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Oxidation of NAD by O 2

Oxidation of NAD by O 2. NADH 2 + 1/2 O 2    -->  NAD + H 2 O Δ G o = -53 kcal/mole If coupled directly to ADP  ATP (7 kcal cost), 46 kcal/mole waste, and heat So the electrons on NADH (and FADH 2 ) are not passed directly to oxygen, but to intermediate carriers,

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Oxidation of NAD by O 2

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  1. Oxidation of NAD by O2 NADH2 + 1/2 O2   -->  NAD + H2O ΔGo= -53 kcal/mole If coupled directly to ADP  ATP (7 kcal cost),46 kcal/mole waste, and heat So the electrons on NADH (and FADH2) are not passed directly to oxygen, but to intermediate carriers, Each transfer step involves a smaller packet of free negative energy change (release)

  2. Iron-sulfur protein NADH2 H heme Ubiquinone; Coenzyme Q H Handout 8-3

  3. Oxidativephosphorylation Handout 8-4

  4. Nelson and Cox, Principles of Biochemistry

  5. Schematic idea of H+ being pumped out nal Handout 8-4

  6. crista + ADP + + + + + + + ATP FoF1 Complex: Oxidative phosphorylation (ATP formation) Handout 8-4

  7. Chemiosmotic theory (Mitchell hypothesis) Proton motive force (pmf) Chemical gradient Electrical gradient Electrochemical gradient Peter Mitchell 1961 (without knowing mechanism) Water-pump-dam analogy 3 exampels of evidence supporting themitchell hypothesis:

  8. H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ pH drops pH rises [H+]  [H+↓] NADH NADH H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Experiment 1 Artificial phospholipid membrane H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ETC Complex I’s

  9. Experiment 2 H + H + H + H + H+ H H H + H + H H + + H + H + H + H + H + H + H + H+ H + H + H + H + H + H + H + ATP H+ H + H + H+ H + H + H + H+ H + H + H H + H + Experiment 2 ADP + Pi Artificially produced mitochondrial membrane vesicle ATP is formed from ADP + Pi

  10. Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation Experiment 3 - H+ - + H+ DNP’s -OH is weakly acidic in this environment (pH7) DNP can easily permeate the mitochondrial inner membrane Outside the mitochondrion, where the H+ concentration is high, DNP picks up a proton After diffusing inside, where the H+ concentration low, it gives up the proton. So it ferries protons from regions of high concentration to regions of low concentration, thus destroying the proton gradient. Electron transport chain goes merrily on and on, but no gradient is formed and no ATP is produced.

  11. The mechanism of ATP formation: The ATP synthetase (or ATP synthase) The F0F1 complex: outside inside Gamma subunit: cam ATP synthetase

  12. ATP synthetase inside outside

  13. Alpha+beta Gamma Three conformational states of the a-b subunit: L, T, and O Handout 8-5

  14. Motor experiment

  15. Actin labeled by tagging it with fluorescent molecules Attached to the gamma subunit Actin is a muscle protein polymer Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature, 386, 299 - 302. Testing the ATP synthetase motor model by running it in reverse (no H+ gradient, add ATP)

  16. Run reaction in reverse, add ATP, drive counter-clockwise rotation of cam 4 3 2 1 5 ATP ATP hydrolysis    

  17. desktop

  18. View of the c-subunits making up the F0 subunit using atomic force microscopy Norbert Dencher and Andreas Engel Animation of the Fo rotation driven by the influx of H+ ions (“wheels within wheels”).M.E. Girvin

  19. Alpha+beta Gamma Handout 8-5

  20. Hongyun Wang and George Oster

  21. ATP accounting • Each of the 3 ETC complex (I, III, IV) pumps enough H+ ions to allow the formation of 1 ATP. • So 3 ATPs per pair of electrons passing through the full ETC. • So 3 ATPs per 1/2 O2 • So 3 ATPs per NADH2 • But only 2 ATPs per FADH2 (skips complex 1)

  22. Handout 8-1

  23. ATP ATP ATP X Fumarate More favorable ∆GO with FAD ATP generated by the ATP synthetase is called is oxidative phosphorylation, or oxphos

  24. Nelson and Cox, Principles of Biochemistry

  25. OXPHOS: 1 NADH from glycolysis 1 NADH from Krebs entry 3NADH from Krebs 1 FADH2 from Krebs Total: 17 ATP 5 NADH = 15 ATP 1 FADH2 = 2 ATP Substrate level phosphorylation (SLP): 2 ATP 1ATP from Glycolysis 1 ATP (GTP) from Krebs Handout 8-6 Grand total (E. coli): 17 + 2 = 19 per ½ glucose or 38 per 1 glucose Handout labeled 8-6

  26. ATP accounting • 38 ATP/ glucose in E. coli • 36 ATP/glucose in eukaryotes • Cost of bringing in the electrons from NADH from glycolysis into the mitochondrion = 1 ATP per electron pair • So costs 2 ATPs per glucose, subtract from 38 to get 36 net.

  27. Efficiency • 36 ATP/ glucose, worth 7 X 36 = 252 kcal/mole of glucose • ΔGo for the overall reaction glucose + 6 O2→ 6CO2 + 6 H2O: -686 kcal/ mole • Efficiency = 252/686 = 37% • Once again, better than most gasoline engines. • and Energy yield: 36 ATP/ glucose vs. 2 ATP/glucose in fermentation (yet fermentation works) • So with or without oxygen, get energy from glucose

  28. Cellular location (eukaryotes): CYTOPLASM MITOCHONDRIA Handout labeled 8-6

  29. Alternative sources of carbon and energy • Shake = milk: • milk sugar = lactose = disaccharide • = glucose – galactose • beta-galactosidase • +HOH → glucose + galactose • glucose → glycolysis, etc. • galactose •  (3 enzymatic steps) •  • glucose

  30. Alternative sources of carbon and energy Bun = starch = poly-alpha-glucose G-1-P → G-6-P glycolysis

  31. Alternative sources of carbon and energy Lettuce = cellulose = polysaccharide Poly-beta glucose →| (stays as the polysaccharide) We have no enzyme for catabolizing cellulose

  32. Alternative sources of carbon and energy French fries = fat (oil) = triglyceride

  33. (Triglyceride) Lipases (hydrolysis)

  34. Handout 9-1 left

  35. +NAD + NADH2 glycerol phosphate glycolysis - O2 +O2 CO2 + H2O ATP DHAP (dihydroxy acetone phosphate) glycerol ???

  36. Glycerol + ATP → glycerol phosphate → DHAP NAD → NADH2 D

  37. +NAD + NADH2 DHAP (dihydroxy acetone phosphate) glycerol phosphate glycolysis +O2 CO2 + H2O ATP glycerol - O2 Glycerol cannot be fermented. E. coli CANNOT grow on glycerol in the absence of air These pathways are real, and they set the rules. No magic is involved

  38. Alternative sources of carbon and energy Hamburger = protein Proteases (e.g., trypsin) →→ 20 AAs Stomach acid (pH1) also helps by denaturing protein making it accessable to proteolytic attack Each of the 20 AA’s has its own catabolic pathway, and ends up in the glycolytic or Krebs cycle pathways But first, the N must be removed:

  39. Handout 9-2 Transamination Glutamic acid alpha-keto-glutaric acid Pyruvate Alanine Oxidative de-amination HOH NAD NADH 2 alpha-keto-glutaric acid Glutamic acid Deamination and transamination of amino acids

  40. PKU (phenylketonuria) E.g., degradation of phenylalanine (6 steps) Phe builds up and gets metabolized to an injurious product (phenyl pyruvate) transaminase Products = Fumaric acid → Krebs and Acetoacetate → 2 Acetyl-CoA → Krebs

  41. Anabolism ATP GLYCOGEN ATP ATP FATS ATP AMINO ACIDS PROTEINS You are what you eat Catabolism Anabolism Anabolism ATP STARCH GLYCOGEN glucose GLYCOLYSIS ATP pyruvate ATP ATP ATP ATP FATS FATS FATS FATS FATS acetyl-CoA KREBS O.A. -K.G. -K.G. -K.G. ATP ATP ATP ATP AMINO ACIDS ATP AMINO ACIDS AMINO ACIDS AMINO ACIDS AMINO ACIDS E.T.C. NAD NAD NAD PROTEINS PROTEINS PROTEINS PROTEINS PROTEINS NADH 2 ATP O H O 2 2 Handout 9-2

  42. Handout 9-2 Handout 9-2

  43. Biosynthesis of monomers E.g., • Fatty acids (acetyl CoA from Krebs cycle) • Amino acids (Serine: 3-phospho-glyceric acid from glycolysis)

  44. Handout 9-1 Start at bottom P P Handout 9-1 right

  45. Handout 9-3 Phosphoester group (Glycolytic intermediate) Glutamate is the amino donor hydrolysis Handout 9-3

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