Electron Transport and Oxidative. Phosphorylation

Transcription

1 Electron Transport and Oxidative Phosphorylation

2 Electron-transport chain electron- Definition: The set of proteins and small molecules involved in the orderly sequence of transfer to oxygen within the inner mitochondrial membranes. 2

3 Mitochondria outer membrane relatively permeable inner membrane permeable only to those things with specific transporters Impermeable to NADH and FADH 2 Permeable to pyruvate

4 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 FADH 2 / glucose

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6 Electron Transport Chain Groups of redox proteins On inner mitochondrial membrane Binding sites for NADH and FADH 2 On matrix side of membrane Electrons transferred to redox proteins NADH reoxidized to NAD + FADH 2 reoxidized to FAD

7 Respiratory chain consists of 3 proton pumps linked by two mobile electron carriers. -These are NADH Q reductase, cytochrome reductase and cytochrome oxidase. The mobile carriers are ubiquinone (Q) and Cytochrome c - Electrons are carried from NADH-Q reductase to cytochrome reductase by ubiquinone (Q). Ubiquinone also carries electrons from FADH 2 to cytochrome reductase. - Cytochrome c, a small protein shuttles electrons between cytochrome reductase to cytochrome oxidase - NADH-Q reductase, succinate-q reductase, cytochrome reductase, and cytochrome oxidase are also called Complex I, II, III, and IV, respectively. 7

8 Phosphorylation at the respiratory chain level, is the process in which ATP is formed as a result of transfer of the electrons to O 2 by a series of electron carriers NADH and FADH 2 formed in glycolysis, fatty acid oxidation and CAC are energy rich molecules, each contains a pair of electrons with high transfer potential.. When these electrons are donated to molecular O 2, energy is liberated which can be used to generate ATP. The flow of these electrons through protein complexes located in the inner membrane of the mitochondria leads to pumping of protons out of the mitochondrial matrix. A proton-motive force is generated consisting of a ph gradient and a transmembrane electric potential. ATP is synthesized by ATP synthase when protons flow back to the mitochondrial matrix through an enzyme complex 8

9 4 Complexes proteins in specific order Transfers 2 electrons in specific order Proteins localized in complexes Embedded in membrane Ease of electron transfer Electrons ultimately reduce oxygen to water 2 H e - + ½ O 2 -- H 2 O

10 Electron Transport Chain

11 Complex 1 Has NADH binding site NADH reductase activity NADH - NAD + NADH ---> FMN--->FeS---> ubiquinone ubiquinone ---> ubiquinone H 2 4 H + pumped/nadh

12 NADH-Q reductase (Complex I). The electrons of NADH enter the chain at NADH-Q reductase (NADH dehydrogenase or Complex I). Electrons are then shunted to Coenzyme Q or ubiquinone (Q) -Flow of electrons from NADH through NADH-Q reductase to ubiquinone (Q) leads to the pumping of H + from matrix to the cytosolic side of the inner mitochondrial membranes Complex I is inhibited by amytal ( a barbiturate), rotenone (a plant product commonly used as an insecticide) and piericidin (an antibiotic) Ubiquinone is also entry point for electrons from FADH 2 of the succinate-q reductase complex (Complex II) 12

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14 Complex II Succinate dehydrogenase (Complex II). Is not a proton pump because less energy is released by the flow of electrons succinate ---FAD ubiquinone Contains coenzyme Q FADH 2 binding site FAD reductase activity FADH 2 -- FAD

15 Complex III Electron flow from ubiquinone to Cytochrome C through cytochrome reductase (Complex III) The flow of a pair of electrons through the complex to Cytochrome C leads to the effective transfer of H + to the cytosolic side Complex III is inhibited by dimercaprol and antimycin A ubiquinone - ubiquinone ox while cyt C gets reduced Also contains cytochromes b proton pump 4H + Adds to gradient 8 H + / NADH 4 H + / FADH 2

16 Cytochrome Oxidase (Complex IV) Catalyze the transfer of electrons from cytochrome c to molecular oxygen Protons are translocated when a pair of electrons flows through the oxidase. Electrons are funnelled into O 2 to completely reduce it to H 2 O and pump protons from the matrix to the cytosolic side of the inner mitochondrial membrane.. Complex IV is inhibited by cyanide, carbon monoxide, azide ATP is synthesized when protons flow back into the mitochondrial matrix through pores associated with ATP synthase 16

17 Complex IV reduction of oxygen cytochrome oxidase cyt a+a3 red ---> oxidized state oxygen ---> water 2 H e - + ½ O H 2 O transfers e - one at a time to oxygen Pumps 2H + out Total of 10 H + / NADH Total of 6 H + / FADH 2

18 Totals Proton gradient created as electrons transferred to oxygen forming water 10 H + / NADH 6 H + / FADH 2

19 Electron Transport Chain

20 Generation of ATP Proton dependant ATP synthase Uses proton gradient to make ATP Protons pumped through channel on enzyme From intermembrane space into matrix ~4 H + / ATP Called chemiosmotic theory

21 ATP synthase complex. The ATP synthase complex consists of a motor (F0) and generator (F1). The proton pore involves the c- ring and the a-protein. The rotary component is the coiled-coil γ- subunit, which is bound to the ε- subunit and to the c-ring. The stationary component is the hexameric α3β3 unit, which is held in place by the δ, b and a- proteins.

22 Binding-change mechanism of ATP synthase. Powered by protons, the rotation of the γ- subunit of ATP synthase induces simultaneous conformational changes in all three αβ-dimers. Each 120 rotation results in ejection of an ATP, binding of ADP and Pi and ATP synthesis.

23 Totals NADH 10 H + X 1 ATP = 2.5 ATP 4 H + FADH 2 6 H + X 1 ATP = 1.5 ATP 4 H +

24 Total ATP from mitochondrial matrix Pyruvate dehydrogenase NADH.2.5 ATP Krebs 3 NADH X 2.5 ATP/NADH.7.5 ATP FADH 2 X 1.5 ATP / FADH ATP GTP X 1 ATP / GTP..1.0 ATP (from a separate reaction) Total (Per glucose = X 2 = 25 ATP).12.5 ATP

25 What about NADH from glycolysis? 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

26 Glycerol phosphate shuttle In muscle and brain Each NADH converted to FADH 2 inside mitochondrion FADH 2 enters later in the electron transport chain Produces 1.5 ATP

27 Total ATP per glucose in muscle and brain Gycerol phosphate shuttle 2 NADH per glucose - 2 FADH 2 2 FADH 2 X 1.5 ATP / FADH ATP 2 ATP in glycoysis 2.0 ATP From pyruvate and Krebs 12.5 ATP X 2 per glucose ATP Total = 30.0 ATP/ glucose

28 Malate Aspartate Shuttle in cytosol In liver and heart NADH oxidized while reducing oxaloacetate to malate Malate dehydrogenase Malate crosses membrane

29 Malate Aspartate Shuttle in matrix Malate reoxidized to oxaloacetate Malate dehydrogenase NAD + reduced to NADH NADH via electron transport yields 2.5 ATP

30 Redox shuttles in the inner mitochondrial membrane

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32 Oxidation and ATP synthesis are coupled by transmembrane proton fluxes Sequence of electron carriers in the respiratory chain. NADH NADH-Q Reductase (I) Q Succinate-Q reductase complex (II) Cytochrome reductase (III) Cyt c Cytochrome oxidase (IV) O 2 32

33 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 ATP Total = 32.0 ATP/ glucose

34 Summary Total ATP / glucose Muscle and brain Uses glycerol phosphate shuttle Heart and liver Uses malate aspartate shuttle 32.0 ATP 30.0 ATP

35 Inhibitors of Oxidative Metabolism Electron Transport System Inhibitors Inhibitors of electron transport selectively inhibit complexes I, III or IV, interrupting the flow of electrons through the respiratory chain. This stops proton pumping, ATP synthesis, and oxygen uptake.

36 Rotenone and amobarbital block electron transfer in NADH-Q reductase. Antimycin A interferes with electron flow in cytochrome reductase. CN, hydrogen sulfide, and CO block electron flow in cytochrome oxidase Uncouplers like 2,4-dinitrophenol dissociate oxidation in the respiratory chain from phosphorylation. They dissipate the proton gradient by carryng protons across the inner mitochondrial membrane Electron transport from NADH to O 2 proceeds normally but ATP is not formed. Oxidation of brown fat in newborn mammals serves not to produce ATP but to generate heat to keep the new born warm Mitochondria of brown fat have a unique protein in their inner membrane called thermogenin (uncoupling protein) Thermogenin provides a path for the protons to return to the matrix without ATP formation As a result the energy of oxidation is not conserved by ATP formation, but is dissipated as heat The same principle applies in hibernating animals 36

37 Rotenone inhibits complex I (NADH-Q reductase) Rotenone, a common insecticide, and some barbiturates (e.g. amytal) inhibit complex I. Because malate and lactate are oxidized by NAD +, their oxidation will be decreased by rotenone. Substrates yielding FADH 2 can still be oxidized, because complex I is bypassed and electrons are donated to ubiquinone. Rotenone inhibition of complex I causes reduction of all components prior to the point of inhibition, because they cannot be oxidized, whereas those after the point of inhibition become fully oxidized.

38 Antimycin A inhibits complex III (QH 2 -cytochrome c reductase) The inhibition of complex III by antimycin A prevents transfer of electrons from either complex I or FADH 2 -containingflavoproteins to cytochrome c. In this case, components preceding complex III become fully reduced, and those after it become oxidized.

39 Cyanide and carbon monoxide inhibit complex IV Azide (N 3 ), cyanide (CN ), and carbon monoxide (CO) inhibit complex IV (cytochrome c oxidase) Because complex IV is the terminal electron transfer complex, its inhibition cannot be bypassed. All components preceding complex IV become reduced, oxygen cannot be reduced, none of the complexes can pump protons, and ATP is not synthesized. Cyanide and carbon monoxide also bind to hemoglobin, and it cannot carry oxygen. In these poisonings, both the ability to transport oxygen and to synthesize ATP are impaired.

40 Inhibitors of ATP Synthase Oligomycin inhibits respiration but, in contrast to electron transport inhibitors, it is not a direct inhibitor of the electron transport system. It inhibits the proton channel of ATP synthase. It causes an accumulation of protons outside the mitochondrion, because the proton pumping system is still intact but the proton channel is blocked.

41 Inhibitors of the ADP ATP Translocase Most ATP is synthesized in the mitochondrion, but used in the cytosol for biosynthetic reactions. Newly synthesized mitochondrial ATP and spent cytosolic ADP are exchanged by a mitochondrial ADP ATP translocase, representing about 10% of the protein in the inner mitochondrial membrane. The translocase can be inhibited by unusual plant and mold toxins, such as bongkrekic acid and atractyloside.

42 1. How many proton pumps are in the electron transport chain (respiratory chain)? 2. How many mobile electron carriers are in the respiratory chain? 3. Name the mobile electron carriers in the electron transport chain. 4. Which complex is inhibited by rotenone? 5. Which enzyme complex is not a proton pump? 6. Which enzyme complex is inhibited by dimercaprol? 7. Which enzyme complex is inhibited by cyanide? 8. What are uncouplers? 9. How does thermogenin generate heat? 10. Where is the electron transport chain found? 42

43 End!