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Lecture 29
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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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Page 917
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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-hydroxyacylCoA
NAD+-dependent dehydrogenation of bhydroxyacyl-CoA by 3-L-hydroxyacyl-CoA
dehydrogense (HAD) to form -ketoacylCoA.
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
Binding Change Mechanism
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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
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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
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.
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.
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
H
NO2
O2N
OH
NO2
O 2N
O
Introduction: Lipids
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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).
Fatty Acid Structure
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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).
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:
1. Zigzag line shows
hydrocarbon chain.
2. Structure showing all
carbon atoms.
3. Space filling structure actual shape.
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.
Important Features of Fatty Acid Cell Membrane
Lipid Components
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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:
1.
Palmitate - (16) - saturated
2.
Stearate (18) - saturated
3.
Oleate (18) - unsaturated, 1 double bond
4.
Linoleate (18) - unsaturated, 2 double bonds
5.
Linolenate (18) - unsaturated, 3 double bonds
6.
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.
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 mixed
triacylglycerol components (contain more
than one fatty acid).
• Butter
11% C4-C12
Small side chains melt at lower
temp. give flexibility at room temp.
10% C14
26% C26
11% C18
Unsaturated also melt at lower temp.
40% unsat C16 + C18 (olive oil approx. 80% unsaturated).
Soap
Soap molecules aggregate in water to form micelles,
dissolve greasy, oily compounds in water.
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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).
Figure 9.3 The commercial hydrogenation of vegetable
oils often leads to trans acids
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How to prepare oleomargarine.
Trans fatty acids may increase blood cholesterol
levels.
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.
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
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
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The membrane lipids are composed of the
glycerophospholipids and sphingolipids, which have
polar and nonpolar regions.
Figure 9.7
Glycerophospholipids
Phosphatidic acid
4 major
glycerophospholipids
are polar/charged:
Other examples of phosphoglycerol derivatives
• Can be in sugars (glycolipids)
• Diphosphatidyl glycerol
O
O
RCO-CH2
O
CH2-OCR
O
RCO-CH
CH-OCR
O
OH
O
CH2-O-P-O-CH2-CH-CH2-O-P-O-CH2
O-
glycerol
O-
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.
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.
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.
The molecular structure common to all steroids
Steroid structures have four fused rings, A, B, C, and D.
Figure 9-10b Cholesterol molecular structure - a steroid
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Cholesterol has a polar head group (OH) and a
nonpolar tail.
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Cholesterol and ester derivatives are abundant in
(blood) plasma proteins called lipoproteins.
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Lipoproteins transport cholesterol to tissues for use
in cell membranes and hormone precursors.
Figure 9-10 The molecular structure common to all steroids
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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
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Cholesterol ester formed between the cholesterol
hydroxyl group and a fatty acid with a long
aliphatic side chain.
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This is a common modification of cholesterol
under physiological 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
Abundant in petroleum (crude
oil) deposits, suggesting a
prokaryotic (bacterial) role in
formation of oil deposits
(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.
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.
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Bile salts are stored in the gallbladder, secreted into
intestines to solubilize, adsorb dietary fats.
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Bile salts have carboxylic acid groups that ionize at
physiological pH (>pH 5.0)
Terpenes
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Terpene class of lipids includes all molecules
biosynthesized from isoprenes (including cholesterol).
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Important terpenes include beta-carotene, lycopene,
squalene.
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Terpenes often have strong odors.
Eicosanoids
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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).
•PGE2
induces wakefulness
•PGD2
promotes sleep
Table 9.4 Common fat-soluble vitamins (terpenes)
Figure 9.14 Two insect pheromones
Housefly
attractant
Honeybee queen
attractant
Key Functions of Cell Membranes
• 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.
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.
Lipid aggregates
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In aqueous solutions, amphiphilic molecules form micelles.
• Have a hydrophobic interior to eliminate contacts
between water and the hydrophobic tails.
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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 bilayers
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Glycerolipids and sphingolipids form bilayers
Structural basis for biological membranes
Impermeable to most polar substances
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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.
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Figure 11–11 Amphipathic phospholipids form a bilayer in water.
(A) Schematic drawing of a
phospholipid 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.
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.