1. Mitochondrial Electron Transport The cheetah, whose capacity for aerobic metabolism makes it one of the fastest animals

2. Glucose Pyruvate Acetyl Co A Fatty Acids Amino Acids Citric acid cycle supplies NADH and FADH 2 to the electron transport chain

3. Reduced coenzymes NADH and FADH 2 are formed in matrix from: (1) Oxidative decarboxilation of pyruvate to acetyl CoA (2) Aerobic oxidation of acetyl CoA by the citric acid cycle (3) Oxidation of fatty acids and amino acids The NADH and FADH 2 are energy-rich molecules because each contains a pair of electrons having a high transfer potential.

4. The reduced and oxidized forms of NAD

5. The reduced and oxidized forms of FAD

6. Electrons of NADH or FADH 2 are used to reduce molecular oxygen to water. A large amount of free energy is liberated. The electrons from NADH and FADH 2 are not transported directly to O 2 but are transferred through series of electron carriers that undergo reversible reduction and oxidation.

7. The flow of electrons through carriers leads to the pumping of protons out of the mitochondrial matrix. The resulting distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a protonmotive force.

8. ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex ATP synthase. The oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane. 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.

9. OXIDATIVE PHOSPHORYLATION IN EUKARYOTES TAKES PLACE IN MITOCHONDRIA Two membranes: outer membrane inner membrane (folded into cristae) Two compartments: (1) the intermembrane space (2) the matrix Inner mitochondrial membrane: Electron transport chain ATP synthase Mitochondrial matrix: Pyruvate dehydrogenase complex Citric acid cycle Fatty acid oxidation Location of mitochondrial complexes The outer membrane is permeable to small molecules and ions because it contains pore-forming protein (porin). The inner membrane is impermeable to ions and polar molecules. Contains transporters (translocases).

10. THE ELECTRON TRANSPORT CHAIN Series of enzyme complexes (electron carriers) embedded in the inner mitochondrial membrane, which oxidize NADH 2 and FADH 2 and transport electrons to oxygen is called respiratory electron-transport chain (ETC). The sequence of electron carriers in ETC cyt b NADH FMN Fe-S Co-Q Fe-S cyt c 1 cyt c cyt a cyt a 3 O 2 succinate FAD Fe-S

11. High-Energy Electrons: Redox Potentials and Free-Energy Changes In oxidative phosphorylation, the electron transfer potential of NADH or FADH 2 is converted into the phosphoryl transfer potential of ATP. Phosphoryl transfer potential is  G°' (energy released during the hydrolysis of activated phos- phate compound).  G°' for ATP = -7.3 kcal mol -1 Electron transfer potential is expressed as E' o, the (also called redox potential, reduction potential, or oxidation-reduction potential).

12. E' o (reduction potential) is a measure of how easily a compound can be reduced (how easily it can accept electron). All compounds are compared to reduction potential of hydrogen wich is 0.0 V. The larger the value of E' o of a carrier in ETC the better it functions as an electron acceptor (oxidizing factor). Electrons flow through the ETC components spontaneously in the direction of increasing reduction potentials. E' o of NADH = -0.32 volts (strong reducing agent) E' o of O 2 = +0.82 volts (strong oxidizing agent) cyt b NADH FMN Fe-S Co-Q Fe-S cyt c 1 cyt c cyt a cyt a 3 O 2 succinate FAD Fe-S

14. Important characteristic of ETC is the amount of energy released upon electron transfer from one carrier to another. This energy can be calculated using the formula:  G o ’=-nF  E’ o n – number of electrons transferred from one carrier to another; F – the Faraday constant (23.06 kcal/volt mol);  E’ o – the difference in reduction potential between two carriers. When two electrons pass from NADH to O 2 :  G o ’=-2*96,5*(+0,82-(-0,32)) = -52.6 kcal/mol

15. Components of electron- transport chain are arranged in the inner membrane of mitochondria in packages called respiratory assemblies (complexes). THE RESPIRATORY CHAIN CONSISTS OF FOUR COMPLEXES cyt b NADH FMN Fe-S Co-Q Fe-S cyt c 1 cyt c cyt a cyt a 3 O 2 succinate FAD Fe-S I III II IV I II III IV

17. The energy is released not in a single step of electron transfer but in incremental amount at each complex. 26.8 Energy released at three specific steps in the chain is collected in form of transmembrane proton gradient and used to drive the synthesis of ATP.

18. Complexes I-IV Mobile coenzymes: ubiquinone (Q) and cytochrome c serve as links between ETC complexes Complex IV reduces O 2 to water

19. Transfers electrons from NADH to Co Q (ubiquinone) Consist of: - enzyme NADH dehydrogenase (FMN - prosthetic group) - iron-sulfur clusters. NADH reduces FMN to FMNH 2. Electrons from FMNH 2 pass to a Fe-S clusters. Fe-S proteins convey electrons to ubiquinone. QH 2 is formed. Complex I (NADH-ubiquinone oxidoreductase) The flow of two electrons from NADH to coenzym Q leads to the pumping of four hydrogen ions out of the matrix.

20. matrix NADH-Q oxidoreductase - an enormous enzyme consisting of 34 polypeptide chains. L-shaped (horizontal arm lying in the membrane and a vertical arm that projects into the matrix). FMN NADH Iron ions in Fe-S complexes cycle between Fe 2+ or Fe 3+ states. Iron-sulfur clusters contains two or four iron ions and two or four inorganic sulfides. Clusters are coordinated by four cysteine residues. Fe-S

21. Complex II (succinate-ubiquinon oxidoreductase) Transfers electrons from succinate to Co Q. Form 1 consist of: - enzyme succinate dehydrogenase (FAD – prosthetic group) - iron-sulfur clusters. Succinate reduces FAD to FADH 2. Then electrons pass to Fe-S proteins which reduce Q to QH 2 Form 2 and 3 contains enzymes acyl-CoA dehydrogenase (oxidation of fatty acids) and glycerol phosphate dehydrogenase (oxidation of glycerol) which direct the transfer of electrons from acyl CoA to Fe-S proteins. Complex II does not contribute to proton gradient.

22. Ubiquinone Q: - lipid soluble molecule, - smallest and most hydrophobic of all the carriers - diffuses within the lipid bilayer - accepts electrons from I and II complexes and passes them to complex III. All electrons must pass through the ubiquinone (Q)- ubiquinole (QH 2 ) pair.

23. Complex III (ubiquinol-cytochrome c oxidoreductase) Transfers electrons from ubiquinol to cytochrome c. Consist of: cytochrome b, Fe-S clusters and cytochrome c 1. Cytochromes – electron transferring proteins containing a heme prosthetic group (Fe 2+  Fe 3+ ). Oxidation of one QH 2 is accompanied by the translocation of 4 H + across the inner mitochondrial membrane. Two H + are from the matrix, two from QH 2

24. Q-cytochrome c oxidoreductase is a dimer. Each monomer contains 11 subunits. Q-cytochrome c oxidoreductase contains three hemes: two b-type hemes within cytochrome b, and one c-type heme within cytochrome c 1. Enzyme also contains an iron-sulfur protein with an 2Fe-2S center.

25. Q cycle  two molecules of QH 2 are oxidized to form two molecules of Q,  one molecule of Q is reduced to QH 2,  two molecules of cytochrome c are reduced,  four protons are released on the cytoplasmic side,  two protons are removed from the mitochondrial matrix

26. Complex IV (cytochrome c oxidase) Transfers electrons from cytochrome c to O 2. Composed of: cytochromes a and a 3. Catalyzes a four-electron reduction of molecular oxygen (O 2 ) to water (H 2 O): O 2 + 4e - + 4H +  2H 2 O Translocates 2H + into the intermembrane space

27. Cytochrome c oxidase consists of 13 subunits and contains two hemes (two iron atom) and three copper ions, arranged as two copper centers.

28. The Catalytic Cycle of Cytochrome c Oxidise

29. The four protons used for the production of two molecules of water come from the matrix. The consumption of these four protons contributes to the proton gradient. Cytochrome c oxidase pumps four additional protons from the matrix to the cytoplasmic side of the membrane in the course of each reaction cycle (mechanism under study). Totally eight protons are removed from the matrix in one reaction cycle (4 electrons)

30. Cellular Defense Against Reactive Oxygen Species If oxygen accepts four electrons - two molecules of H 2 O are produced single electron - superoxide anion (O 2.- ) two electrons – peroxide (O 2 2- ). O 2.-, O 2 2- and, particularly, their reaction products are harmful to cell components - reactive oxygen species or ROS. DEFENSE superoxide dismutase (manganese-containing version in mitochondria and a copper-zinc-dependent in cytosol) O 2.- + O 2.- + 2H + = H 2 O 2 + O 2 catalase H 2 O 2 + H 2 O 2 = O 2 + 2 H 2 O antioxidant vitamins: vitamins E and C reduced glutathione