of electron transport also inhibits ATP synthesis.

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19. Ionophores Ionophores are termed because of their ability to form complex with certain cations and facilitate their transport across the mitochondrial membrane  So ionophores are lipophilic. e. g: Valinomycin: allows penetration of K + across the mitochondrial membrane and then discharges the membrane potential between outside and the inside ( i.e: does not affect the pH potential). Nigericin: also acts as ionophore for K + but in exchange with H +. It therefore abolishes the pH gradient.