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  1. Lecture 29 • Oxidative phosphorylation/Uncoupled electron transport • Fatty acids • Quiz on Friday 12-3-11 (beta-oxidation) full class • Quiz on Wednesday 12-7-10 (fatty acid synthesis)-20 min quiz

  2. Formation of a trans  double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). • Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA • NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA. • C-C bond cleavage by -ketoacyl-CoA thiolase (KT) Page 917

  3. ATP Synthase • Proton diffusion through the protein drivesATP synthesis! • Two parts: F1 and F0

  4. Racker & Stoeckenius confirmed Mitchell’s hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin

  5. Binding Change Mechanism • ADP + Pi <-> ATP + H2O • In catalytic site Keq = 1 • ATP formation is easy step • But once ATP is formed, it binds very tightly to catalytic site (binding constant = 10-12M) • Proton induced conformation change weakens affinity of active site for ATP (binding constant = 10-5)

  6. Binding Change Mechanism • Different conformation at 3 catalytic sites • Conformation changes due to proton influx • ADP + Pi bind to L (loose) site • Proton (energy) driven conformational change (loose site) causes substrates to bind more tightly (T). • ATP is formed in tight-site. • ATP is released from the O (open) site. • Requires influx of three protons to get one ATP

  7. ATPase is a Rotating Motor • Bound  subunits to glass slide • Attached a fluroescent actin chain to  subunit. • Hydrolysis of ATP to ADP + Pi cause filament to rotate 120o per ATP.

  8. How does proton flow cause rotation?

  9. Active Transport of ATP, ADP and Pi Across Mitochondrial Inner Membrane • ATP is synthesized in the matrix • Need to export for use in other cell compartments • ADP and Pi must be imported into the matrix from the cytosol so more ATP can be made. • Require the use of transporters

  10. Transport of ATP, ADP and Pi • Adenine nucleotide translocator = ADP/ATP antiport. • Exchange of ATP for ADP causes a change in  due to net export of –1 charge • Some of the energy generated from the proton gradient (PMF) is used here • Pi is imported into the matrix with a proton using a symport. • Because negative charge on the phosphate is canceled by positive charge on proton no effect on , but effects pH and therefore PMF.

  11. Transport of ATP, ADP and Pi • NRG required to export 1 ATP and import 1 ADP and 1 Pi = NRG generated from influx of one proton. • Influx of three protons required by ATPase to form 1 ATP molecule. • Need the influx of a total of 4 protons for each ATP made.

  12. P/O Ratio • The ratio of ATPs formed per oxygens reduced • e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen • 4 H+ flow back into matrix per ATP to cytosol • 10/4 = 2.5 for electrons entering as NADH • For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen • 6/4 = 1.5 for electrons entering as succinate

  13. Regulation of Oxidative Phosphorylation • ADP is required for respiration (oxygen consumption through ETC) to occur. • At low ADP levels oxidative phosphorylation low. • ADP levels reflect rate of ATP consumption and energy state of the cell. • Intramolecular ATP/ADP ratios also impt. • At high ATP/ADP, ATP acts as an allosteric inhibitor for Complex IV (cytochrome oxidase) • Inhibition is reversed by increasing ADP levels.

  14. Uncouplers • Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient • Uncouplers are hydrophobic molecules with a dissociable proton • They shuttle back and forth across the membrane, carrying protons to dissipate the gradient • w/o oxidative-phosphorylation energy lost as heat • Dinitrophenol once used as diet drug, people ran 107oF temperatures

  15. Introduction: Lipids • Lipids are primary example of biological molecule that assembles into large ordered structures, but are not considered a polymer. • Characteristic solubility behavior: More soluble in nonpolar solvents: diethyl ether methanol, hexane than H2O. • Diversity in chemical structure results in diversity in biological function: • Formation of biological barriers in cells, organelles to large and charged ions and molecules. • Sterol lipids are precursors for many hormones. • Some lipids are signal transduction molecules, b-carotine, retinal, prostaglandins, electron carriers (ubiquinone - CoQ).

  16. Fatty Acid Structure • Fatty acids are a basic component of many lipids. • Structure has polar/ionic region (-COOH / COO-). • Structure also has nonpolar region - unbranched aliphatic hydrocarbon chain. • Polar-nonpolar molecules are called amphiphilic. • Typically bound up in fats (triacylglycerols - ester bond with C3 glycerol alcohols).

  17. Figure 9.1 The structures of saturated fatty acid Octadecanoic acid, • a saturated acid. • Saturated fatty acids are more flexible. • Three types of chemical structures: • Zigzag line shows hydrocarbon chain. • Structure showing all carbon atoms. • Space filling structure - actual shape.

  18. Figure 9.1 The structures of an unsaturated fatty acid 9-octadecenoic acid, • an unsaturated acid. • Unsaturated fatty acids nearly always cis • More rigid than saturated fatty acids.

  19. Important Features of Fatty Acid Cell Membrane Lipid Components • Fatty acids are essential to membrane structure and function. • Are components of all membrane lipids exceptsterols. • Hydrophobic tails form non-polar barrier to diffusion of polar solutes through membrane. • Most fatty acids are 12-20 carbon atoms in length; optimal size range for formation of bilayer structures. • 16-18 carbon versions are especially common: • Palmitate - (16) - saturated • Stearate (18) - saturated • Oleate (18) - unsaturated, 1 double bond • Linoleate (18) - unsaturated, 2 double bonds • Linolenate (18) - unsaturated, 3 double bonds • Arachidonate (20) - unsaturated, 4 double bonds • Note: Almost all double bonds are in cis configuration, with a sharp bend or kink in each tail, prevents tight packing in membrane.

  20. Table 7-2 Structures of Some Common Fatty Acids Note bend(s) in unsaturated structures

  21. Table 9.1 Structures of common fatty acids

  22. Nonpolar triacylglycerol lipids - storage and membrane lipids Two primary biological roles: Energy metabolism -storage - nonpolar. Heat Insulation Adipocyte cells - animal cells that store fat for metabolic fuel.

  23. Figure 9.2 Stepwise addition of fatty acids to glycerols to make triacl glycerols (fats) Ester bonds hold the acyl groups to the glycerol backbone.

  24. Fatty acid storage • Major form of storage is in the triacylglycerol form (triglyceride, neutral lipid). • Not structural in this form. • Most natural fats contain a mixture of mixedtriacylglycerol components (contain more than one fatty acid). • Butter 11% C4-C12 10% C14 26% C26 11% C18 40% unsat C16 + C18 Small side chains melt at lower temp. give flexibility at room temp. Unsaturated also melt at lower temp. (olive oil approx. 80% unsaturated).

  25. Soap Soap molecules aggregate in water to form micelles, dissolve greasy, oily compounds in water. • How to make soap: • Caveman method: • Boil animal fat with wood ash (lye). • Or cremate bodies. • Mix fatty acid with NaOH or KOH to form Na+ or K+ salt. • Mg++ and Ca++ salts are insoluble-why bath soaps don’t work in hard water. • Saponification: mix fat (triacylglycerols) with NaOH to form glycerol and soap (heat).

  26. Figure 9.3 The commercial hydrogenation of vegetable oils often leads to trans acids • How to prepare oleomargarine. • Trans fatty acids may increase blood cholesterol levels.

  27. Figure 9.3 The commercial hydrogenation of vegetable oils often leads to trans acids “Margarine got a boost in the 1970s when studies came out indicating that dietary cholesterol and saturated fat raise LDL (low-density lipoprotein) cholesterol levels in the blood and increase the incidence of heart disease. Made with vegetable oils, margarine has no cholesterol and less saturated fat than butter. Naturally, margarine advertisers began to trumpet the health benefits of eating margarine. However, further research on the trans fatty acids found in partially hydrogenated oils took many by surprise (C&EN, Sept. 22, 2003, page 33). Trans fatty acids, though unsaturated, were found to also increase LDL ("bad" cholesterol) levels in the blood. In the U.S., the Food & Drug Administration will require labeling of trans fats on all products by 2006. In anticipation of the U.S. regulation, many margarine makers have found alternative fats and oils or alternative forms of hydrogenation that minimize the creation of trans fatty acids.” From August 16, 2004 Chemical and Engineering News 82:33, p. 24 “WHAT'S THAT STUFF? MARGARINE”

  28. Common Oils and Fats - Triacylglycerols Fats are from animal tissue - mainly saturated- solids at room temperature. Oils are from plant seeds - mainly unsaturated

  29. Waxes Waxes function as protective coatings for plant leaves, skin lubrication, “waterproofing” feathers of birds Consist of fatty acid ester of long chain alcohol.

  30. Polar Lipids • Differ from triglycerides since they have one or more polar head groups. • 2 main types: glycerol or sphingosine based • Main class are phospholipids • involved in membrane structure • Amphipathic

  31. Fat molecules are hydrophobic, whereas phospholipids are amphipathic. (A) Triacylglycerol, a fat molecule, is entirely hydrophobic. (B) Phospholipids such as phosphatidyl-ethanolamine are amphipathic, containing both hydrophobic and hydrophilic portions. The hydrophobic parts are shaded red, and the hydrophilic parts are shaded blue and green. (The third hydrophobic tail of the triacylglycerol molecule is drawn here facing upward for comparison with the phospholipid, although normally it is depicted facing down.)

  32. Examples of polar lipids - Glycerophospholipids and Sphingolipids • The membrane lipids are composed of the glycerophospholipids and sphingolipids, which have polar and nonpolar regions.

  33. Figure 9.7 Glycero-phospholipids Phosphatidic acid 4 major glycerophospholipids are polar/charged:

  34. O O RCO-CH2 CH2-OCR O O Other examples of phosphoglycerol derivatives • Can be in sugars (glycolipids) • Diphosphatidyl glycerol O RCO-CH CH-OCR OH O CH2-O-P-O-CH2-CH-CH2-O-P-O-CH2 O- O- glycerol

  35. Figure 9.8 Sphingolipid Structure sphingosine Fatty acid Polar head 4 major sphingolipids are: Found in nerve cell membranes brain, nervous system. Tay-Sachs disease: accumulation of ganglioside in brain and spleen causes death by age 4.

  36. Sphingolipids • Sphingomyelin (also considered a phospholipid) • Important in myelin sheath of nerve cell membrane. • Cerebrosides are glycolipids - found in brain and other tissues. • Sugar attached at P. • Ganglosides are sphingolipids with several sugars as head groups. Terminal sugar is sialic acid.

  37. Lipid/membrane consitutents that cannot be saponified. • Triacylglycerides, phospholipids, and sphingolipids can be saponified (hydrolyzed with OH-) • Some cannot be saponified: steroids (cholesterol-based) and terpenes.

  38. The molecular structure common to all steroids Steroid structures have four fused rings, A, B, C, and D.

  39. Figure 9-10b Cholesterol molecular structure - a steroid • Cholesterol has a polar head group (OH) and a nonpolar tail. • Cholesterol and ester derivatives are abundant in (blood) plasma proteins called lipoproteins. • Lipoproteins transport cholesterol to tissues for use in cell membranes and hormone precursors.

  40. Figure 9-10 The molecular structure common to all steroids • C30 Cholesterol molecule is derived from 5-carbon isoprene subunit. Cholesterol is traditionally only associated with animal cells, but derivatives are also identified in plants.

  41. Figure 9-10 (c) A cholesterol fatty acid ester • Cholesterol ester formed between the cholesterol hydroxyl group and a fatty acid with a long aliphatic side chain. • This is a common modification of cholesterol under physiological conditions.

  42. Figure 7-6 Structure of the Cholesterol, a Component of Mammalian Cell Membranes (c) The most common membrane sterols are cholesterol in animals and several related phytosterols in plants.

  43. Cholesterol and Eggs in the diet From Jennifer Moll,Your Guide to Cholesterol. About fifteen years ago, egg consumption was discouraged by many health care practitioners because of their high cholesterol content. The average intact egg contains about 210 mg of cholesterol, whereas the recommended intake of cholesterol is 300 mg. However, a study published in the Journal of the American Medical Association, in addition to several other studies, refute this. This study looked at the effects of egg consumption in 100,000 men and women, and concluded that eggs alone do not contribute to high cholesterol. In fact, when cholesterol was omitted from the diet of these subjects, their total cholesterol levels decreased only by 1%. What researchers did discover was that individuals who consumed eggs also consumed bacon, ham, butter, and other food products that could contribute to high cholesterol levels. Not only do these foods have high cholesterol, they also contain high amounts of saturated fats and trans-fats--both of which contribute to high cholesterol levels and atherosclerosis. Given these studies and the fact that eggs are an excellent source of nutrition, the American Heart Association now recommends that you can eat one egg a day, as opposed to three or four per week it previously allowed. Eggs are a rich source of protein, containing the essential amino acids required by your body. In addition to protein, eggs also contain many vitamins, minerals, and a fatty molecule called lecithin, which aids in transporting and metabolizing fats in the body .It is cautioned that if you do consume one egg a day, you might need to watch your total cholesterol levels since too much cholesterol could raise your LDL levels.

  44. Figure 7-8 The Structure of Hopanoids Abundant in petroleum (crude oil) deposits, suggesting a prokaryotic (bacterial) role in formation of oil deposits A hopanoid, one of a class of sterol-like molecules that appear to function in the plasma membranes of at least some prokaryotes as sterols do in the membranes of eukaryotic cells. The structure of cholesterol, for comparison. A weakly hydrophilic side chain (-CH2OH or -OH) protrudes from each molecule.