1. B 12 revisited

2. Oxidation of Propionyl-CoA Most dietary fatty acids are even-numbered Many plants and some marine organisms also synthesize odd-numbered fatty acids Propionyl-CoA forms from  -oxidation of odd- numbered fatty acids Bacterial metabolism in the rumen of ruminants also produces propionyl-CoA

3. Figure 25-20The rearrangement catalyzed by methylmalonyl-CoA mutase. Page 923

4. Figure 25-21 Structure of 5’-deoxyadenosyl- cobalamin (coenzyme B 12 ). Page 923

5. Page 926 Proposed mechanism of methylmalonyl- CoA mutase. Homolytic cleavage Each product gets 1 electron from the bond Cobalt acts as a reversible free radical generator! Adenosyl radical abstracts H from substrate

6. Oxidative Phosphorylation Coupling of reduction of O 2 with ATP production –Substrate level phosphorylation? –High energy intermediate structure/state? –Something else?

7. Figure 22-12 Electron micrographs of mouse liver mitochondria. (a) In the actively respiring state. (b) In the resting state. Page 806

8. Chemiosmotic Theory How to make an unfavorable ADP + P i = ATP possible? Phosphorylation of ADP is not a result of a direct reaction between ADP and some high energy phosphate carrier Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient The electrochemical gradient is established by transporting protons against the electrochemical gradient during the electron transport

10. Chemiosmotic Energy Coupling Requires Membranes The proton gradient needed for ATP synthesis can be stably established across a topologically closed membrane –Plasma membrane in bacteria –Cristae membrane in mitochondria –Thylakoid membrane in chloroplasts Membrane must contain proteins that couple the “downhill” flow of electrons in the electron transfer chain with the “uphill” flow of protons across the membrane Membrane must contain a protein that couples the “downhill” flow of proton to the phosphorylation of ADP

12. Figure 22-3Freeze-fracture and freeze-etch electron micrographs of the inner and outer mitochondrial membranes. Page 799

13. How could you identify and reconstruct the ETC? Intact mitochondria Submitochondrial particles Identify components: –P–Pyridine-linked DH –F–Flavin-linked DH –I–Iron-sulfur proteins –C–Cytochromes –U–Ubiquinone

14. Coenzyme Q or Ubiquinone Ubiquinone is a lipid-soluble conjugated dicarbonyl compound that readily accepts electrons Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side

17. How do they fit together? Redox potentials Visualize redox by UV/vis INHIBITORS!!!

18. Cytochrome c Absorbs Visible Light Intense Soret band near 400 nm absorbs blue light and gives cytochrome c an intense red color Cytochromes are sometimes named by the position of their longest-wavelength peak

21. Iron-Sulfur Centers Found in several proteins of electron transport chain, including NADH:ubiquinone oxidoreductase Transfers one electron at a time

24. Figure 22-9 The mitochondrial electron-transport chain. Page 803

26. In the presence of antimycin A and an electron donor, is Cyt b in its oxidized or reduced state?

28. Separation of functional complexes of the respiratory chain.

29. Figure 22-14The mitochondrial electron-transport chain. Page 808

30. Path of electrons from NADH, succinate, fatty acyl–CoA, and glycerol 3-phosphate to ubiquinone

31. NADH:Ubiquinone Oxidoreductase a.k.a. Complex I One of the largest macro-molecular assemblies in the mammalian cell Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes NADH binding site in the matrix side Non-covalently bound flavin mononucleotide (FMN) accepts two electrons from NADH Several iron-sulfur centers pass one electron at the time toward the ubiquinone binding site

32. NADH:ubiquinone oxidoreductase (Complex I).

33. Structure of NADH:Ubiquinone Oxidoreducase The complete macromolecular assembly can be seen in electron microscopy. Part of the bacterial protein has been crystallized but the 3D structure of the membrane-spanning domain remains unknown

34. NADH:Ubiquinone Oxidoreducase is a Proton Pump Transfer of two electrons from NADH to uniquinone is accompanied by a transfer of protons from the matrix (N) to the inter-membrane space (P) Experiments suggest that about four protons are transported per one NADH NADH + Q + 5H + N = NAD + + QH 2 + 4 H + P Reduced coenzyme Q picks up two protons Despite 50 years of study, it is still unknown how the four other protons are transported across the membrane

35. Succinate Dehydrogenase a.k.a. Complex II FAD accepts two electrons from succinate Electrons are passed, one at a time, via iron- sulfur centers to ubiquinone that becomes reduced QH 2

37. Structure of Complex II (succinate dehydrogenase). = path of e - transfer Heme b protects against rogue electrons forming reactive oxygen species

38. Cytochrome bc 1 Complex a.k.a. Complex III Uses two electrons from QH 2 to reduce two molecules of cytochrome c

39. Cytochrome bc1 complex (Complex III). a dimer of identical monomers, each with 11 different subunits. Complex has two distinct binding sites for ubiquinone, QN and QP. The interface between monomers forms two caverns, each containing a QP site from one monomer and a QN site from the other. The ubiquinone intermediates move within these sheltered caverns.

40. The Q Cycle Experimentally, four protons are transported across the membrane per two electrons that reach CytC Two of the four protons come from QH 2 The Q cycle provides a good (but complicated) model that explains how two additional protons are picked up from the matrix

41. Animation of Q cycle http://www.life.illinois.edu/crofts/qcycle_mo del.htmlhttp://www.life.illinois.edu/crofts/qcycle_mo del.html http://www.macromol.uni- osnabrueck.de/BC1_complex.php

43. The Q cycle, shown in two stages

44. Cytochrome c Cytochrome c is a soluble heme-containing protein in the intermembrane space Heme iron can be either ferrous(Fe 3+, oxidized) or ferric(Fe 2+, reduced) Cytochrome c carries a single electron from the cytochrome bc 1 complex to cytochrome oxidase

45. Cytochrome Oxidase a.k.a. Complex IV Mammalian cytochrome oxidase is a membrane protein with 13 subunits Contains two heme groups Contains copper ions –Two ions (Cu A ) form a binuclear center –Another ion (Cu B ) bonded to heme forms Fe- Cu center

47. Cytochrome Oxidase Passes Electrons to O 2 Four electrons are used to reduce one oxygen molecule into two water molecules Four protons are picked up from the matrix in this process Four additional protons are passed from the matrix to the inter-membrane space by an unknown mechanism

49. Summary of the Electron Flow in the Respiratory Chain

51. Proton-motive Force The proteins in the electron transport chain created the electrochemical proton gradient by one of the three means: –actively transported protons across the membrane via poorly understood mechanisms –passed electrons to coenzyme Q that picked up protons from the matrix –took electrons from QH 2 and released the protons to the inter-membrane side

56. The inner mitochondrial membrane separates two compartments of different [H + ], resulting in differences in chemical concentration (ΔpH) and charge distribution (Δ  ) across the membrane.

57. Chemiosmotic Model for ATP Synthesis Electron transport sets up a proton-motive force Energy of proton-motive force drives synthesis of ATP

58. Energy Calculator http://bcs.whfreeman.com/lehninger5e/pages/bcs- main.asp?v=&s=19000&n=00040&i=19040.01&o=|00610|00580|005 90|00510|00540|00600|00550|00570|00630|00010|00020|00030|000 40|00070|00080|00090|00100|01000|02000|03000|04000|05000|060 00|07000|08000|09000|10000|11000|12000|13000|14000|15000|160 00|17000|18000|19000|20000|21000|22000|23000|24000|25000|260 00|27000|28000|99000|

61. Mitochondrial ATP Synthase Complex The proton-motive force causes rotation of the central shaft  This causes a conformational change within all the three  pairs The conformational change in one of the three pairs promotes condensation of ADP and P i into ATP

63. Figure 22-43Model of the E. coli F 1 F 0 –ATPase. Page 832

72. Rotational Catalysis

76. Movies http://atom.chem.wwu.edu/sacahill/472/atp %20synthase.movhttp://atom.chem.wwu.edu/sacahill/472/atp %20synthase.mov http://atom.chem.wwu.edu/sacahill/472/atp %20synthase2.movhttp://atom.chem.wwu.edu/sacahill/472/atp %20synthase2.mov http://atom.chem.wwu.edu/sacahill/472/rot arymech.movhttp://atom.chem.wwu.edu/sacahill/472/rot arymech.mov

81. Figure 22-46Uncoupling of oxidative phosphorylation. Page 834

82. Figure 22-47Mechanism of hormonally induced uncoupling of oxidative phosphorylation in brown fat mitochondria. Page 835

83. ATP Yield From Glucose

85. Let’s Sing! Lyrics?

88. Light Energy is Converted to ATP in Plant Chloroplasts

91. Various Pigments Harvest the Light Energy The energy is transferred to the photosynthetic reaction center

94. Light-Induced Redox Reactions and Electron Transfer Cause Acidification of Lumen The proton-motive force across the thylakoid membrane drives the synthesis of ATP

96. Flow of Protons: Mitochondria, Chloroplasts, Bacteria According to endosymbiotic theory, mitochondria and chloroplasts arose from entrapped bacteria Bacterial cytosol became mitochondrial matrix and chloroplast stroma