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

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1. Formation of a trans double bond by dehydrogenation by acyl-CoA dehydrogenase (AD).

2. Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA

3. NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA.

4. C-C bond cleavage by -ketoacyl-CoA thiolase (KT)

ATP Synthase

• Proton diffusion through the protein drives ATP synthesis!

• Two parts: F1 and F0

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

• 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)

Binding Change Mechanism

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

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.

How does proton flow cause rotation?

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

• 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.

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.

Transport of ATP, ADP and Pi

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

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.

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

O2N OH

NO2 H

O2N O

NO2

• 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:

1. Formation of biological barriers in cells, organelles to large and charged ions and molecules.

2. Sterol lipids are precursors for many hormones.

3. Some lipids are signal transduction molecules, -carotine, retinal, prostaglandins, electron carriers (ubiquinone - CoQ).

Introduction: Lipids

• Fatty acids are a basic component of many lipids.

1. Structure has polar/ionic region (-COOH / COO).

2. 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).

Fatty Acid Structure

Octadecanoic acid,

- a saturated acid.

Saturated fatty acids are more flexible.

• Three types of chemical structures:1. Zigzag line shows

hydrocarbon chain.

2. Structure showing all carbon atoms.

3. Space filling structure - actual shape.

Figure 9.1 The structures of saturated fatty acid

9-octadecenoic acid,

- an unsaturated acid.Unsaturated fatty acids

nearly always cis

More rigid than saturated fatty acids.

Figure 9.1 The structures of an unsaturated fatty acid

Important Features of Fatty Acid Cell Membrane Lipid Components

• Fatty acids are essential to membrane structure and function.• Are components of all membrane lipids except sterols.• 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.

Table 7-2 Structures of Some Common Fatty Acids

Note bend(s) in unsaturated structures

Table 9.1 Structures of common fatty acids

Nonpolar triacylglycerol lipids - storage and membrane lipids

Two primary biological roles:

1. Energy metabolism -storage - nonpolar.

2. Heat Insulation

Adipocyte cells - animal cells that store fat for metabolic fuel.

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.

• Major form of storage is in the triacylglycerol form (triglyceride, neutral lipid).

• Not structural in this form.• Most natural fats contain a mixture of mixed

triacylglycerol components (contain more than one fatty acid).

• Butter

Fatty acid storage

11% C4-C1210% C1426% C2611% C1840% 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).

Soap molecules aggregate in water to form micelles,

dissolve greasy, oily compounds in water.

Soap

• How to make soap:• Caveman method:

1. Boil animal fat with

wood ash (lye).

2. 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).

• How to prepare oleomargarine.How to prepare oleomargarine.• Trans fatty acids may increase blood cholesterol Trans fatty acids may increase blood cholesterol

levels.levels.

Figure 9.3 The commercial hydrogenation of vegetable oils often leads to trans acids

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”

Common Oils and Fats - Triacylglycerols

Fats are from animal tissue - mainly saturated- solids at room temperature.

Oils are from plant seeds - mainly unsaturated

Waxes

Waxes function as protective coatings for plant leaves, skin lubrication, “waterproofing” feathers of birds

Consist of fatty acid ester of long chain alcohol.

• 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

Polar Lipids

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.)

Examples of polar lipids - Glycerophospholipids and Sphingolipids

• The membrane lipids are composed of the glycerophospholipids and sphingolipids, which have polar and nonpolar regions.

Figure 9.7 Glycero-phospholipids

Phosphatidic acid

4 major glycerophospholipids are polar/charged:

• Can be in sugars (glycolipids)• Diphosphatidyl glycerol

Other examples of phosphoglycerol derivatives

RCO-CH2

O

CH2-OCR

O

RCO-CH

O

CH2-O-P-O-CH2-CH-CH2-O-P-O-CH2

OH

O

CH-OCRO O

O- O-glycerol

Figure 9.8 Sphingolipid Structure

4 major sphingolipids are:

Found in nerve cell membranesbrain, nervous system.

Tay-Sachs disease: accumulation of ganglioside in brain and spleen causes death by age 4.

Fatty acid

sphingosine

Polar head

• 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.

Sphingolipids

• Triacylglycerides, phospholipids, and sphingolipids can be saponified (hydrolyzed with OH-)

• Some cannot be saponified: steroids (cholesterol-based) and terpenes.

Lipid/membrane consitutents that cannot be saponified.

The molecular structure common to all steroids

Steroid structures have four fused rings, A, B, C, and Steroid structures have four fused rings, A, B, C, and D. D.

Figure 9-10b Cholesterol molecular structure - a steroid

• Cholesterol has a polar head group (OH) and Cholesterol has a polar head group (OH) and a nonpolar tail.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.

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.

Figure 9-10 (c) A cholesterol fatty acid ester

• Cholesterol ester formed between the Cholesterol ester formed between the cholesterol hydroxyl group and a fatty cholesterol hydroxyl group and a fatty acid with a long aliphatic side chain.acid with a long aliphatic side chain.

• This is a common modification of This is a common modification of cholesterol under physiological cholesterol under physiological conditions.conditions.

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.

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.

Figure 7-8 The Structure of Hopanoids

(a) 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.

(b) The structure of cholesterol, for comparison. A weakly hydrophilic side chain (-CH2OH or -OH) protrudes from each molecule.

Abundant in petroleum (crude oil) deposits, suggesting a prokaryotic (bacterial) role information of oil deposits

Figure 9-11(b-d) Structures of bioactive products produced from cholesterol.

b) Estradiol - a female sex hormone.

c) Testosterone - a male sex hormone.

d) Cortisol - a regulator of glucose metabolism.

Figure 9-11 (e,f) Structures of bile salts produced from cholesterol.

e) Cholate - a bile salt derived from cholic acid.

f) Glycholate - a bile salt derived from glycholic acid.

• Bile salts are stored in the gallbladder, secreted into intestines to solubilize, adsorb dietary fats.

• Bile salts have carboxylic acid groups that ionize at physiological pH (>pH 5.0)

Terpenes• Terpene class of lipids includes all molecules

biosynthesized from isoprenes (including cholesterol).

• Important terpenes include beta-carotene, lycopene, squalene.

• Terpenes often have strong odors.

Eicosanoids• Three classes of the Eicosanoid class of lipids:

1. Prostaglandins - isolated from prostrate gland, found in nearly all tissues.

2. Thromboxanes - 6-membered rings with oxygen - may help in blood clotting.

3. Leukotrienes - isolated from leukocytes, cause contraction of smooth muscle.

• All are derived from polyunsaturated 20-carbon fatty acid, arachidonate (20:4 5,8,11,14).

Three classes of the Eicosanoid lipids are all derived from polyunsaturated 20-carbon fatty acid, arachidonate (20:4 5,8,11,14).

•PGEPGE22 induces wakefulness induces wakefulness

•PGDPGD22 promotes sleep promotes sleep

Table 9.4 Common fat-soluble vitamins (terpenes)

Figure 9.14 Two insect pheromones

Housefly attractant

Honeybee queen attractant

• Cell membranes have at least five distinct functional roles:

1. Define the boundaries of the cell and its

organelles.

2. Serve as locations for specific functions.

3. Provide for and regulate transport

processes.

4. Contain the receptors needed to detect

external signals.

5. Provide mechanisms for cell-to-cell

contact, communication, and adhesion.

Key Functions of Cell Membranes

Figure 11–5 A typical membrane lipid molecule has a hydrophilic head and hydrophobic tails.

Figure 9.9 Assembly of polar lipids into lipid bilayer structures (sheets)

Hydrophobic interactions provide stabilizing energy to hold the bilayer together.

• In aqueous solutions, amphiphilic molecules form micelles.

• Have a hydrophobic interior to eliminate contacts between water and the hydrophobic tails.

• Micelles form only after a critical micelle concentration is reached (dependent on the amphiphile). For short tails (dodecyl sulfate) need higher conc. (1 mM) whereas for longer hydrophobic tails in biological lipids need a lower concentration (<10-6)

Lipid aggregates

• Glycerolipids and sphingolipids form bilayers

• Structural basis for biological membranes

• Impermeable to most polar substances

• A suspension of phospholipids can be disrupted using sonciation to form liposomes-closed self-sealing lipid vesicles bounded by a single-bilayer.

• Biological membranes are lipid bilayers with which proteins are associated.

Lipid bilayers

Figure 11–11 Amphipathic phospholipids form a bilayer in water.

(B) Computer simulation showing the phospholipid molecules (red heads and orange tails) and the surrounding water molecules (blue) in a cross section of a lipid bilayer.

(A) Schematic drawing of a phospholipid bilayer in water.

Figure 11–12 Phospholipid bilayers spontaneously close in on themselves to form sealed compartments.

The closed structure is stable because it avoids the exposure of the hydrophobic hydrocarbon tails to water, which would be energetically unfavorable.