Note for lecture 9, 2010

1. Slides or areas with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam. Note for lecture 9, 2010 Links to movies: Rotation of  actin filament arm Animation 1Animation 2Animation 3

2. ATP debt paid in full • 2 ATP • + 4 ATP • = + 2 ATP “glycolysis” ends here Handout 7-2

3. 1 glucose + 2 ADP + 2 Pi + 2 NAD 2 pyruvate + 2 ATP + 2 NADH2 ΔGo = -18 kcal/mole So overall reaction goes essentially completely to the right.

4. (Handout 7-3) Handout 7-4b

5. ΔG = ΔGo + RTln([products] • [reactants]) pull pull Handout 7-4b

6. 1) First way was: a coupled reaction (i.e., a different reaction) . One of two ways the cell solves the problem of getting a reaction to go in the desired directionGlucose + ATP  glucose-6-P04 + ADP, ΔGo = -3.4 kcal/mole 2) The second way: Removal of the product of an energetically unfavorable reaction Uses a favorable downstream reaction “Pulls” the unfavorable reaction Operates on the second term of the ΔG equation. ΔG = ΔGo + RTln([products]/[reactants]) The second way the cell gets a reaction to go in the desired direction:

7. So glucose  pyruvic acid ADP  ATP, as long as we have plenty of glucose Are we all set? No…. What about the NAD?.. We left it burdened with those electrons. Soon all of the NAD will be in the form of NADH2 Very soon Glycolysis will screech to a halt !! Need an oxidizing agent in plentiful supply to keep taking those electron off the NADH2, to regenerate NAD so we can continue to run glucose through the glycolytic pathway.

8. 1) Oxygen Defer (and not always present, actually) 2) Pyruvate, our end-product of glycolysis In E. coli, humans: Pyruvate  lactate, NADH2  NAD, coupled In Yeast: Pyruvate  ethanol + CO2 Oxidizing agents around for NAD:

10. Glucose ATP ATP excreted Handout 7-1b

11. yeast E. coli H C=O | CH3 Acetaldehyde detail humans

12. Lactate fermentation Ethanolic fermentation Mutually exclusive, depends on organism Other types, less common fermentations, exist (e.g., propionic acid fermentation, going on in Swiss cheese) Fermentation: anaerobiosis (no oxygen)

13. glucose--> 2 lactates, without considering the couplings for the formation of ATP's (no energy harnessing): ΔGo = -45 kcal/moleSo 45 kcal/mole to work with. Out of this comes 2 ATPs, worth 14 kcal/mol. So the efficiency is about 14/45 = ~30% Where did the other 31/45 kcal/mole go? Wasted as HEAT. The efficiency of fermentation

14. Since 2 ATPs ARE produced, taking them into account, for the reaction: Glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP ΔGo = -31 kcal/mole (45-14) Very favorable. All the way to the right. Keep bringing in glucose, keep spewing out lactate, Make all the ATP you want. Fermentation goes all the way to the right glucose--> 2 lactates, without considering the couplings for the formation of ATP's (no energy harnessing): ΔGo = -45 kcal/mole kcal/moleOut of this comes 2 ATPs, worth 14 kcal/mol. So the efficiency is about 14/45 = ~30% That’s fermentation.

15. - O2 glycolysis +O2 ? CO2 + H2O Gl;ycerol as an alternative sole carbon and energy source for E. coli ATP +NAD + NADH2 DHAP (dihydroxy acetone phosphate) glycerol phosphate glycerol and ADP + Pi  ATP

16. Glycerol + ATP → glycerol phosphate → DHAP NAD → NADH2

17. +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. Stoichiometry of chemical reactions must be obeyed. No magic is involved

18. Complete oxidation of glucose, Much more ATP But nature’s solution is a bit complicated. The fate of pyruvate is now different Energy yield But all this spewing of lactate turns out to be wasteful. Using oxygen as an oxidizing agent glucose could be completely oxidized, to: … CO2 That is, burned. How much energy released then? Glucose + 6 O2 6 CO2 + 6 H2O ΔGo = -686kcal/mole ! Compared to -45 to lactate (both w/o ATP production considered)

19. Acetyl-CoA Score: Per glucose A 2 NADH 2 NADH 2 ATP 2 CO2 Handout 8-1

20. Acetyl-CoA O || CH3 - C –OH + Co-enzyme A  Acetyl ~CoA Acetic acid, acetate Acetate group Pantothenic acid (vitamin B5)

21. Acetyl-CoA Per glucose B 2 oxaloacetate 2 NADH2 NADH 2 NADH 2 NADH 2 ATP 2 CO2 2 CO2 2 CO2 6 CO2

22. GTP + ADP  GDP + ATP ΔGo = ~0 G= guanine (instead of adenine in ATP) GTP is energetically equivalent to ATP

23. Acetyl-CoA Per glucose B 2 oxaloacetate 2 NADH2 NADH 2 NADH 2 NADH 2 ATP 2 CO2 2 CO2 2 CO2 6 CO2

24. FAD = flavin adenine dinucleotide Business end (flavin) ribose adenine ribose FAD + 2H. FADH2

25. D Acetyl-CoA Per glucose oxaloacetate 2 NADH 2 NADH 2 NADH 2 NADH 2 FADH2 2 NADH 2 ATP 2 ATP 2 CO2 2 CO2 2 CO2 Note label is in OA after one turn of cycle, half the time on top, half on bottom. So no CO2 from Ac-CoA after just one turn. (CO2 in first turn from OA). Succinic dehydrogenase

26. E Per glucose 2 NADH 2 NADH 2 NADH 2 NADH 2 FADH2 2 NADH 2 ATP 2 ATP Glucose + 6 O2 6 CO2 + 6 H2O : By glycolysis plus one turn of the Krebs Cycle: 1 glucose (6C)  2 pyruvate (3C)  6 CO2 2 X 5 NADH2 and 2 X 1 FADH2 produced per glucose 4 ATPs per glucose NADH2 and FADH2 still must be reoxidized …. No oxygen yet to be consumed No water produced yet Paltry increase in ATP so far 2 CO2 2 CO2 2 CO2

27. 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) Oxidation of NAD by O2

28. Iron-sulfur protein Handout 8-3 10 NADH2 ~ Free energy heme Ubiquinone, or Coenzyme Q Cytochromes are proteins Up to 50 C’s long

29. Oxidativephosphorylation Handout 8-4

30. H+ ions (protons) are pumped out as the electrons are transferred (outside) I II III IV Nelson and Cox, Principles of Biochemistry

31. Schematic idea of H+ being pumped out Relaxation back Conformationalchange Handout 8-4

32. Proton flow back in via mass action FoF1 Complex: Oxidative phosphorylation (ATP formation) Handout 8-4

33. Artificial phospholipid membrane H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ETC Complex I’s pH drops pH rises NADH NADH Slides with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam. H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+

34. ADP + Pi Slides with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam. Artificially produced mitochondrial membrane vesiclewith ADP and Pi trapped inside ATP is formed from ADP + Pi

35. Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation Slides with a blue background are subjects that were skipped in lecture. They are included here just for your interest and will not be included in any exam.  - + H+ DNP’s -OH is weakly acidic in this environment 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.

36. Nobel Prize 1978 Chemiosmotic theory Proton motive force (pmf) Chemical gradient Electrical gradient Electrochemical gradient Peter Mitchell 1961 (without knowing mechanism) Water-pump-dam analogy Some evidence:

37. crista + ADP + + + + + + + ATP What about E. coli? Cell membrane houses all components the inside the outside

38. The mechanism of ATP formation: The ATP synthetase (or ATP synthase) The F0F1 complex: outside inside Gamma subunit: cam the inside the outside Gamma subunit is inserted inside ATP synthetase

39. ATP synthetase inside outside Flow of protons turns the C-subnunit wheel. C-subunits turn the gamma cam

40. Outside Inside Chloroplasts Mitochondria Outside Inside

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

42. Alpha+beta Gamma (top view) ADP Pi Three conformational states of the a-b subunit: L, T, and O Handout 8-5

43. Movie link

45. Motor experiment Attach the big arm (MW 42K) Detach the C-subunits

46. 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)

47. Run reaction in reverse: add ATP, drive counter-clockwise rotation of cam 4 3 2 1 5 Here the cam has no driving motor (c) attached any more Start here ATP ATP hydrolysis     x counter-clockwise

48. Movie link

49. 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) ATP accounting

50. Handout 8-1