Plan for today

 Finish up with electron transport chain and oxidative

phosphorylation
 We’re almost done looking at carbohydrate metabolism!

 Then – lipids and membranes.

 Carbohydrate metabolism so far:
 Anabolism (synthesis of larger carbohydrates) and Catabolism
(breakdown of glycogen, glucose, other sugars)
 After glycolysis: Pyruvate activated to enter TCA cycle as acetyl CoA
 Citric acid cycle: Started with a 2-carbon acetyl CoA, which combined with
a 4-carbon molecule oxaloacetate (the limiting reagent of TCA cycle) to
form citrate – 6C
 This is then oxidized, giving reduced cofactors NADH and FADH2
 Cycle continues, and can be replenished by anaplerotic reactions
 Which were our regulation points? What was the sign of their delta G?
 Electrons from TCAC carried by reduced cofactors to e- transport chain
 Peter Mitchell’s chemiosmotic hypothesis: These electrons used to generate
a proton gradient, to use to make ATP.
 4 complexes in chain, and 2 electron carriers: UQ/ Co-q or Ubiquinone in
membrane, Cytochrome C in innermembrane space
 Accounting: How many high-energy
molecules have we produced so far from
one molecule of glucose?
From glycolysis From activating From TCA cycle Total up until
pyruvate end of TCA cycle

ATP (or 2 0 2 4
GTP)
NADH 2 2 6 10

NADPH 0 0 0 0

FADH2 0 0 2 2
Electron Transport Chain and
Oxidative Phosphorylation
Chapter 10 continued
 Overall flow of electrons from NADH to O2 :
Electrons given to complex I  UQ  III  cyt c  IV  O2

NADH +H+ + ½ O2 → H2O + NAD+ ΔGo’ ~ -220kJ/mol

 3 steps release enough energy to generate a proton concentration

gradient across the inner mitochondrial membrane.
 Overall flow of electrons from FADH2 to O2 :
Electrons given to complex II  UQ  III  cyt c  IV  O2

FADH2 + ½ O2 → H2O + FAD ΔGo’ ~ -150kJ/mol

 Transfer of 2 e- to CoQ
 2 steps release enough energy to generate a proton concentration
gradient across the inner mitochondrial membrane.

Innermembrane
space

 Matrix
A side note: Compartmentalization of the cell creates a need
for shuttles - to bring NADH from cytosol into mitochondria
NADH from glycolysis can’t directly enter electron transport, so we have
shuttle systems to indirectly bring in their reducing potential.

Fast but inefficient in muscle & brain: More efficient in kidney, liver,
glycerol phosphate shuttle heart: malate shuttle
Inner membrane

matrix

Inner space
Energy Relationship in ETC
Delta G = -nFE

 What do we do with all this free energy?

 Make a proton gradient to make ATP.
 ATP Synthase (F1Fo-ATPase)
a protein complex in the inner mitochondrial membrane;
uses a proton gradient to catalyze synthesis of ATP
 Conformational coupling 2. Protons can only pass through
1. As electrons pass through the specialized channels (such as F1Fo
ETC, a proton gradient arises and ATPase)
we develop a proton-motive force 3. The energy from these pumps is
used to make ATP from ADP and
Pi.
 ATP Synthase (F1Fo-ATPase)
intermembrane 1- Fo unit:
space
- Transmembrane channel for protons

2- F1 unit:
- Projects into the matrix
- ATP synthesis from ADP + Pi

Three forms of energy here that ATP

synthase interconverts between!
1) H+ gradient
2) Mechanical energy
3) Chemical bonds/
chemical energy
mitochondrial ATP synthase does this using
matrix
“Conformational Coupling”
Conformational coupling:
An Fo slice parallel to the membrane:

The energy yielded from the proton gradient is conserved in the

form of a conformational change in the ATP synthase
 Proton movement through Fo part of ATP synthase causes
structural change (counterclockwise rotation of axle)
 This axle movement causes structural changes in F1 parts
 Three conformational states in F1:
 Open; low affinity for ATP / Loose binding to ADP / Tight binding
(catalytically active)
ATP synthesis cycle:
1. ATP synthesis begins with binding of ADP and Pi to the L
site.
2. Axle turns from proton pumping, converting LT and allowing
T site to catalyze ADP+PiATP
3. Axle turns from proton pumping, converting TO and
releasing ATP
ATP synthase in motion…
 http://www.youtube.com/watch?v=PjdPTY1wHdQ
The Mitochondrion at 1,000,000x magnification
(by David Goodsell)
How much ATP do we get from
Oxidative phosphorylation?
 the P/O ratio:
 ratio ATP produced / amount oxygen consumed.
 Oxidation of NADH:
2 e- transferred through Complexes I, III, and IV:
P/O ratio ~ 2.5ATP PER NADH
 Oxidation of FADH2:
2 e- transferred through Complexes II, III, and IV:
P/O ratio ~ 1.5 ATP per FADH2
G3P shuttle  FADH2  1.4 ATP per NADH
Malate shuttle  NADH  2.25 ATP per NADH
ATP synthesis from oxidation of one molecule of glucose
NADH FADH2 ATP
Glycolysis 2 0 2
Pyruvate activation 2 0 0
Citric acid cycle 6 2 2

Recall: P/O ratio for NADH is 2.5; P/O ratio for FADH2 is 1.5
Oxidative Phosphorylation ATP

2 Pyruvate activation NADH 5,

6 Citric acid cycle NADH
2 Citric acid cycle FADH2
2 glycolytic NADH (via two shuttles)
TOTAL
SUM TOTAL ATP, GLUCOSE TO CO2+H2O
Regulation of Oxidative Phosphorylation
 Depends on the need for ATP.
 ↑ with ADP level
 ↑ with rate of catabolic reactions (catabolism of glucose; FA)
 ↑ in presence of NADH and FADH2
 ↑ with O2 consumption

 Regulated by level of transmembrane proton gradient.

 Regulated by availability of NADH, FADH2.

 Depends on the presence of ADP and Pi in the matrix.

Disrupting electron transport and/or ATP
synthesis: Cellular respiration inhibitors
I
• Electron transport inhibitors
•Rotenone
•Antimycin
•Cyanide

• ATP synthase inhibitor

•Oligomycin – binds Fo
Disrupting oxidative phosphorylation:
Uncouplers
 Dissipating the proton gradient causes an
uncoupling of oxidation and phosphorylation
 Example: dinitrophenol
 lipophilic, weak acids functional groups
 carries protons across the inner mitochondrial membrane ↓ [H+]
gradient; ↓ oxidative phosphorylation
Energy of the proton gradient is lost as heat.

 “Diet pill” (1940): effective in reducing weight but caused many fatalities.

 Hibernating animals (and newborn human): use uncoupling

proteins (UPC) to maintain body heat (brown adipose tissues).
 Aerobic respiration - The price to pay
 Incomplete reduction of O2 creates reactive by-products that are
dangerous (free radicals).
 If there aren’t enough electrons going through ETC, we can get
problems.
 Free radicals arising from oxidative metabolism appear to be
partially responsible for the aging process.
 Neurodegenerative conditions such as Parkinson's, Alzheimer's, and
Huntington's diseases are associated with mitochondrial oxidative
damage.
 Antioxidants pick up free radicals and so have protective effect!
Review questions
 How can reduction potentials be used to determine electron
flow between electron carriers?
 What is the final electron acceptor of the respiratory chain?

 How are electrons delivered to Complex, I, II, III and IV?

 How is the proton gradient formed and how is it used to

synthesized ATP?
 How is oxidative phosphorylation regulated? What can
disrupt/decouple this?
 How is cytosolic NADH processed?
 Back to map of our course:

Lipids Proteins

Carbohydrates Nucleic Acids

 For each, we’ll study structures; reactions; energy; synthesis and breakdown

 We’ve now started looking at metabolism – breaking down nutrients for

ATP, and synthesizing larger molecules

 Next - Lipids: Structure from small to big; breakdown; synthesis.

Lipids and Membranes

Chapter 11
Objectives

 Understand the structures and function of

lipids
 Review the structure and function of biological
membranes
 Lipids as a class of biomolecules
 Unlike other biomolecules, lipids have no structural unity
 Lipids are classified based on an operational definition:
 Substances from living organisms that dissolve well in non-polar
solvents.
Lipids include (but are not limited to):
 Fatty acids
 Molecules built up from fatty acids: eicosanoids, triacylglycerols,
sphingolipids, phosphoacylglycerols, glycolipids, waxes
 Isoprenoids: terpenes and steroids
 Also - lipid-soluble vitamins, such as A, E, D and K.
Fatty acids
 Carboxylic acids (usually between 14 and 22 carbons).
Recall/compare amino
acids nomenclature:

 Unbranched hydrocarbon chain with an even number of carbon atoms.

 Acyl chain can be saturated All c-c bonds are single
or unsaturated Some double bonds (c=c)

 Most C-C bonds are single bonds; any double bonds are usually cis.
 If more than one C=C, double bonds are not conjugated

 Fatty acid nomenclature specifically indicates the location of the double bond.
 FAs are common component of other lipids (ester or amide linkages).
Saturated fatty acids

Melting point ___ with ________

Unsaturated fatty acids

Melting point _increases_ with increased number of double bonds

Draw the structure of cis, cis, cis-Δ9,12,15-octadecadienoic acid. (linolenic acid)

This can also be written as ______________.

______________________ fatty acids have been
implicated in improved health benefits.
Fatty acid conformations

Single bonds can The double bond

Omega – 9 – fatty acid
wiggle cannot wiggle
Straight chain
– cannot bend
and does not
 Eicosanoids
 Powerful hormone-like molecules
 Many of them derived from arachidonic acid or eicosapentaenoic acid
 Three main types of Eicosanoids: Leukotrienes.

Prostaglandins

Thrombin
Waxes
 Ester made from a long fatty acid and a long-chain alcohol.
 Serve as protective coatings for both plants and animals
 Triacylglycerols
(aka Triglycerides)
 3 fatty acids esterified to the hydroxyl
groups of D-glycerol
 Main constituent of animal/vegetable
fats; a compact form of energy storage
1) Highly reduced
2) Pack well; no H20
Phospholipids D-glycerol with fatty acids esterified at C1 & C2 and
a phosphate-containing head group esterified to C3.

 Amphiphilic
 Major lipid constituents of biological membranes.
 When present in sufficient quantities, they form a bilayer
(premise of biological membranes)
Sphingolipids sphingosine
Ceramide

 Amphiphilic
 Constituent of biological membrane

 Derived from Sphingosine: a long-chain

nitrogen-containing alcohol (R2 = OH), no fatty acid.
 Ceramides: Fatty acyl linked to primary amino group
of sphingosine.
 R2 group can vary:
 Sphingomyelin: ceramide esterified to a
phosphocholine or phosphoethanolamide at R2.
Fatty acid chain
 Glycosphingolipids: ceramide with a glycoside
bond to a simple or complex sugar at R2

Glycosphingolipid
Phospholipids &
Sphnigolipids
have diverse Phopsholipids

structures
Both at headgroup and at
the fatty acid chain. This
contributes to the amazing
diversity and function of
membranes
Sphingolipids
Isoprenoids  Repeating five carbon units of isoprene units

Two sub-classes:
Terpenes Steroids
 Classified by number of isoprene units  Derivatives of triterpenes
(monoterpenes: 2 isoprene units, so C10H16)
 Four fused rings
 Found in essential oils of plants
 Found in all eukaryotes, small
number of bacteria
 Precursor of steroid hormones
& Vitamin D

vitamin D
 Cholesterol: Most abundant animal steroid
(major component of biological membranes).
 Weakly amphiphilic due to hydroxyl group on C3.
Lipoproteins
 ‘Lipoprotein’ can technically mean any lipid-protein covalent attachment,
but generally refers to a lipid-protein particle held together by H-bonds
 These complexes carry triglycerides/cholesterol through the blood
 Classified by density; can range from 5-1000nm

This is LDL – Low Density Lipoprotein  Specific

(indicated by presence of Apolipoprotein B100) apolipoproteins
bind specific cell
surface receptors at
the appropriate
tissues, allowing the
lipoprotein to drop
off the cargo
 What you need to know
about lipids
 Recognize structures of different types of lipids (fatty acids,
eicosanoids, waxes, triglycerides, phospholipids, sphingolipids,
isoprenoids [terpenes/steroids]); and lipoprotein particles
 Draw a fatty acid given the chemical name
(eg. 18:2Δ9,12 or 18:3ω-6 or cis,cis, cis-Δ9,12-octadecadienoic acid)
 Predict relative melting temperatures of different fatty acids
 Know relevant function of general types of lipids
Next: Plasma Membranes
 Overall structure
 As influenced by lipid saturation and fatty-acid chain length
 The fluid mosaic model and lipid rafts
 Membrane proteins and transport across membranes
 What is membrane/protein research about now?
Different membranes with different roles
What do membranes do?
 The plasma membrane –
a lipid bilayer
Major lipid constituents:

What is the major driving force for bilayer formation?

Enthalpic – sequestering hydrophobic groups – all kinds of solvation
networks going on
Lipid shape/type impacts bilayer properties
 Shape of lipid (FAs
and/or head group) can
introduce local curvature
to the bilayer

Cholesterol presence;
degree of FA saturation;
length of FA:
all influence fluidity
 Not all lipids have
only 2 tails!
(shape can be
complicated)

Kdo2-Lipid A
(Side note: lipids also form other biologically
relevant structures in water solvent)
 Lipids are amphiphilic molecules – in water solvent, sequester
hydrophobic parts away from hydrophilic; thermodynamics at work!
The fluid mosaic model of the membrane
 Mosaic: Lipids and proteins exist side by side without forming any
intermediate substances.
 Fluid: lateral movement of both proteins & lipids.

Historical view

Controversial:
Lipid rafts (?)

 In all these views:

 Integral and peripheral membrane
proteins are present
More current –
 Lipid composition can vary between
More crowded!
leaflets
Membrane Proteins
 Peripheral proteins
on one side only

 Integral proteins
 Go all the way through

 Lipid-anchored proteins
(covalent via amino groups and
Cys).
 N- Myristoylation: irreversible
 S-palmitoylation: reversible

Something to consider – the identity/types of amino acids at specific

points in the membrane protein: 1) at the middle of the membrane; 2)
at the solvent-exposed regions; 3) at the interface region?
Membrane proteins: what do they do?
Transport
Catalysis
Communication – receptors
Organization / structural

What do they look like?

Transport Across Membranes

 Passive transport: molecules

move down concentration
gradient, no energy needed
 Simple Diffusion
 Facilitated Diffusion
 Active transport: molecules
move against concentration
gradient, energy needed
 Primary
 Secondary
 Where is current membrane /
membrane-protein research going?
 The idea of curvature affecting signalling, membrane organization, …
 The “myth” (?) of the lipid raft?
 How do lipids and proteins move beween membrane leaflets?
 Protein structures and folding in membranes – re-thinking ideas from soluble proteins
 How to isolate and study membrane proteins?
 What are the functions of many membrane proteins?
 LOTS more! This is a really open, exciting field right now.
Functional categorization of the E. coli
inner membrane proteome

A field for you?

Summary
 What structural features distinguish triacyglycerols,
phospholipids, sphingolipids and cholesterol?
 How do chain length and saturation influence the overall
structure of a lipid bilayer?
 What are the similarities and differences between
integral, peripheral and lipid-linked membrane proteins?
 Describe the fluid mosaic model of membrane structure.

 What are the different types of transport system?