Paper-IV- BI 104T: Bioenergetics and Cell Biology

Unit – I: Bioenergetics
1. Elements of importance in biochemistry (H, C, N, O, P, S), types and energy of bonds
and interactions (ionic, covalent, coordinate, H-bonds, van der Waals, hydrophobic
interactions)
2. Laws of thermodynamics, Gibbs free energy, relevance of entropy and enthalpy in
biological systems and reactions; first and second-order reactions
3. Log and ln scales in biological processes (exponential growth curves, radioactive
decay)
4. Biological oxidation, high energy compounds
5. High energy bonds, redox and phosphate potential
6. Structure of membrane, forces stabilizing membranes
7. Formation of ion gradients across a membrane (proton gradients in organelles), role
of transporters and channels
8. ETC in mitochondria and chloroplasts, un-couplers and inhibitors of energy transfer
9. Polarization of cell, resting potential, action potential, propagation of impulse
BIOENERGETICS

References to follow:
1. Lehninger principle of Biochemistry by
Nelson and Cox.
2. Biochemistry by Berg, Tymoczko and
Stryer.
3. Biochemistry by Voet and Voet.
 Why to study Bioenergetics??
• Living organisms must work to stay alive, to grow and to reproduce
• All living organisms have the ability to produce energy and to channel it into biological
work
• Living organisms carry out energy transductions, conversions of one form of energy to
another form
• Biological energy transductions obey the same physical laws that govern all other
natural processes
• Bioenergetics is the quantitative study of the energy transductions that occur in living
cells and of the nature and function of the chemical process underlying these
transductions
• Modern organisms use the chemical energy in fuels (carbonhydrates, lipids) to bring
about the synthesis of complex macromolecules from simple precursors
• They also convert the chemical energy into concentration gradients and electrical
gradients, into motion and heat, and, in a few organisms into light (fireflies, some
deep-sea fishes)
 Periodic Table showing biologically
important elements
IA 0
1

7
 The Six Most Abundant Elements of Life
 Most biological molecules are made from covalent
combinations of six important elements, whose
chemical symbols are CHNOPS.
 Biological molecules, or biomolecules, are built by joining
these atoms through covalent bonds.
 Although more than 25 types of elements can be found in
biomolecules, six elements are most common. These are
called the CHNOPS elements; the letters stand for the
chemical abbreviations of carbon, hydrogen, nitrogen,
oxygen, phosphorus, and sulfur.
                                                                                                            
,N
 Valency of the elements
 Each element has a characteristic valence that determines the number
of covalent bonds it can form. The ability of an atom to combine with
other atoms depends on the number of electrons in the outer shells of
the atoms.
 Some elements, the so-called noble gases, have complete outer shells
and do not share electrons. Many elements will share electrons with
other elements, such that each element completes its outer electron
shell capacity.
 A shared electron pair is called a covalent bond. The number of
covalent bonds that each element can form is called its valency.
                                                                                                                                                             
 Organic molecules contain C & H.
 Methane (CH4), glucose (C6H12O6) are organic .
 Water (H2O), carbon dioxide (CO2) are inorganic .

Organic molecules are typically derived from living things

Special features of the element Carbon:
• can form bonds with up to 4 other atoms
• can form complex linear, branched, ringed structures
• bonds tend to be relatively non-polar, stable
 Important Functional Groups
All biological molecules are basically carbon skeletons
with various functional groups attached

–H (hydrogen: nonpolar “default” group)

–OH, -SH (hydroxyl, sulphydril: polar)
–COOH or –COO- (carboxyl: acidic)
–NH2 or –NH3 + (amino: basic)
–H2PO4 or –PO4 2- (phosphate: acidic)
–CH3 , -CH2CH3(methyl, ethyl: non-polar)
Intermolecular forces and Chemical BONDING
 Intermolecular forces
Why do some solids dissolve in water Why are some substances gases
but others do not? at room temperature, but
others are liquid or solid?

What gives metals the ability to conduct electricity, what makes

non-metals brittle?
 Overview
• There are 2 types of attraction in
molecules:
1. intramolecular bonds
2. intermolecular forces

• Intermolecular forces (IMF) have to do

with the attraction between molecules (vs.
the attraction between atoms in a
molecule)
 Intermolecular Forces

Intramolecular = strong
Intermolecular = weak

They do control physical properties such as boiling and melting

points, vapor pressure, and viscosity
Types of Intermolecular Forces

Van der Waals Forces

• Dipole-dipole interactions
• Ion-Dipole interactions
• London dispersion forces

Hydrogen bonding
Dipole-Dipole Interactions

Molecules that have

permanent dipoles are
attracted to each other.
 Ion-Dipole Interactions
Attractive forces between an ion and a polar molecule
Ion-Dipole Interaction

The larger the charge the stronger the force

 London Dispersion Forces

Occasionally electrons wind up on the same side

of the atom.
 London Dispersion Forces

At that instant, the helium atom is polar.

 London Dispersion Forces

This polar helium atom then induces a dipole

on a neighboring helium atom.
 London Dispersion Forces

London dispersion forces, or dispersion forces, are

attractions between an instantaneous dipole and
an induced dipole.
 London Dispersion Forces

• These forces are present in all molecules, whether

they are polar or nonpolar.
• The tendency of an electron cloud to distort in
this way is called polarizability.
 Hydrogen Bonding

• The dipole-dipole interactions

experienced when H is bonded to N,
O, or F are unusually strong.
• We call these interactions hydrogen
bonds.
 Hydrogen “Bonding”
• Strong polar
attraction
– Like magnets

• Occurs ONLY
between H of one
molecule and N, O,
F of another
H “bond”
 Properties of H-Bonding
H is shared between
2 atoms of OXYGEN or NITROGEN or H-Bonding in
FLUORINE of 2 different molecules Water molecule

• Nitrogen, Oxygen and

Fluorine
– Small atoms with strong
nuclear charges
– very high electronegativities
Hydrogen Bonding: Water
 Basics of Chemistry – Atomic Structure

Atomic Structure
– Nucleus
• Protons (+)
and Neutrons
(0)
– Both weight
1.0 amu
– Energy Levels
• Electrons (-)
– <1 amu
(1/2000)
 Atoms make up Elements

Same types of atoms (Fe, Cu, etc.) Elements form compounds and molecules

Not all atoms of the same kind are alike - Isotopes

 Electrons Interact to form Chemical Bonds
Valence Electrons – Atomic Number
– Atomic number: number of protons = number of electrons
– Valence: most outer shell electrons that react with other atoms

Ions: When an atom gains or loses an electron (Na+, Cl-)

Metals and Nonmetals and Formation of Ions

– Metals: tend to lose electrons and form Positive ions or cations
– Nonmetals: gain electrons and form negative ions or anions

Ion Attraction
– Electrostatic attraction
– Forms Ionic Bonds
 Ionic and Covalent Bonds
1. Ionic Bonds
– Formed via electrostatic attraction
• Metals (cations) and Nonmetal (anions)
• Electronegativity
– Crystalline solids
– Salts: NaCl; MgCl2 (Electrolytes)
– Importance of ions/electrolytes

2. Covalent Bonds
– Similar electronegativity
– Nonmetals
– Forms molecules
• Importance of molecules and covalent
compounds
 Covalent Compounds – Polarity
Covalent compounds
– Slight charges are formed during the
sharing of electrons
– Equal Sharing “nonpolar”
• No slight charges formed
– Unequal Sharing “polar”
• Slight differences in EN – charges
• Forms two poles or “dipole”

• Water – Polar Covalent Compound

– Universal Solvent
– Chemical reactions
 Properties of Ionic Compounds
• Hard solid @ 22oC
• High melting point
• Nonconductors of electricity in solid phase
• Good conductors in liquid phase or
dissolved in water (aq)
 Properties of Covalent Substances

• Low m.p. and b.p.

• Relatively soft solids as compared
to ionic compounds
• Nonconductors of electricity in any
phase
 Types of Covalent Bonds
NON-Polar bonds
–Electrons shared evenly in the bond
–E-neg difference is very less and becomes zero
for identical atoms. For e.g. diatomic molecules
O2, N2, H2.

Polar bond
–Electrons unevenly shared
–E-neg difference between zero and 2.0
 Non-polar molecules
• Sometimes the bonds within a H
molecule are polar and yet the Methane
molecule is non-polar because its (CH4) H C H
shape is symmetrical.
H
Polar molecules (Dipoles)
• Not equal on all sides Hydrogen chloride
– Polar bond between 2 atoms (HCl)
makes a polar molecule
 -
– asymmetrical shape of +
H Cl 
molecule
 Metallic bonding

– Occurs between like atoms of

a metal in the free state It’s the mobile
– Valence e- are mobile (move electrons that enable
freely among all metal atoms) metals to conduct
– Positive ions in a sea of electricity
electrons

• Metallic characteristics
– High mp temps, ductile, malleable,
shiny
– Hard substances
– Good conductors of heat and
electricity as (s) and (l)
Thermodynamics in Biology

• Laws of thermodynamics
• Quantitative relationships
among free energy,
enthalpy and entropy
• Describe the special role
of ATP in biological
energy exchanges
 Laws of thermodynamics
• Biological energy transductions obey the laws of
thermodynamics

• 1. For any physical or chemical change, the

total amount of energy in the universe remains
constant; energy may changed from or it may
be transported from one region to another, but
it can not be created or destroyed
• 2. The universe always tends toward increasing
disorder: in all natural processes the entropy of
the universe increases
Living organism as a Thermodynamic System
• The reacting system may be an organism, a cell or
two reacting compounds. The reacting system and its
surroundings together constitute the universe.
• In the laboratory some chemical or physical processes
can be carried out in closed systems and no material
or energy is exchanged with the surroundings
• However living organisms are open systems. They
exchange both material and energy with their
surroundings
• Living systems are never at equilibrium with their
surroundings
 Concept of Free Energy
• Gibbs free energy (G): G expresses the
amount of energy capable of doing work
during a reaction at constant temperature
and pressure.
• When a reaction proceeds with the release
of free energy (that is, when the system
changes so as to posses less free energy) ΔG
has a negative value and the reaction is said
to be exergonic
• In endergonic reactions, the system gains
free energy and ΔG is positive
• The unit of ΔG is joules/mole or
calories/mole
 Concept of Enthalpy
• Enthalpy (H): H is the heat content of the
reacting system. H reflects the number and kinds
of chemical bounds in the reactants and
products.
• When a chemical reaction releases heat, it is said
to be exothermic, the heat content of the
products is less than that of the reactants and ΔH
has a negative value
• Reacting systems that take up heat from their
surroundings are endothermic and have positive
values of ΔH
• The unit of ΔH is joules/mole or calories/mole
 Concept of Entropy
• Entropy (S): S is a quantitative expression for the randomness or
a disorder in a system

• Whenever a chemical reaction results in an increase in the

number of molecules the entropy of the surroundings increases

• Whenever a solid substance is converted into liquid or gaseous

forms the entropy of the surroundings increases. Because this
transformations allow the molecule more freedom for movement

• The unit of ΔS is joules/mole. Kelvin

 Relation between Free Energy, Enthalpy and
Entropy

 Under the constant temperature and pressure changes in

free energy, enthalpy and entropy in biological systems are
related to each other by the equation
ΔG= ΔH - TΔS
ΔG= (Gproducts– Greactives); ΔH= (Hproducts – Hreactives)
T= Absolute temperature; ΔS= (Sproducts – Sreactives)

 Living organisms preserve their internal order by taking

free energy from their surroundings in the form of
nutrients or sunlight and returning to their surroundings an
equal amount of energy as heat and entropy
 Demonstrative Example of Application of
Thermodynamics:The oxidation of glucose

Increase in
the Entropy
 Cells require sources of free energy- but How?

• Living organisms acquire free energy from

nutrient molecules. Cells transform this free
energy into ATP and other energy-rich
compounds
• They are capable of providing energy for
biological work at constant temparature.
• The composition of a reacting system tends to
continue changing until equilibrium is reached.
• At the equilibrium the rates of the forward and
reverse reactions are equal and no further
change occurs in the system.
 Description of the Equilibrium Constant

• The Keq is defined by the molar concentrations of

products and reactants at equilibrium
aA+bB cC+ dD

[C]c [D]d
• Keq = [This ratio is also known
Mass-action ratio, Q]
[A]a [B]b
 Standard Free Energy and its Properties
• When a reacting system is not at equilibrium, the tendency to move toward
the equilibrium represents a driving force expressed as free energy change
(ΔG).
• Under standard conditions (250C), when reactants and products are initially
at the 1 M concentrations the force driving the system toward equilibrium is
defined as the standard free energy change (ΔG0)
• Physical constants based on this biochemical standard state are called
standard transformed constants and written as ΔG'0 and K'eq to distinguish
them from the untransformed constants which are used by chemists.
Where, ΔG'0 = ΔG'0 products– ΔG'0 reactives
• When ΔG'0 is negative, the products contain less free energy than the
reactants and the reaction will proceed spontaneously under standard
conditions
• When ΔG'0 is positive, the products contain more free energy than the
reactants and the reaction will tend to go in the reverse direction under
standard conditions
 Actual free energy change
• Actual free energy change (ΔG) is a function of
reactant and product concentrations and of the
temparature prevailing during the reaction
which will not necessarily match the standard
conditions as defined before
• ΔG of any reaction proceeding spontaneously
toward its equilibrium is always negative, become
less negative as the reaction proceeds, and is zero
at he point of equilibrium, indicating that no more
work can be done by the reaction
• ΔG and ΔG'0 for a reaction like that
A+B C+D
is written as
[C] [D]
ΔG=ΔG'0 + RTln
[A] [B]
• An example:
• A+B C+D

• Reaction is taking place at the standard

temparature and pressure
• But the concentrations of A,B,C and D are not equal
and none of them at the 1M concentration
• In order to determine actual ΔG under these non-
standard concentrations as the reaction proceeds
from left to right, we enter the actual
concentrations of A,B,C and D in this equation.
• Rest of the terms in the equation (R,T, ΔG'0 ) are
standard values
• When the reaction is at equilibrium there is no force
driving the reaction in either direction and ΔG is
zero, thus equation reduces to
[C] [D]
• 0= ΔG'0 + RTln
[A] [B]

• ΔG'0 = -RT lnK'eq ΔG'0

 Bio-energetic advantages of Coupling of Reaction

• The criteria for spontaneity of a reaction is the value

of ΔG not ΔG'0

• Standard free energy changes are additive.

• In the case of two sequential chemical reactions,

• A B ΔG'01
• B C ΔG'02
• Since the two reactions are sequential, we can
write the overall reaction as

A C ΔG'0total

• The ΔG'0 values of sequential reactions are

additive.

ΔG'0total = ΔG'01 + ΔG'02

• A B ΔG'01
• B C ΔG'02

• Sum: A C ΔG'01 + ΔG'02

• This is how a thermodynamically unfavorable

(endergonic) reaction can be driven in the forward
direction by coupling it to a highly exergonic
reaction through a common intermediate
• The main rule in biochemical reactions in
living organisms:

• All endergonic reactions are coupled to an

exergonic reaction.
• There is an energy cycle in cells that links
anabolic and catabolic reactions.
 The first step of glycolysis

Phosphorylation of Glucose involves two chemical

reactions:

Reaction 1:
• Glucose + Pi Glucose 6-phosphate+H2O
ΔG'0 =13.8 kj/mol

• ΔG'0 >0 reaction is not spontaneous

Reaction 2: Hydrolysis of ATP - very exergonic cellular
reaction

ATP + H2O ADP + Pi

ΔG'0 = -30.5 kj/mol

ΔG'0 < 0 reaction is spontaneous

 These two reactions share the common intermediates H2O
and Pi and may be expressed as sequential reactions:
Glucose + Pi Glucose 6-phosphate+H2O
ATP + H2O ADP + Pi

Sum: Glucose+ATP Glucose 6-phosphate+ADP

The overall standard free energy changes:

• ΔG'0 =13.8 kj/mol + (-30.5 kj/mol)= -16.7 kj/mol

Overall reaction is exergonic

• Energy stored in ATP is used to drive to synthesis
of glycose 6-phosphate, eventhough its
formation from glucose and Pi is endergonic.
• This strategy works only if compounds such as
ATP are continuously available.
 Multiplicative nature of Equilibrium constants

The equilibrium constant for the two coupled reactions is,

Although the G values for two reactions that sum to a third are additive, the Keq for
a reaction that is the sum of two reactions is the product of their individual Keq
values. Equilibrium constants are multiplicative.
 Rate Equations and
Order of Reactions
 Rate Equations and Order of Reactions
 Zeroth, First and Second Order
Reactions
 Rate Equations and Order of
Reactions

For the reaction aA + bB  cC + dD

Rate  k[A]x[B]y
where x and y are the orders of reaction with
respect to A and B
 x and y can be positive or negative integers or
fractional
 x  y is the overall order of reaction
 Important things to note……

For multi-step reactions,

 x, y have no direct relation to the stoichiometric

coefficients and,
 can ONLY be determined experimentally

For single-step reactions (elementary reactions),

x = a and y = b
For the reaction aA + bB  cC + dD

Rate  k[A]x[B]y
x = 0  zero order w.r.t. A
x = 1  first order w.r.t. A
x = 2  second order w.r.t. A

y = 0  zero order w.r.t. B

y = 1  first order w.r.t. B
y = 2  second order w.r.t. B
For the reaction aA + bB  cC + dD
Describe the reaction with the following rate law

Rate  k[B]2
The reaction is zero order w.r.t. A and
second order w.r.t. B.
For the reaction aA + bB  cC + dD

Rate  k[A]x[B]y

k is the rate constant

• Temperature-dependent
• Can only be determined from experiments
For the reaction aA + bB  cC + dD

Rate  k[A]x[B]y
3 1
rate mol dm s
k x y
 3 x 3 y
[A] [B] (mol dm ) (mol dm )
units of k : -
mol dm3 s1/(mol dm3)x+y or,
mol dm3 min1 /(mol dm3)x+y
For the reaction aA + bB  cC + dD

Rate  k[A]0[B]0

units of k
= mol dm3 s1/(mol dm3)0+0
= mol dm3 s1
= units of rate
 For the reaction aA + bB  cC + dD

Rate  k[A][B]0

units of k
= mol dm3 s1/(mol dm3)1+0
= s1
For the reaction aA + bB  cC + dD

Rate  k[A][B]

units of k
= mol dm3 s1/(mol dm3)1+1
= mol1 dm3 s1
The overall order of reaction can be
deduced from the units of k
For the reaction
aA + bB + cC + …  products

Rate  k[A]x[B]y[C]z…
units of k : -
mol dm3 s1/(mol dm3)x+y+z+…
Determination of rate equations

To determine a rate equation is to find k, x, y,

z,…

Rate  k[A]x[B]y[C]z…
Hydrolysis of high
energy phosphate
bonds
 Phosphoryl group transfer and ATP
• Living cells obtain free energy in a chemical form by the catabolism of nutrient
molecules.
• They use that energy to make ATP from ADP and Pi .

ATP donates some of its chemical energy to

1. Endergonic processes such as the synthesis of metabolic intermediates and
macromolecules from smaller precursors
2. The transport of substances across membranes against concentration gradients
3. Mechanical motion (muscle contraction)

• This donation of energy from ATP can occur in the two forms

• A) ATP ADP+ Pi or

• B) ATP AMP+ 2 Pi
•
 The free energy change for ATP hydrolysis is
large and negative
• The hydrolytic cleavage of the terminal phosphoanhydride
bond in ATP separates one of the three negatively charged
phosphates and thus relieves some of the electrostatic
repulsion in ATP
• Released Pi is stabilized by the formation of several resonance
forms not possible in ATP
• ADP2- is the other product of hydrolysis and it immediately
ionizes, releasing H+ into a medium of very low [H+] ( ̴ 10-7 M)
• Because the concentrations of the products ot ATP hydrolysis
are far below the concentrations at equilibrium, mass action
favors the hydrolysis in the cell
Biochemical Reaction for ATP hydrolysis
Note>
• Although the hydrolysis of ATP is

highly exergonic (ΔG'0 = -30,5

kj/mol), the ATP is stable at pH 7,

because the activation energy

for ATP hydrolysis is relatively

high.

• Rapid hydrolysis of ATP occurs

only when catalyzed by an

enzyme
 Mechanism of ATP hydrolysis
• The free energy change for ATP hydrolysis is -30,5 kj/mol under
standard conditions but the actual free energy change (ΔG) of
ATP hydrolysis in living cells is very different.
• The cellular concentrations of ATP, ADP and Pi are not same and
are much lower than the 1 M standard conditions.

Mg2+ in the cytosol binds

to ATP and ADP and for
most enzymatic reactions
that involve ATP as
phosphorly group donor,
the true substrate is
MgATP-2. The relevant ΔG'0
is therefore that for
MgATP-2 hydrolysis.
 Importance of Coupling

The downward motion of an object releases potential energy that can do

mechanical work. The potential energy made available by spontaneous
downward motion, an exergonic process (pink),
can be coupled to the endergonic upward movement of another object
(blue).
 Energy Co-ordinate
 The exergonic reaction has a large, negative free-energy change (G2), and the
endergonic reaction has a smaller, positive free energy change (G1).
 The third reaction accomplishes the sum of reactions 1 and 2, and the free-energy
change, G3, is the arithmetic sum of G1 and G2.
 As G3 is negative, the overall reaction is exergonic and proceeds spontaneously.
 Resonance vs Tautomerism
 Resonance structures are different representations of the same structure. 
 The atoms have the same connectivity, but they differ in the arrangement of their
lone pairs and double bonds.

 Tautomerization involves a change in connectivity of the atoms yielding two different

constitutional isomers. 
 Tautomerization is an actual chemical reaction that can take place. 
 The two tautomers will have different reactivity, boiling points, melting points as they
are two unique molecules. 
Compounds have large free energy change

• Phosphorylated compounds
• Thioesters (Acetyl-CoA)
 Phosphorylated compounds
• Phosphoenolpyruvate
• 1,3-bisphosphoglycerate
• Phosphocreatine
• ADP
• ATP
• AMP
• PPi
• Glucose 1-phosphate
• Fructose 6-phosphate
• Glucose 6-phosphate
 Phosphoenolpyruvate
• Phosphoenolpyruvate contains a phosphate ester bond that undergoes hydrolysis to
yield the enol form of pyruvate

• The enol form of pyruvate can immediately tautomerize to the more stable keto form of
pyruvate. Because phosphoenolpyruvate has only one form (enol) and the product,
pyruvate, has two possible forms, the product is more stabilized relative to the
reactant.

• This is the greatest contributing factor to the high standard free energy change of
hydrolysis of phosphoenolpyruvate (ΔG'0 = -61.9 kj/mol)
 1,3-bisphosphoglycerate (1,3-BPG)
• 1,3-BPG contains an anhydride bond between the carboxyl
group at C-1 and phosphoric acid.
• Hydrolysis of this acyl phosphate is accompanied by a large,
negative, standard free energy change (ΔG'0 = -49.3 kj/mol)
• This large, negative ΔG'0 can, again, be explained by resonance
stability of the carboxylate form favouring the forward reaction.
 Phosphocreatine
• In the phosphocreatine, the P-N bond can be hydrolyzed to
generate free creatine and Pi.
• The release of Pi and the resonance stabilization of creatine
favor the forward reaction. The standard free energy change
of phosphocreatine is large and negative (ΔG'0 = -49.3 kj/mol).
• Pi is also resonance stabilised
 Thioesters (e.g. Acetyl coenzyme A or Acetyl-CoA)
• In thioesters a sulfur atom replaces
the usual oxygen in the ester bond
having large, negative standard
free energy change of hydrolysis.
• Acetyl-coA is one of many
thioesters important in
metabolism. The acyl group in
these compounds is activated for
trans-acylation, condensation or
oxidation-reduction reactions.
• Hydrolysis of the ester bond
generates a carboxylic acid which
can ionize and assume several
resonance forms.
• ΔG'0 = -31.4 kj/mol for acetyl-CoA
hydrolysis
 Summary for hydrolysis reactions
• For hydrolysis reactions with large, negative standard free
energy changes, the products are more stable than the
reactants for one or more of the following reasons:
• 1. The bond strain in reactants due to electrostatic repulsion
is relieved by charge separation, as for ATP
• 2. The products are stabilized by ionization, as for ATP, acyl
phosphates, thioesters.
• 3. The products are stabilized by isomerization
(tautomerization) as for phosphoenolpyruvate
• 4. The products are stabilized by resonance as for creatine
released from phosphocreatine, carboxylate ion released
from acyl phosphates and thioesters and phosphate released
from anhydride or ester linkages
• The phosphate compounds found in living
organisms can be arbitrarily divided into two groups
based on their standard free energy changes of
hydrolysis.
• ‘High-energy’ compounds have a ΔG'0 of hydrolysis
more negative than -25 kj/mol.
• ‘low-energy’ compounds have a less negative ΔG'0.
• Based on this criterion, ATP, with a ΔG'0 of
hydrolysis of -30 kj/mol is a high-energy compound;
glucose 6-phosphate is a low-energy compound
(ΔG'0 = -13,8 kj/mol)
 What is actually meant by ‘high-energy
phosphate bond’ ?
• The term ‘high-energy phosphate bond’ was used by biochemists to
describe P-O bond broken in hydrolysis reactions for a long time. But it is
incorrect and misleading as it wrongly suggests that the bond itself
contains the energy.
• In fact, the breaking of all chemical bonds requires an input of energy.
The free energy released by hydrolysis of phosphate compounds does
not come from the specific bond that is broken.
• It results from the products of the reaction having a lower free energy
content than the reactants
• As is evident from the additivity of free energy changes of sequential
reactions, any phosphorylated compound can be synthesized by
coupling the synthesis to the breakdown of another phosphorylated
compound with a more negative standard free energy change of
hydrolysis.
• Example:
• ΔG'0
• PEP + H2O Pyruvate + Pi -61,9
• ADP+ Pi ATP+ H2O +30,5

• PEP + ADP Pyruvate + ATP -31,4

• Clevage of Pi from PEP releases more energy than is needed to

drive to condensation of Pi with ADP, the direct donation of a
phosphoryl group from PEP to ADP is thermodynamically feasible.
• Notice that while the overall reaction above is represented as the
algebraic sum of first two reactions, the overall reaction (third)
does not involve Pi ; PEP donates a phosphoryl group directly to
ADP.
ATP serves as the universal energy currency
• Phosphorylated compounds have a high or low phosphoryl group
transfer potential, on the basis of their standard free energy changes
of hydrolysis
• Much of catabolism is directed toward the synthesis of high-energy
phosphate compounds, but their formation is not an end in itself;
they are the means of activating a wide variety of compounds for
further chemical transformation.
• The transfer of a phosphoryl group to a compound effectively puts
free energy into that compound, so that it has more free energy to
give up during subsequent metabolic transformations.
• Because of its intermediate position on the scale of group transfer
potential, ATP can carry energy from high-energy phosphate
compounds produced by catabolism to compounds such as glucose,
converting them into more reactive species.
• Although in aqueous solution ATP is thermo-dynamically unstable
and is therefore a good phosphoryl group donor, it is kinetically
stable.
• Because of high activation energies required for uncatalyzed
reaction ATP does not spontaneously donate phosphoryl groups
to water or to the other potential acceptors in the cell.
• ATP hydrolysis occurs only when specific enzymes which lower
the energy of activation are present
• The cell is therefore able to regulate the disposition of the energy
carried by ATP by regulating the various enzymes that act on ATP
• Each of the three phosphates of ATP is susceptible to nucleophilic
attack and each position of attack yields a different type of
product
 ATP hydrolysis occurs
only when specific
enzymes which lower
the energy of
activation are present.
 The cell is therefore
able to regulate the
disposition of the
energy carried by ATP
by regulating the
various enzymes that
act on ATP .
Attack at the Attack at the Attack at the α-
 Each of the three
- phosphate ϐ-phosphate phosphate
phosphates of ATP is displaces ADP displaces displaces PPi and
susceptible to and transfers AMP and transfers
nucleophilic attack and Pi transfers PPi adenylate
each position of attack
yields a different type
of product.
 Examples
• Fatty acid activation reactions during the fatty
acid synthesis and /or oxidation
• Amino acid activation reactions during the
protein synthesis
• DNA and RNA synthesis
• Direct hydrolysis of ATP (ATP ADP+Pi ) is the
source of energy in the conformational changes
that produce muscle contraction and transport an
ion or a molecule across a membrane against
concentration gradient (An example: Na+- K+
exchange by Na+ K+ ATPase)
 Transphosphorylations between nucleotides
• Till now we have focused on ATP as the cell’s energy currency and
donor of phosphoryl groups, all other nucleoside triphosphates
(GTP, UTP, CTP) and all the deoxynucleoside triphosphates (dATP,
dGTP, dTTP and dCTP) are energetically equivalent to ATP.
• The free energy changes associated with hydrolysis of their
phosphoanhydride linkages are very nearly identical with those for
ATP.
• In preparation for their various biological roles, these other
nucleotides are generated as the nucleoside triphosphate (NTP)
forms by phosphoryl group transfer to the corresponding
nucleoside diphosphates (NDPs) and monophosphates (NMPs)
• ATP is the primary high-energy phosphate compound produced by
catabolism in the processes of glycolysis, oxidative phosphorylation.
Several enzymes carry phosphoryl groups from ATP to the other
nucleotides
• Nucleoside diphosphate kinases, found in all cells, catalyzes the reaction
Mg2+
ATP+NDP(or dNDP) ADP+NTP(or dNDP)

• Although this reaction is fully reversible the relatively high ATP/ADP ratio in
cells normally drives the reaction to the right, with the net formation of
NTPs and dNTPs

• Phosphoryl group transfers from ATP result in an accumulation of ADP.

For example, when muscle is concracting vigorously ADP accumulates and
interferes with ATP-dependent contraction.

• During periods of intense demand for ATP, the cell lowers the ADP
concentration, and at the same time acquires ATP, by the action of
adenylate kinase:

Mg2+
2ADP ATP + AMP
• This reaction is fully reversible, so after the intense demand for ATP ends, the enzyme can
recycle AMP by converting it to ADP which can then be phosphorylated to ATP in
mitochondria

• A similar enzyme guanylate kinase, converts GMP to GDP at the expense of ATP. By-
pathways such as these, energy conserved in the catabolic production of ATP is used to
supply the cell with all required NTPs and dNTPs

• Phosphocreatine (PCr) serves as a ready source of phosphoryl groups for the quick
synthesis of ATP from ADP. The phosphocreatine concentration in skeletal muscle is
considerably higher than those in the other tissues. The enzyme creatine kinase catalyzes
the reversible reaction.
Mg2+
• ADP+ PCr ATP+Cr ΔG'0 = -12.5 kj/mol

• When a sudden demand for energy depletes ATP, the PCr reservoir is used to replenish ATP
at a rate faster than ATP can be synthesized by catabolic pathways
• When the demand for energy slackens ATP produced by catabolism is used to replenish the
PCr reservoir by reversal of the creatine kinase reaction
 SUMMARY
• ATP is the chemical link between catabolism and
anabolism. The exergonic conversion of ATP coupled
to many endergonic processes in living organisms
• Cells also contain some high-energy compounds
which have a high phosphorylation potential, like
ATP. They are good donors of phosphoryl groups.
 Oxidation-Reduction reactions in biological
systems
 Transfer of phosphoryl group is a central feature of metabolism
and in energy transfer (due to the tendency of ATP to get
hydrolyzed desperately).

 An equally important reaction mechanism to transfer free energy

in biological systems is the transfer of electron in oxidation
reduction reactions (due to tendency of some atoms to accept
electron desperately).

 Oxygen is the strongest electron acceptor in biological systems,

due to it very high electro negativity and hence it is the strongest
oxidizing agent.

 Fluorine is the strongest oxidizing agent but it is present in trace

amount in living system.
 Key Components of Redox Pathway
1. Oxidizing ability: capacity to accept electrons
depend on the electro negativity of the atom.

2. Flow of electrons can be used to do useful

work as is done in battery operated motors,
the electromotive force (EMF).

3. In living systems, electron flow from various

electron carrier to oxygen and the EMF
generated is utilized for various energy
transduction reactions.
Oxidation states of Carbon in various compounds
Oxidation states of Carbon in various compounds – contd.
 Electrons are transferred from one molecule (electron donor) to another
(electron acceptor) in one of the four different ways

1. Direct electron transfers: Fe2+ + Cu2+ = Fe3+ + Cu+

2. As hydrogen atom: AH2 = A + 2e- + 2H+

B + 2e- + 2H+ = BH2
--------------------------------
AH2 + B = A + BH2

3. As hydride ion (:H-): As in case of NAD-linked dehydrogenases involving

transfer of two electron.

4. Through direct combination with oxygen: Covalent incorporation of

oxygen into an organic molecule.

In biological systems oxidation is often referred as dehydrogenation.

 Standard reduction potential (Eo)

 It is the electric potential

generated by a redox reaction
against hydrogen electrode,
when the concentration of
reduced and oxidized species
are at 1 M concentration.

 Reduction potential of a half-

cell depends on the activity
of reduced and oxidized
species which is
approximated by their
concentrations.
 Nernst Equation

E = Eo + RT/nf ln [electron acceptor]/[electron donor]

N= number of electrons transferred, f= Faraday’s constant.

At 25 oC, the equation is;

E = Eo + 0.026V/n ln [electron acceptor]/[electron donor]

Standard reduction potential is used for free energy

change calculation as follows:
Δ G = -nf Δ E or Δ Go = -nf Δ Eo
 The General Nernst Equation
The general Nernst equation correlates the Gibb's Free Energy DG and the EMF
of a chemical system known as the galvanic cell.

For the reaction:

aA+bB=cC+dD

[C]c [D]d
and Q = ---------
[A]a [B]b

It has been shown that, ΔG =  Δ G° + R T ln Q 

and , Δ G = - n F Δ E

Therefore,
- n F Δ E = - n F Δ E° + R T ln Q

where R, T, Q and F are the gas constant (8.314 J mol-1 K-1), temperature (in K),
reaction quotient, and Faraday constant (96485 C) respectively. 
Thus, we have

RT [C]c [D]d
Δ E = Δ E° - ------- ln ----------
nF [A]a [B]b

This is known as the Nernst equation.

The equation allows us to calculate the cell potential of any galvanic cell for any
concentrations.

Relationship between equilibrium and the Gibb's free energy :

When a system is at equilibrium,  Δ E = 0, and Qeq = K.

R T [C]c [D]d
Δ E° = ----- ln ---------, (for equilibrium concentrations)
n F [A]a [B]b

Thus, the equilibrium constant and  Δ E° are related.

 Important values of constants in the Nernst
Equation

2.303 is a conversion factor from natural log to log10

R = Gas constant, 8.135 J K-1 mol-1

T = temperature in K (273 + temp in oC)

z = valency of ion (i.e. for Na+ is plus one, Ca2+ is plus two and Cl- is minus one)

F = Faraday’s constant, 9.684 x 104 C mol-1

 The Nernst Equation at 298 K
At any specific temperature, the Nernst equation derived above can be reduced into a simple
form.

For example, at the standard condition of 298 K (25°), the Nernst equation becomes

0.0592 V [C]c [D]d

Δ E = Δ E° - ------------ log ---------
n [A]a [B]b

To be noted that: log is the logarithm function based 10, and ln, the natural logrithm
function.

Example:
For the cell, Zn | Zn2+ || H+ | H2 | Pt

we have a net chemical reaction of Zn(s) + 2 H+ = Zn2+ + H2(g)

and the standard cell potential  Δ E° = 0.763.

If the concentrations of the ions are not 1.0 M, and the H2 pressure is not 1.0 atm, then the
 0.0592 V P(H2) [Zn2+]
Δ E = Δ E° - ------------- log ------------
n [H+]2
with n = 2 in this case, because the reaction involves 2 electrons.

 The numerical value is 0.0592 only when T = 298 K. This constant is temperature
dependent. Note that the reactivity of the solid Zn is taken as 1.
 If the H2 pressure is 1 atm, the term P(H2) may also be omitted.
 The expression for the argument of the log function follows the same rules as those for
the expression of equilibrium constants and reaction quotients.

When a cell is at equilibrium,  Δ E = 0.00 and the expression becomes an equilibrium
constant K, which bears the following relationship:

n Δ E°
log K = ----------
0.0592 where  Δ E° is the difference of standard potentials of the half cells
involved.

 A battery containing any voltage is not at equilibrium.

 The Nernst equation also indicates that you can build a battery simply by using the same
material for both cells, but by using different concentrations.
Example 1:
Calculate the EMF of the cell
Zn(s) | Zn2+ (0.024 M) || Zn2+ (2.4 M) | Zn(s)Solution

Zn2+ (2.4 M) + 2 e = Zn Reduction

Zn = Zn2+ (0.024 M) + 2 e Oxidation
------------------------------------------------------------------------
Zn2+ (2.4 M) = Zn2+ (0.024 M) Δ E° = 0.00 - - Net reaction

Using the Nernst equation:

0.0592 (0.024)
Δ E = 0.00 - ----------- log --------
2 (2.4)

= (-0.296)(-2.0)

= 0.0592 V
Example 2 (Role of reaction stoichiometry)
Show that the voltage of an electric cell is unaffected by multiplying the reaction equation by
a positive number.

Assume that we have the cell

Mg | Mg2+ || Ag+ | Ag and the reaction is: Mg + 2 Ag+ = Mg2+ + 2 Ag

Using the Nernst equation

0.0592 [Mg2+]
Δ E = Δ E° - ---------- log --------
2 [Ag+]2

If we multiply the equation of reaction by 2, we will have2 Mg + 4 Ag + = 2 Mg2+ + 4 Ag

Hence, there are 4 electrons involved in this equation, and n = 4 in the Nernst equation:

0.0592 [Mg2+]2
Δ E = Δ E° - ---------- log --------
4 [Ag+]4
which can be simplified as
0.0592 [Mg2+]
Δ E = Δ E° - ---------- log --------
2 [Ag+]2 Thus, the cell potential  Δ E is not affected.
 Home Task
The standard cell potential Δ E° for the following reaction is -0.353 V.

Fe + Zn2+ = Zn + Fe2+

If a piece of iron is placed in a 1 M Zn2+ solution, what is the equilibrium concentration of

Fe2+?
NADH and NADPH are soluble electron carriers that act
with dehydrogenases.

NAD+ + 2e- + 2H+ = NADH + H+

CH3CH2OH + NAD+ = CH3CHO + NADH + H+

Flavin nucleotides are tightly
bound to flavoproteins
1. The standard cell potential Δ E° for the reaction, Fe + Zn2+ = Zn + Fe2+is -0.353 V.
If a piece of iron is placed in a 1 M Zn2+ solution, what is the equilibrium concentration of
Fe2+?

Solution

The equilibrium constant K may be calculated using

K = 10   Δ
(n E°)/0.0592 
    = 10-11.93
    = 1.2x10-12
    = [Fe2+]/[Zn2+].

Since [Zn2+] = 1 M,

it is evident that
        [Fe2+] = 1.2E-12 M.
Biomembrane
Biological membranes
Define the external boundaries of cells
Regulate the molecular traffic across the boundary
Divide the internal space of eukaryotic cells into discrete
compartments to segregate processes and components
Organize complex reaction sequences and are central to
both biological energy conservation and cell-to-cell
communication

Three-layer (trilaminar)
structure, 50-80 Å thick

120
Biological activities of biological membranes

Flexibility- permits shape changes that accompany cell

growth and movement
Ability to break and reseal- two membranes can fuse
(exocytosis); a single membrane-enclosed compartment
can undergo fission to yield two sealed compartments
(endocytosis or cell division)
Selectively permeable- retain certain compounds and ions
within cells or specific cellular compartments, while
excluding others.
The Composition and Architecture of
Membranes
Each Type of Membrane Has Characteristic
Lipids and Proteins:
Lipid compostion of the plasma membrane and
organelle membranes of a rat hepatocyte

123
 Glycerophospholipids
They are similar to triacylglycerols, but have one ester bond replaced
with an amino alcohol phosphate ester

O H2C O C R2

R1 C O CH O

H2C O P O X

O
glycerophospholipid

X= any polar group, e.g. serine, choline, ethanolamine or inositol.

• The 2 fatty acids tend to be non-identical. They may differ in length
and/or the presence/absence of double bonds.
• Glycerophospholipids are the main class of phospholipids
• Glycerophospholipids are the main lipid component of cell
membranes.
 Major Glycerophospholipids in Plasma
Membrane
Phosphatidyl ethanolamine
O

O H2C O C R2
Phosphatidyl choline
R1 C O CH O

H2C O P O X

O
Phosphatidyl Serine
glycerophospholipid
X = Inositol Phosphatidyl Inositol
 O

O H2C O C R2

R1 C O CH O

H2C O P O
X = Inositol
O H

OH OH
H OH
OH H
phosphatidyl- H H
inositol
H OH

Phosphatidylinositol, with inositol as polar head group,

is one glycerophospholipid.
In addition to being a membrane lipid,
phosphatidylinositol has roles in cell signaling.
 Sphingolipids
Sphingolipids are derivatives of the long hydrocarbon amino alcohol
sphingosine.
OH OH
OH OH
H H
H2C C CH H2C C CH

H3N+ CH NH CH

HC O C HC

(CH2 )12 R (CH2 )12

sphingosine CH3 ceramide CH3

• The amino group of sphingosine can form an amide bond with a

fatty acid carboxyl, to yield a ceramide.
 Complex Sphingolipids
• Sphingomyelin In complex sphingolipids, a polar “head
• Cerebroside group" is esterified to the terminal hydroxyl
• Ganglioside of the sphingosine moiety of the ceramide. 

 Sphingomyelin CH3 O
H2 H2 
This is a ceramide with a H3C N+
C C O P O
phosphocholine or CH3 O OH
phosphethanolamine head group phosphocholine H
esterified to terminal hydroxyl H2C C CH

group. sphingosine NH CH

Sphingomyelins are common O C HC

constituent of plasma membranes fatty acid R (CH2 )12
in animal cells. These are major
Sphingomyelin
component of membranous myelin CH3

sheath that surrounds nerve cell

axons.
 A cerebroside is a sphingolipid (ceramide) with a monosaccharide
such as glucose or galactose as polar head group.
CH2OH

OH O
H OH
OH H O
H
H H H2C C CH

H OH NH CH

O C HC

R (CH2 )12
cerebroside with
-galactose head group CH3

 A ganglioside is a ceramide with a polar head group that is a complex

oligosaccharide, including the acidic sugar derivative sialic acid.
Cerebrosides and gangliosides, collectively called glycosphingolipids,
are commonly found in the outer leaflet of the plasma membrane
bilayer, with their sugar chains extending out from the cell surface.
These play important role in cell-cell recognition.
Phospholipids are amphiphilic molecules, due to presence of
hydrophobic and hydrophilic parts
Each glycerophospholipid includes
 a polar region: glycerol, carbonyl O of fatty acids, Pi, &
the polar head group (X)
 non-polar hydrocarbon tails of fatty acids (R1, R2).
O

O H2C O C R2

R1 C O CH O

H2C O P O X

O
glycerophospholipid Schematic representation of a
phospholipid
Sphingolipids and Cholesterol Cluster Together
in Membrane Rafts
Micro-domains (rafts) in the plasma membrane
 Lipid Rafts
 Lipid rafts are microdomains on membrane, whose composition
differs from the rest of the membrane.
 Complex sphingolipids tend to separate out from
glycerophospholipids & co-localize with cholesterol in lipid rafts.
CH3 O
H2 H2 
H3C N+ C C O P O

CH3 O OH
phosphocholine H
H2C C CH
sphingosine NH CH

O C HC HO
fatty acid Cholesterol
R (CH2 )12
Sphingomyelin CH3

 Hydrogen bonding between the hydroxyl group of cholesterol

and the amide group of sphingomyelin may in part account for
the observed affinity of cholesterol for sphingomyelin in raft
domains.
All Biological Membranes Share Some
Fundamental Properties
Fluid mosaic model for membrane structure

133
 A Lipid Bilayer Is the Basic Structural
Element of Membranes
Amphipathic lipid aggregates that form in water

Ex: free fatty acids Ex: glycerophospholipids

lysophospholipids sphingolipids
detergents (SDS)
 Asymmetric distribution of phospholipids
between the inner and outer monolayers of the
erythrocyte plasma membrane
Peripheral Membrane
Proteins Are Easily
Solubilized

Peripheral and integral

membrane proteins

136
Transbilayer disposition of
glycophorin in an
erythrocyte

137
Integral Proteins Are
Held in the
Membrane by
Hydrophobic
Interactions with
Lipids

Integral membrane
proteins

138
The Topology of an
Integral Membrane
Protein Can Be
Predicted from Its
Sequence

Hydrophobic plots

*Unbroken sequences
of more than 20
hydrophobic residues

*10-20% of all proteins:

integral membrane
proteins
139
Tyr and Trp residues of membrane proteins
clustering at the water-lipid interface

Tyr: orange
Trp: red
Charged residues (Lys, Arg, Glu, Asp): blue
140
Membrane proteins with β-barrel structure

141
Covalently Attached
Lipids Anchor Some
Membrane Proteins

Lipid-linked
membrane proteins

142
Amphiphilic lipid molecules self assemble in water to
form complexes in which polar regions are in contact with
water and hydrophobic regions away from water.

Depending on the lipid, possible molecular arrangements:

 Micelle structures.
 Bilayer. This is the most stable configuration for
amphipathic lipids with a cylindrical shape, such as
phospholipids.
 Membrane fluidity
Gel Phase
 At low temperatures, the membrane
bilayer is in a highly ordered gel
phase, with the acyl chains stretched
out (very few gauche conformations)
and tightly packed, and with a small
cross-sectional area per lipid.
 Consequently, the membrane bilayer liquid crystal Gel crystal
is thicker, more laterally compressed,
and less permeable to water and
solutes.
Liquid Phase
 As the temperature is raised, a phase transition temperature (Tm) is reached, and
the acyl chains cooperatively melt.
 There is an increase in the number of gauche conformations, and the bilayer is
now in the fluid, liquid crystalline, or liquid-disordered (ld) phase.
 In this phase, the acyl chains are mobile, the head groups are well hydrated, and
the bilayer is thinner and less densely packed, allowing easier percolation of
solutes into or through the bilayer.
 Cholesterol
Cholesterol is an important constituent of cell membranes, has a rigid
ring system and a short branched hydrocarbon tail.

Cholesterol is largely
hydrophobic.
But it has one polar group, a
hydroxyl, making it
amphiphilic. HO
Cholesterol
 Effect of Cholesterol on Membrane

HO
Cholesterol Cholesterol
in membrane
• Cholesterol inserts into bilayer membranes with its hydroxyl group oriented
toward the aqueous phase & its hydrophobic ring system adjacent to fatty
acid chains of phospholipids.
• The OH group of cholesterol forms hydrogen bonds with polar phospholipid
head groups.

• Interaction with the relatively rigid cholesterol decreases the mobility of

hydrocarbon tails of phospholipids.
• But the presence of cholesterol in a phospholipid membrane interferes with
close packing of fatty acid tails in the crystalline state, and thus inhibits
transition to the crystal state.
Two strategies by which phase changes of membrane
lipids are avoided:
 Cholesterol is abundant in membranes, such as
plasma membranes, that include many lipids with
long-chain saturated fatty acids.
In the absence of cholesterol, such membranes would
crystallize at physiological temperatures.
 The inner mitochondrial membrane lacks cholesterol,
but includes many phospholipids whose fatty acids
have one or more double bonds, which lower the
melting point to below physiological temperature.
 Movements of Lipid molecules in Bilayer
Lateral mobility/ lateral diffusion: Lipid molecules readily exchange
places with their neighbors within a monolayer/one leaflet (~107 times
per second).
Rotation: Individual lipid molecules rotate very rapidly about their long
axis (Flexible hydrocarbon chains).
Flip Flop: Migration of lipid molecules from one leaflet to another
(from one half of a bilayer to the other). Flip-flop would require the
polar head-group of a lipid to traverse the hydrophobic core of the
membrane and therefore is normally very slow (< 1 per month).
Transbilayer
Movement of Lipids
Requires Catalysis

Motion of single
phospholipids in a
bilayer

“Flippase”

149
 Membrane Dynamics
Acyl Group in the Bilayer Interior are Ordered
to Varying Degrees

Relatively low temp:

semisolid gel phase

Intermediate temp:
liquid-ordered state

Relatively high temp:

liquid-disordered state

150
 Certain Integral Proteins Mediate Cell-Cell
Interactions and Adhesion
Four examples of integral protein types that function
in cell-cell interactions.
Membrane Fusion Is
Central to Many Biological
Processes
Specific fusion of two
membranes requires that:
(1) they recognize each other
(2) their surfaces become
closely apposed
(3) their bilayer structures
become locally disrupted
(4) their bilayer fused to form a
single continuous bilayer
(5) triggered at the appropriate
time or by a specific signal 152
Solute Transport across Membranes
Passive Transport Is
Facilitated by
Membrane Proteins

Energy changes
accompanying passage of
a hydrophilic solute
through the lipid bilayer of
a biological membrane

154
Classification of transporters

155
The Glucose Transporter of Erythrocytes
Mediates Passive Transport
Proposed structure of GLUT1

156
Proposed structure of GLUT1-

Helical wheel and side-by-side interactions

157
Kinetics of glucose transport into erythrocytes

158
Model of glucose transport
into erythrocytes by GLUT1

159
Regulation by insulin of glucose transport by
GLUT4 into a myocyte

160
The Chloride-Bicarbonate Exchanger Catalyzes
Electrochemical Cotransport of Anions across the
Plasma Membrane
Three general classes of transport systems
 Transport Through Membrane

The relative permeability of a synthetic lipid bilayer to different classes

of molecules.
The smaller uncharged molecules rapidly diffuses across the bilayer.
 Types of Membrane Transport
Membrane Transport

Non-mediated Mediated
(Requires
transport protein

Passive Mediated Active

Transport protein
 Ionophores
Ionophores are lipid-soluble organic molecule that transport ions across
a hydrophobic cell membrane DOWN the concentration gradient.

Carrier Ionophore:
• Increases the permeability of specific
ions –selectivity
• Diffusion through membrane
• Releasing ion to other side
• Sensitive to temperature

Eg: Valinomycin for K+, FCCP for H+,

A23187 for Ca2+, Mg2+

Channel formers:
• Form channel or pores spanning the membrane,
through which ions can transport
• Temperature insensitive
Eg: gramicidin for K+
Valinomycin, a peptide ionophore that binds K +
 Passive Transport
The free energy change of a solute is given by,
μi is the chemical potential of A and μ0i is the chemical potential under standard state
[Note: This is only valid for dilute solution. Under real condition, [A] should be replaced by activity
of A, aA = γ[A]]
FOR UNCHARGED SPECIES

A (out) ΔμA= μA (in) –μA (out) = RT ln{[A]in/[A]out}

If [A] out > [A]in , ΔμA is negative, spontaneous
inward flow of A

A (in)
FOR CHARGED SPECIES
ΔμA = RT ln{[A]in/[A]out} + ZFΔV
• When a cell uses no energy to move particles across a membrane
passive transport occurs
• Particles go DOWN their concentration gradient in accord with the 2nd
Law of Thermodynamics.
 Gradient of chemical potential as driving force in
transport
The driving force for non-mediated passive transport of A through the
membrane depends on its electrochemical potential gradient

Where, JA = flux of A (rate of passage per unit area),

x is the distance,
dμA/dx = electrochemical potential gradient
UA = mobility of the solute (velocity per unit force)
Nernst-Plank Equation

For an uncharged species,

Fick’s First law of diffusion

A substance diffuses in the direction
that eliminates its concentration
gradient i.e d[A]/dx, at a rate
proportional to the magnitude of the
concentration gradient.

Where, DA = UART= Diffusion coefficient

 Permeability Coefficient
For a membrane with thickness x,

JA
Slope = PA

[A]out-[A]in

PA = DA/x = membrane’s permeability co-efficient for A

• Measure of a solute’s tendency to transfer from

aqueous outside to non-polar membrane core
Passive-Mediated Transport or Facilitated diffusion
• Flow of solute molecules occur DOWN the concentration gradient
with the involvement of specific carrier proteins

Example: Glucose transporters across erythrocyte membrane

Characteristics
• Exhibit high speed and selectivity Jmax
Permeability coeff for D-glucose >>
that of D-mannitol in intact RBC

J Glucose
• Exhibit saturation kinetics
½ Jmax

• Susceptibility to competitive inhibition

6-O-benzyl-D-galactose is a [Glucose]
competitive inhibitor

• Susceptibility to chemical inactivation

 Active Transport
ΔμA= μA (in) –μA (out) = RT ln{[A]in/[A]out} + ZFΔV

If ΔμA is positive, requires an energy input for inward flow of A

ATP driven Active Transport

(Na+-K+)-ATPase of plasma membrane:

Pumps Na+ out of cell while K+ inside the cell with concomitant
hydrolysis of ATP.

3Na+ (in) +2K+ (out) + ATP + H2O = 3Na+ (out) +2K+ (in) + ADP + Pi
 Active Transport
• Generation of resting membrane potential
• Controls cell volume
• Provides free energy for active transport of glucose and amino acids

E1
E2
 Examples of ATP Driven Active Transport
Ca2+-ATPase or Ca2+Pump

• Present on plasma membrane, ER membrane

• Pumps out Ca2+ at the expense of ATP hydrolysis.
• Kinetic mechanism similar to Na+-K+ pump

Low [H+]
H -K -ATPase of Gastric mucosoa
+ +

• Present on parietal cell membrane of

gastric mucus in mammals.
• Pumps out H+ and pump in K+ at the
expense of ATP hydrolysis.
• Kinetic mechanism similar to Na+-K+
pump
• Electroneutral-Antiport

High [H+]
 Examples of Ion Gradient Driven Active Transport

Dissipation of electrochemical potential gradient to drive a membrane

transport

Na+-Glucose Symport
[Na+]
• Present on the brush
border cells of intestinal
epithelium
• Accumulates glucose
from intestinal lumen in a
Na+ dependent manner
• The energy comes from
the [Na+] gradient
established by Na+-K+
pump.
[Glucose]
High

Gradient of concentration
 Examples of Ion Gradient Driven Active Transport

Lactose Permease High [H+] Low [H+]

• Present in gram negative

bacteria such as E. coli.
• Proton gradient is
established as a result of
oxidative metabolism
• This gradient is utilized to
co-transport one H+ and
one lactose molecule

• Addition of cyanide blocks oxidative metabolism-inhibits H+ gradient

generation and transport
• Addition of 2,4-dinitrophenol dissipates proton gradient and inhibit
transport
 Ion Channel
 High selectivity
 Faster transport than carrier protein
 Transport down the concentration gradient
 Switch between ‘Open’ and ‘Closed’ conformations
 Transition between two conformations highly regulated
(i) Ligand gated: transition switched by specific ligand binding
(ii) Voltage gated: transition switched by electrical potential across the
membrane
(iii) Mechanically gated: transition induced by mechanical stress
 Examples of Various Ion Channels

Family Representative Subfamilies

Voltage-gated Voltage-gated Na+ channel
cation channels Voltage-gated K+ channels
Voltage-gated Ca2+ channel
Neurotransmitter Acetyl choline gated cation channel
gated ion channel Glutamate gated Ca2+ channel
 Membrane Potential
Membrane potential arises due to difference in electrical charge across the
membrane
Origin of Membrane potential
• Electrogenic pumps (Na+-K+ Pump)
• Passive transport (mainly K+ leakage channels)
 Nernst Equation for Ion Flow across Membrane
Resting membrane potential
ΔμA= μA (in) –μA (out) = RT ln{[A]in/[A]out} + ZFΔV

Free energy change Free energy change

per mole of the solute per mole of the solute
moved across the moved across the
membrane due to membrane due to
concentration gradient voltage gradient

At equilibrium, these two influences balances each other and there is no net
flow of ion,
Under such condition, ΔμA = 0

At 37°C, the equation becomes volts

 Calculate ΔVK+ and ΔVNa+ at 37°C , if [K+]out = 5 mM, [K+]in = 140 mM, [Na+]out = 150mM,
[Na+] = 15 mM
Active Transport Results in Solute Movement against
a Concentration or Electrochemical Gradient

180
P-Type ATPases Undergo Phosphorylation
during Catalytic Cycles
Na+K+ ATPase

181
Postulated mechanism
of Na+ and K+ transport
by the Na+K+ ATPase

182
Postulated mechanism
of Na+ and K+ transport
by the Na+K+ ATPase

183
F-Type ATPase Are
Reversible, ATP-
driven Proton Pumps

Structure of the FoF1

ATPase/ATP
synthase

184
Reversibility of F-
type ATPase

185
Mucus lining the surface of the lungs traps bactieria

186
A Defective Ion Channel in Cystic Fibrosis
Topology of cystic fibrosis transmembrane
conductance regulator (CFTR)

187
Ion Gradients Provide the Energy for Secondary
Active Transport
Lactose uptake in E. coli

189
Structure of the lactose transporter (lactose
permease) of E. coli

190
Glucose transport in intestinal epithelial cells

191
Resting Potentials
and
Action Potentials
 Resting Membrane Potential

• Neurons have a selectively permeable membrane

• During resting conditions membrane is:
– permeable to potassium (K+) (channels are open)
– impermeable to sodium (Na+) (channels are closed)
• Diffusion force pushes K+ out (concentration gradient)
• This creates a positively charged extra-cellular space.
• Electrostatic force pushes K+ in
• Thus, there is a ‘dynamic equilibrium’ with zero net movement
of ions.
• The resting membrane potential is negative (- 60mv)
Cell Membrane

Copyright © Allyn & Bacon 2004

 Resting Membrane Potential
OUTSIDE Na+ Cl-
K+

Electrostatic Force Force of Diffusion

+++++++++++++++++++++++++++++++++++++++++++

open Closed 3Na/2K no open

channel channel pump channel channel

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - -

Force of Diffusion Electrostatic Force

INSIDE K +
Na
+ Cl-

- 65 mV
Pr-

K+ = Potassium; Na+ = Sodium; Cl- = Chloride; Pr- = proteins

195
Voltage-dependent ion channel: An ion channel that opens or
closes according to the value of the membrane potential.
Depolarization: Reduction (toward zero) of the membrane
potential of a cell from its normal resting potential. Normal
depolarizing events are termed EPSPs – excitatory post-synaptic
potentials – and result from opening of Na+ channels.
Hyperpolarization: An increase in the membrane potential of a cell,
relative to the normal resting potential. Hyperpolarizing events are
termed IPSPs – inhibitory post-synaptic potentials – and result from
opening of Cl- channels (Cl- higher outside than in, so flow in and
make cell more negative). Also can result from opening K+
channels.
Action potential: The brief electrical impulse that provides the
basis for conduction of information along an axon.
Threshold of excitation: The value of the membrane potential that
must be reached to produce an action potential.
All-or-none law: The principle that once an action potential is
triggered in an axon, it is propagated without decrement to the end
of the fiber.
 Membrane Potentials

2. Excitatory 3. threshold
1. Resting Post- 4.
Potential synaptic Action Inhibitory
potential Potential Post-synaptic
potential

Copyright © Allyn & Bacon 2004

Resting and Action Potentials
 Concentration of Ions and
State of Channels at Rest

-65 mV

Concentrations maintained by Na+/K+ and Ca++ pumps.

 Formation of Potentials
• All potentials result from ions moving across
membranes.
• Two forces on ions: Diffusion (from high to low
concentration); Electrical (toward opposite charge and
away from like charge).
• Each ion that can flow through channels reaches
equilibrium between two forces.
• Equilibrium potential for each ion determined by
Nernst Equation.
• K+ make -ve potentials;
• Na+ make +ve potentials.
 Nernst Equation using real values

• EK+ = +58 mV log10 ([K+] outside/[K+] inside).

(+58 mV for room temperature, squid axon).

• EK+ = 58 mV log10 1/20 = -75 mV.

• ENa+ = 58 mV log10 10/1 = + 58 mV.
• ECl- = -58 mV log10 15 = -68 mV.
• ECa++ = +58 mV log10 10,000 = +220 mV.
 Action Potential Results from Voltage-gated
Na+ Channels

ENa+ = +58 mV 

EK+ = -75 mv 
 Action Potentials
• Only neurons and muscles have action potentials
(not all neurons).
• Due to voltage-gated Na+ channels.
• Most in axons, at initial segment (axon hillock) and
nodes of Ranvier. A few in big dendrites where
depolarizations need a boost.
• Channel ionic currents are studied by voltage clamps
and patch clamps.
 Voltage Clamp
• Used to measure ion currents in squid giant axons
(Hodgkin & Huxley).
• Study single ion by changing ions in axon.
• Hold voltage constant by injecting current with large
electrode. Measured current I.
• Measured Na+ or K+ current during action potential:
INa+ = V/R = K/R ~ Na+ conductance.
• Measure “channel” permeability changes.
• Predicted action potential changes from Nernst Eq
and channel permeabilities.
Single Channels

Study electrical properties,

Ionic properties,
Pharmacology (toxins, agonists, antagonists)
Molecular biology (mutant channels)
The Structure of a K+ Channel Reveals the
Basis of Its Specificity
Structure and function of the K+ channel of
Streptomyces lividans

207
Structure and
function of the K+
channel of
Streptomyces
lividans -

Diagram of the K+
channel in cross
section

208
K+ binding sites in the selectivity pore of the K+
channel

209
The Neuronal Na+ Channel Is a Voltage-Gated
Ion Channel
Voltage-gated Na+ channel of neurons

210
Voltage-gated
Na+ channel of
neurons-

Four domains
are wrapped
about a central
transmembrane
channel

211
Voltage-gated Na+
channel of neurons-

The voltage-sensing
mechanism involves
movement of helix 4
perpendicular to the
plane of the membrane
in response to a
change in potential

212
The Acetylcholine Receptor Is a Ligand-Gated Ion
Channel
Structure of the acetylcholine receptor ion channel

213
Top view of a cross section through the center of M2
helices showing five Leu side chains protruding into
the channel, constricting it to a diameter too small to
allow passage of ions

214
Defective Ion Channels Can Have Adverse
Physiological Consequences

215
ELECTRON TRANSPORT CHAIN
NADH and FADH2, transfer their electrons to a series of compounds
which are associated with the inner mitochondrial membrane.

 The protein/complexes are arranged in order of increasing

electronegativity as described below:
NADH dehydrogenase> ubiquinone (UQ)> succinate dehydrogenase>
cytochrome complex> cytochrome c > cytochrome oxidase complex.

 Each complex is reduced by gaining two electrons from the one

before it and oxidized by donating its two electrons to the one after
it.

 As the electrons are passed they become more stable and therefore
generate free energy that is used to pump protons (3 pumps) into
the intermembrane space from the matrix (active transport).

 Oxygen is the final electron acceptor and it joins with two protons in
the matrix to form water.
 ELECTRON TRANSPORT CHAIN
Steps:
 NADH, from pyruvate oxidation and the Krebs Cycle, gives up its two
electrons to NADH dehydrogenase,
 The mobile carriers UQ and cytochrome c shuttle electrons from one protein
complex to the next until they reach the cytochrome oxidase complex (final
acceptor).
 Each protein complex (3) also acts as a proton pump, using the free energy
released to move protons from the matrix to the intermembrane space.
 At the cytochrome oxidase complex, cytochrome oxidase, catalyzes the
reaction between the electrons, protons and oxygen to form water.
 (2H+ + 1/2O2 --> H2O)

• This process is highly exergonic (giving up free energy of 222kJ/mol). The

chemical potential energy of the electron position is converted to the
electrochemical potential energy of a proton gradient that forms across the
inner mitochondrial membrane.
– High concentration in the intermembrane space, low concentration in the
matrix.
– The proton gradient will be used to produce ATP.
ELECTRON TRANSPORT CHAIN and ATP SYNTHASE
 ELECTRON TRANSPORT CHAIN

• This process is highly exergonic (giving up free energy

of 222kJ/mol). The chemical potential energy of the
electron position is converted to the electrochemical
potential energy of a proton gradient that forms
across the inner mitochondrial membrane.

– High concentration in the intermembrane space,

low concentration in the matrix.
– The proton gradient will be used to produced ATP.
 ELECTRON TRANSPORT CHAIN
• FADH2 skips the first protein compound (starts at UQ). This means
that NADH oxidation pumps three protons into the
intermembrane space, while FADH2 oxidation pumps only two
protons.

• Three ATP are formed from NADH while two ATP are formed
from FADH2..

• Once NADH and FADH2 are oxidized they pick up more H+ in

glycolysis, pyruvate oxidation, or the Kreb's cycle (recycling of
electron carriers).

• There are many copies of the ETC along the cristae, therefore lots
of ATP can be produced.
 CHEMIOSMOSIS and OXIDATIVE ATP SYNTHESIS
(Oxidative Phosphorylation)

• There is an electrochemical gradient across the

intermembrane space. (More protons outside than in the
matrix).
• Two parts: difference in charge and a difference in
concentration.
• The inner membrane is impermeable to protons.
• The protons are forced through special proton channels that
are coupled with ATP synthase (ATPase).
• The electrochemical gradient produces a proton-motive force
(PMF) that moves the protons through this ATPase complex.
• Each time a proton comes through the ATPase complex, the
free energy of the electrochemical gradient is reduced and
this energy is used to create ATP from ADP + Pi in the matrix.
 CHEMIOSMOSIS and OXIDATIVE ATP
SYNTHESIS
(Oxidative Phosphorylation)
.
• The continual production of ATP is dependent on the
maintenance of a proton reservoir in the intermembrane
space.
• This depends on the continued movement of electrons and
that depends on the availability of oxygen.
• Therefore we need oxygen to prevent the ETC from being
“clogged up” and we need food to provide the glucose that
provides electrons for the ETC.
Chemiosmotic
Mechanism

Electron
transport chain
sets up an H+
gradient (proton
motive force).
Energy of the
PMF is harnessed
to make ATP.
 Mitochondrion

 Double membrane, with

inner membrane very
impermeable

 TCA occurs in the matrix

 ETC in the inner

membrane
Electron transport chain
 ETC carriers: Coenzyme Q

 Mobile
electron
carrier within
the bilayer

 1- or 2-
electron
acceptor
ETC carriers: FMN

 Prosthetic group
of ETC protein
(complex I)

 1- or 2-electron
acceptor
 ETC carriers: Cytochromes
 Heme proteins

 Most are integral

proteins

 Cytochrome c is a
soluble peripheral
protein
ETC carriers: Iron-sulfur proteins
 ETC carriers: Copper centers

 CuA center  CuB center

Electron Transport Chain: Proteins
Electron Transport Chain
Complex I
 Complex I catalyzes two simultaneous and obligately
coupled processes:

(1) the exergonic transfer to ubiquinone of a hydride ion

from NADH and a proton from the matrix, expressed by

(2) the endergonic transfer of four protons from the

matrix to the intermembrane space.

Complex I is therefore a proton pump driven by the energy of

electron transfer, and the reaction it catalyzes is vectorial:
It moves protons in a specific direction from one location (the
matrix, which becomes negatively charged with the departure of
protons) to another (the intermembrane space, which becomes
positively charged).
Alternate Entries
Alternate Entries

Complex II
(aka succinate
dehydrogenase)
Alternate Entries
Alternate Entries
Electron Transport Chain
Complex III
Complex III
Electron Transport Chain
Complex IV
Complex IV
 Electron Transport Chain

Succinate
dehydrogenase

 Electrons flow from carriers with low to

high reduction potential.

 This is energetically downhill.

Per electron
pair: Electron Transport Chain
4 H+ in 4 H+ out

Succinate
dehydrogenase

4 H+ in 4 H+ out

 Some carriers pump protons.

2 H+ in 2 H+ out
Flow direction
Flow direction
Inhibitors of electron transport
Electron transport
chain and ATP
synthesis in
Chloroplast
Solar energy as the
ultimate source of all
biological energy.

Photosynthetic organisms
use the energy of sunlight
to manufacture glucose and
other organic products,
which heterotrophic cells
use as energy and carbon
sources.
The light reactions of
photosynthesis generate energy
rich NADPH and ATP at the
expense of solar energy.

These products are used in the

carbon assimilation reactions,
which occur in light or darkness,
to reduce CO2 to form trioses and
more complex compounds (such
as glucose) derived from trioses.
 Chloroplast

Electron Micrograph
Schematic diagram
 Chloroplast is surrounded by two membranes, an outer membrane that is
permeable to small molecules and ions, and an inner membrane that encloses the
internal compartment. This compartment contains many flattened, membrane-
surrounded vesicles or sacs, the thylakoids, usually arranged in stacks called grana.
 Embedded in the thylakoid membranes (commonly called lamellae) are the
photosynthetic pigments and the enzyme complexes that carry out the light
reactions and ATP synthesis. The stroma (the aqueous phase enclosed by the inner
membrane) contains most of the enzymes required for the carbon assimilation
reactions.
 Hill Reaction
In 1937 Robert Hill found that when leaf extracts containing
chloroplasts were illuminated, they
(1) evolved O2 and
(2) reduced a nonbiological electron acceptor added to the
medium,
A = Hill reagent
According to the Hill reaction:

Severo Ochoa showed that NADP is the

biological electron acceptor in
chloroplasts, according to the equation
 Electromagnetic spectrum

Visible light is electromagnetic radiation of wavelengths 400 to 700 nm, ranging

from violet to red. The energy of a single photon (a quantum of light) is greater at
the violet end of the spectrum than at the red end; shorter wavelength (and higher
frequency) corresponds to higher energy. The energy, E, in a “mole” of photons (1
einstein, or 6 X1023 photons) of visible light is 170 to 300 kJ, as given by the Planck
equation:
where h is Planck’s constant (6.626 X 10-34 Js.
These amounts of energy are almost an order of magnitude greater than the 30
to 50 kJ required to synthesize a mole of ATP from ADP and Pi.
A molecule that has absorbed a photon is in
an excited state is generally unstable. Photochemical
An electron lifted into a higher-energy
orbital usually returns rapidly to its normal
significance
lower-energy orbital; the excited molecule
decays to the stable ground state, giving up
the absorbed quantum as light or heat or
using it to do chemical work.
Light emission accompanying decay of
excited molecules (called fluorescence) is
always at a longer wavelength (lower
energy) than that of the absorbed light.
An alternative mode of decay important in
photosynthesis involves direct transfer of
excitation energy from an excited molecule
to a neighbouring molecule.
Just as the photon is a quantum of light
energy, so the exciton is a quantum of
energy passed from an excited molecule to
another molecule in a process called exciton
transfer.
 Chlorophyll – why does it fit the bill?
 The most important light-absorbing pigments in the thylakoid membranes are the
chlorophylls, green pigments with polycyclic, planar structures resembling the
protoporphyrin of hemoglobin , except that Mg2+, not Fe2+, occupies the central
position.
 All chlorophylls have a long phytol side chain, esterified to a carboxyl-group
substituent in ring IV, and chlorophylls also have a fifth five membered ring not
present in heme.
 The heterocyclic five-ring system that surrounds the Mg2+ has an extended
polyene structure, with alternating single and double bonds. Such polyenes
characteristically show strong absorption in the visible region of the spectrum;
the chlorophylls have unusually high molar extinction coefficients and are
therefore particularly well-suited for absorbing visible light during
photosynthesis.
 Chloroplasts always contain both chlorophyll a and chlorophyll b. Although both
are green, their absorption spectra are sufficiently different that they
complement each other’s range of light absorption in the visible region.
 Chlorophyll is always associated with specific binding proteins, forming light-
harvesting complexes (LHCs) in which chlorophyll molecules are fixed in relation
to each other, to other protein complexes, and to the membrane.
Primary and secondary
photopigments
Chlorophylls a and b and
bacteriochlorophyll are the
primary gatherers of light energy.

Phycoerythrobilin and
phycocyanobilin (phycobilins)
are the antenna pigments in
cyanobacteria and red algae.

Carotene (a carotenoid)

Lutein (a xanthophyll)
The areas shaded pink are the conjugated systems (alternating single and double
 Absorption of visible light by photopigments

Plants are green because their pigments absorb light from the red and blue regions of the
spectrum, leaving primarily green light to be reflected or transmitted. Compare the
absorption spectra of the pigments with the spectrum of sunlight reaching the earth’s
surface; the combination of chlorophylls (a and b) and accessory pigments enables plants to
harvest most of the energy available in sunlight.
 A phycobilisome.
A light-harvesting complex, LHCII.

The functional unit is an LHC trimer, with 36 Found in cyanobacteria and red algae,
chlorophyll and 6 lutein molecules. phycobilin pigments bound to specific
Shown here is a monomer, viewed in the proteins form complexes called
phycoerythrin (PE), phycocyanin (PC),
plane of the membrane, with its three
and allophycocyanin (AP). The energy of
transmembrane -helical segments, seven photons absorbed by PE or PC is
chlorophyll a molecules (green), five conveyed through AP (a phycocyanobilin
chlorophyll b molecules (red), and two binding protein) to chlorophyll a of the
molecules of the accessory pigment lutein reaction center by exciton transfer, a
(yellow), which form an internal cross-brace. process discussed in the text.
  Results of a classic experiment
Englemann’s performed by T. W. Englemann in 1882 to
determine the wavelength of light that is
Experiment most effective in supporting
photosynthesis.
 Englemann placed cells of a filamentous
photosynthetic alga on a microscope
slide and illuminated them with light
from a prism, so that one part of the
filament received mainly blue light,
another part yellow, another red.
 To determine which algal cells carried
out photosynthesis most actively,
Englemann also placed on the
microscope slide bacteria known to
migrate toward regions of high O2
concentration.
 After a period of illumination, the
distribution of bacteria showed highest
O2 levels (produced by photosynthesis)
in the regions illuminated with violet and
red light.
I. Results of a similar experiment that used modern techniques (an oxygen
electrode) for the measurement of O2 production.
II. An action spectrum (as shown here) describes the relative rate of
photosynthesis for illumination with a constant number of photons of different
wavelengths.
III. An action spectrum is useful because, by comparison with absorption spectra,
it suggests which pigments can channel energy into photosynthesis.
Organization of photosystems in
the thylakoid membrane

Photosystems are tightly packed

in the thylakoid membrane, with
several hundred antenna
chlorophylls and accessory
pigments surrounding a
photoreaction center.

Absorption of a photon by any of

the antenna chlorophylls leads to
excitation of the reaction center
by exciton transfer (black arrow).
Also embedded in the thylakoid
membrane are the cytochrome
b6f complex and ATP synthase
Exciton and electron
transfer
 This generalized scheme
shows conversion of the
energy of an absorbed
photon into separation of
charges at the reaction
center.
 Note that step 1 may repeat
between successive antenna
molecules until the exciton
reaches a reaction-center
chlorophyll.
The asterisk (*) represents
the excited state of an
antenna molecule.
Functional modules of photosynthetic machinery in purple bacteria and green sulfur bacteria.
(a) In purple bacteria, light energy drives electrons from the reaction center P870 through
pheophytin (Pheo), a quinone (Q), and the cytochrome bc1 complex, then through cytochrome
c2 back to the reaction center. Electron flow through the cytochrome bc1 complex causes proton
pumping, creating an electrochemical potential that powers ATP synthesis. (b) Green sulfur
bacteria have two routes for electrons driven by excitation of P840: a cyclic route passes through
a quinone to the cytochrome bc1 complex and back to the reaction center via cytochrome c, and
a noncyclic route from the reaction center through the iron-sulfur protein ferredoxin (Fd), then
to NAD in a reaction catalyzed by ferredoxin:NAD reductase.
 Photoreaction center of the purple bacterium
Rhodopseudomonas viridis.

(a) The system has four components: three subunits, H, M, and L (brown, blue, and gray,
respectively), with a total of 11 transmembrane helical segments, and a fourth protein,
cytochrome c (yellow), associated with the complex at the membrane surface. Subunits L and M
are paired transmembrane proteins that together form a cylindrical structure with roughly
bilateral symmetry about its long axis. Shown as space-filling models (and in (b) as ball-and-stick
structures) are the prosthetic groups that participate in the photochemical events.
Integration of photosystems This “Z scheme” shows the pathway of
I and II in chloroplasts electron transfer from H2O to NADP in
noncyclic photosynthesis.
a. To raise the energy of electrons derived
from H2O to the energy level required to
reduce NADP to NADPH, each electron
must be “lifted” twice (heavy arrows) by
photons absorbed in PSII and PSI.
b. One photon is required per electron in
each photosystem. After excitation, the
high-energy electrons flow “downhill”
through the carrier chains shown.
c. Protons move across the thylakoid
membrane during the water-splitting
reaction and during electron transfer
through the cytochrome b6f complex,
producing the proton gradient that is
central to ATP formation.
d. The dashed arrow is the path of cyclic
electron transfer which involves only PSI;
electrons return via the cyclic pathway to
PSI, instead of reducing NADP to NADPH.
 Cyclic photophosphorylation
 Using an alternative path of light-induced electron flow, plants can vary the ratio of NADPH to
ATP formed in the light; this path is called cyclic electron flow to differentiate it from the
normally unidirectional or noncyclic electron flow from H2O to NADP.

 Cyclic electron flow involves only PSI. Electrons passing from P700 to ferredoxin do not
continue to NADP, but move back through the cytochrome b6 f complex to plastocyanin.

 Like green sulfur bacteria, Plastocyanin donates electrons to P700, which transfers them to
ferredoxin when the plant is illuminated. Thus, in the light, PSI can cause electrons to cycle
continuously out of and back into the reaction center of PSI, each electron propelled around
the cycle by the energy yielded by the absorption of one photon.

 Cyclic electron flow is not accompanied by net formation of NADPH or evolution of O2.
However, it is accompanied by proton pumping by the cytochrome b6 f complex and by
phosphorylation of ADP to ATP, referred to as cyclic photophosphorylation.

The overall equation for cyclic electron flow and photophosphorylation is simply

Importance: By regulating the partitioning of electrons between NADP reduction and cyclic
photophosphorylation, a plant adjusts the ratio of ATP to NADPH produced in the light-
dependent reactions to match its needs for these products in the carbon-assimilation reactions
and other biosynthetic processes.
 Photosystem II of the cyanobacterium Synechococcus elongates

 The monomeric form of the complex shown here has two major transmembrane
proteins, D1 and D2, each with its set of cofactors.
 Although the two subunits are nearly symmetric, electron flow occurs through only one
of the two branches of cofactors, that on the right (on D1).
 The arrows show the path of electron flow from the Mn ion cluster (Mn4, purple) of the
water-splitting enzyme to the quinone PQB (orange).
The photochemical events occur in the sequence indicated by the step numbers.
 The supramolecular complex of PSI and
its associated antenna chlorophylls

A large number of antenna

chlorophylls surround the reaction Reduced phylloquinone is reoxidized as it
center and convey to it (red arrows) passes two electrons, one at a time, to an
the energy of photons they have Fe-S protein (FX) near the N side of the
absorbed. membrane From FX, electrons move
The result is excitation of the pair of through two more Fe-S centers (FA and FB),
chlorophyll molecules that constitute then to the Fe-S protein ferredoxin in the
P700. Excitation of P700 greatly stroma.
decreases its reduction potential, Ferredoxin then donates electrons to
and it passes an electron through NADP(not shown), reducing it to NADPH,
two nearby chlorophylls to one of the forms in which the energy of
phylloquinone (QK; also called A1).. photons is trapped in chloroplasts.
 Equalization of electron flow in PSI and PSII
by modulation of granal stacking

I. A hydrophobic domain of LHCII in one thylakoid lamella inserts into the neighbouring
lamella and closely appresses the two membranes.
II. Accumulation of plastoquinol (not shown) stimulates a protein kinase that
phosphorylates a Thr residue in the hydrophobic domain of LHCII, which reduces its
affinity for the neighboring thylakoid membrane and converts appressed regions (granal
lamellae) to nonappressed regions (stromal lamellae).
III. A specific protein phosphatase reverses this regulatory phosphorylation when the
[PQ]/[PQH2] ratio increases.
Localization of PSI
and PSII in thylakoid
membranes.

Light harvesting complex LHCII and ATP synthase are located in regions of the thylakoid
membrane that are appressed (granal lamellae, in which several membranes are in contact) and
in regions that are not appressed (stromal lamellae) and have ready access to ADP and NADP in
the stroma. Photosystem II is present almost exclusively in the appressed regions, and
photosystem I almost exclusively in nonappressed regions exposed to the stroma. LHCII is the
“adhesive” that holds appressed
lamellae together
 Electron and proton flow through the
cytochrome b6f complex

(a) The crystal structure of the complex reveals the positions of the cofactors involved in
electron transfers. In addition to the hemes of cytochrome b (heme bH and bL; also called heme
bN and bP, respectively, because of their proximity to the N and P sides of the bilayer) and that
of cytochrome f (heme f ), there is a fourth (heme x) near heme bH, and there is a -carotene of
unknown function. Two sites bind plastoquinone: the PQH2 site near the P side of the bilayer,
and the PQ site near the N side. The Fe-S center of the Rieske protein lies just outside the bilayer
on the P side, and the heme f site is on a protein domain that extends well into the thylakoid
lumen. (b) The complex is a homodimer arranged to create a cavern connecting the PQH2 and
PQ sites). This cavern allows plastoquinone movement between the sites of its oxidation and
reduction.
Electron and proton flow through
the cytochrome b6f complex  Plastoquinol (PQH2) formed in
PSII is oxidized by the
cytochrome b6f complex in a
series of steps like those of
the Q cycle in the cytochrome
bc1 complex (Complex III) of
mitochondria.
 One electron from PQH2
passes to the Fe-S center of
the Rieske protein (purple),
the other to heme bL of
cytochrome b6 (green).
 The net effect is passage of
electrons from PQH2 to the
soluble protein plastocyanin,
which carries them to PSI.
 Dual roles of cytochrome b6f
and cytochrome c6 in
cyanobacteria

Cyanobacteria use cytochrome b6f,

cytochrome c6, and plastoquinone
for both oxidative phosphorylation
and photophosphorylation.

(a) In photophosphorylation,
electrons flow (top to bottom) from
water to NADP.
(b) In oxidative phosphorylation,
electrons flow from NADH to O2.
Both processes are accompanied by
proton movement across the
membrane, accomplished by a Q
cycle.
Water-splitting
activity of the
oxygen-
evolving
complex.

Shown here is the process that produces a four-electron oxidizing agent—believed to

be a multinuclear center with several Mn ions—in the water-splitting complex of PSII.
 The sequential absorption of four photons (excitons), each absorption causing the
loss of one electron from the Mn center, produces an oxidizing agent that can
remove four electrons from two molecules of water, producing O2.
 The electrons lost from the Mn center pass one at a time to an oxidized Tyr residue
in a PSII protein, then to P680.
Summing up the Overall reaction of PS II: When PQB
photochemical reactions has acquired two electrons in two
such transfers from PQA and two
protons from the solvent water, it is
in its fully reduced quinol form,
PQBH2.
The Tyr residue loses both a proton and
an electron, generating the electrically
neutral Tyr free radical, .Tyr.

The Tyr radical regains its missing

electron and proton by oxidizing a
cluster of four manganese ions in the
water-splitting complex.

In this state, the Mn complex can

take four electrons from a pair of
water molecules, releasing 4H+
and O2.

As the four protons produced in this reaction are released into the thylakoid lumen, the
oxygen-evolving complex acts as a proton pump, driven by electron transfer.
Proton and electron circuits in thylakoids

 Electrons (blue arrows) move

from H2O through PSII, through
the intermediate chain of
carriers, through PSI, and finally
to NADP.
 Protons (red arrows) are
pumped into the thylakoid
lumen by the flow of electrons
through the carriers linking PSII
and PSI, and re-enter the stroma
through proton channels formed
by the Fo (designated CFo) of
ATP synthase.
 The F1 subunit (CF1) catalyzes
synthesis of ATP.
 Stoichiometry of photophosphorylation
 As electrons move from water to NADP in plant chloroplasts, about 12 H+ move
from the stroma to the thylakoid lumen per four electrons passed (that is, per O2
formed).
 Four of these protons are moved by the oxygen-evolving complex, and up to eight
by the cytochrome b6f complex. The measurable result is a 1,000-fold difference
in proton concentration across the thylakoid membrane (ΔpH = 3).
 The electrochemical potential has two components: a proton concentration
difference (pH) and an electrical potential due to charge separation.
 In chloroplasts, pH is the dominant component; counter-ion movement
apparently dissipates most of the electrical potential.

In illuminated chloroplasts, the energy stored in the proton gradient per mole of protons is:

 So the movement of 12 mol of protons across the thylakoid membrane

represents conservation of about 200 kJ of energy—enough energy to drive the
synthesis of several moles of ATP (ΔG = 30.5 kJ/mol).
 Experimental measurements yield values of about 3 ATP per O2 produced.
 Stoichiometry of photophosphorylation
 At least eight photons must be absorbed to drive four electrons from H2O to
NADPH (one photon per electron at each reaction center).

 The energy in eight photons of visible light is more than enough for the synthesis
of three molecules of ATP.

 ATP synthesis is not the only energy-conserving reaction of photosynthesis in

plants; the NADPH formed in the final electron transfer is (like its close analog
NADH) also energetically rich.

The overall equation for noncyclic photophosphorylation is:

 Comparing ATP synthesis between mitochondria, chloroplast and bacteria

Comparison of the topology of proton movement and ATP synthase orientation in the
membranes of mitochondria, chloroplasts, and the bacterium E. coli. In each case,
orientation of the proton gradient relative to ATP synthase activity is the same.
Scaling
in
Biology
 A scale, in this sense, is a leveled range of values/numbers from
lowest to highest that measures something at regular intervals. A
great example is the classic number line that has numbers lined up
at consistent intervals along a line.

Linear Scale
A linear scale is much like the number line described above. They key to this type of
scale is that the value between two consecutive points on the line does not change
no matter how high or low you are on it.

For instance, on the number line, the distance between the numbers 0 and 1 is 1
unit. The same distance of one unit is between the numbers 100 and 101, or -100
and -101. However you look at it, the distance between the points is constant
(unchanging) regardless of the location on the line.
 What Is a Logarithmic Scale?
A logarithmic scale is much different to the linear one.

On this scale the value between two consecutive points not only
changes, but also has a distinct pattern.

Logarithms or 'logs' are based on exponents.

Exponents are the 'little numbers' that are written as superscripts

next to a base variable or number. For example, in the expressions
2^3 = 8, the number 3 is the exponent.

These numbers multiply the base by itself a designated amount of

times. In 2^3, the exponent tells us that 2 should be multiplie by itself
3 times: 2^3 = 2 x 2 x 2 = 8.
Imagine that we need to measure a really large
quantity of something.

I. If a scientist needs to measure billions or even

trillions of molecule, they might just make a
logarithmic scale with each number (i.e. from 0 to
1) increase representing an increase by a factor of
10.
II. That would mean that going from 0 to 1 means
increasing 10 units, and going from 0 to 2 means
increasing 100 units, because 10^2 = 100.
III. Numbers on a logarithmic scale are representative
of a factor increase in real units.

A great way to visualize this is by looking at the graph of an

exponential function. One of the properties shown in the example
below is that, as x increases, y increases 'exponentially' or by a
greater quantity for every additional unit of x.
• Bacteria divide by Binary Fission.
• This is a form of asexual reproduction.
• Under ideal conditions it can take place every 20
minutes!
• In this way huge numbers of bacteria can be
produced very rapidly.
• Because of this we use a special scale called the
logarithmic scale to represent their numbers.
• In a logarithmic scale each division represents a
unit increase in the value of x in the term 10x.
• Thus:
100 = 0
101 = 10
102 = 100
103 = 1000
104 = 10,000
105 = 100,000 etc

Logarithmic scale

0 1 10 100 1,000 10,000 100,000 1,000,000

 Growth curve for Bacteria

Lag phase Log Phase Stationary Death

Phase
Phase
Number of Bacteria
Logarithmic Scale

Time
The Lag Phase
•After inoculation there is normally a brief
period of adaptation by the cells to the
new conditions.
•Bacteria are producing the enzymes
necessary to digest the nutrients.
•The rate of growth begins to increase
towards the end of this phase.
The Log (Logarithmic / Exponential)
phase
• There is a rapid period of growth during this
phase due to the fact that:
• Bacteria have developed the necessary
enzymes and there are plenty of nutrients.
• There are few waste products being
produced.
• The rate of cell division is currently at its
maximum with the number of bacteria
doubling as often as every 20 minutes.
The Stationary Phase
• The rate of growth levels off during this
period.
• This is because:
• The nutrients are becoming used up.
• The amount of waste produced by the
bacteria themselves is increasing.
• The rate at which new cells are produced
is equal to the rate at which other cells
are dying.
The Death (Decline) Phase
During this phase more bacteria are
dying than are being produced. This is
because:
• Very few nutrients are left.
• Many bacteria are poisoned by the
waste produced by such large numbers
•Thus the rate of growth is falling.
Bioluminescence
Green fluorescent protein
Perrin-Jablonski
diagram
S is singlet and T is triplet.
The S0 state is the ground state and the subscript
numbers identify individual states.
Singlet & Triplet
DS0
 Stokes shift
The Stokes shift is the gap between the maximum of
the first absorption band and the maximum of the
fluorescence spectrum
loss of vibrational energy in the excited
state as heat by collision with solvent

heat
 Example fluorophores

fluorescein

ethidium bromide
bound to DNA.
 Quantum Yield
Quantum Yield = FF
• FF = number of fluorescence quanta emitted divided by
number of quanta absorbed to a singlet excited state
• FF = ratio of photons emitted to photons absorbed
•Quantum yield is the ratio of photons emitted to
photons absorbed by the system:
Instruments
 Intrinsic Fluorescence of Proteins and Peptides

Lifetime
Absorption Fluorescence
ns Wavelength Wavelength
Absorptivity Quantum
nm nm
Tryptophan 2.6 280 5,600 348 0.20

Tyrosine 3.6 274 1,400 303 0.14

Phenylalanine 6.4 257 200 282 0.04

 Green fluorescent protein (abbreviated GFP)
 Isolated from the Pacific jellyfish
Aequorea victoria and now plays
central roles in biochemistry and
cell biology due to its widespread
use as an in vivo reporter of gene
expression, cell lineage, protein ­
protein interactions and protein
trafficking.

 One of the most important

attributes of GFP which makes it so
useful in the life sciences is that the
luminescent chromophore is
formed in vivo, and can thus
generate a labeled cellular
macromolecule without the
difficulties of labeling with
exogenous agents.
Ribbon diagram of the
Green Fluorescent Protein
(GFP) drawn from the wild-
type crystal structure. The
buried chromophore, which
is responsible for GFP's
luminescence, is shown in
full atomic detail.
 The structure of GFP
Eleven-strand beta-barrel wrapped
around a central alpha-helix core.

This central core contains the

chromophore which is spontaneously
formed from a chemical reaction involving
residues Ser 65, Tyr 66, and Gly 67 (SYG)

There is cyclization of the polypeptide

backbone between Ser 65 and Gly 67 to
form a 5-membered ring, followed by
oxidation of Tyr 66.

The high quantum yield of GFP

fluorescence probably arises from the
nearly complete protection of the
fluorophore from quenching water or
oxygen molecules by burial within the
beta-barrel.
 Fluorescence Properties and Their Variation

 Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a minor
one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1, respectively.
 Its emission peak is at 509 nm in the lower green portion of the visible spectrum.
 For wild type GFP, exciting the protein at 395 nm leads to rapid quenching of the
fluorescence with an increase in the 475 nm excitation band. This photoisomerization effect
is prominent with irradiation of GFP by UV light.
 In a wide range of pH, increasing pH leads to a reduction in fluorescence by 395 nm
excitation and an increased sensitivity to 475 nm excitation.
Some examples of
Bioluminescence
 The Electromagnetic Radiation Spectrum

Only green and blue wavelengths pass through water a great distance.
 Light
Penetration
in the
Ocean
What color/wavelength of light will animals use?
What organisms that you know of have
bioluminescence?

Bioluminescence evolved in several kingdoms.

Evolution:
In early evolution, O2 was toxic. Some organisms
were able to convert it to a nontoxic substance,
which had the tendency to produce photons of
light. This may have had a selective advantage to
some organisms.

Not found in freshwater organisms.

 luciferase
Luciferin + O2 oxyluciferin + light
 • Bacterial
• Intrinsic

Photobacterium
 (bacterial)
Light emitting organ
Cephalopod Photophore
Examples of Bacterial Photophores:
• fish, few squid, Pyrosoma (tunicate)

How do they get bacteria?

• organ open to exterior (provide entrance for bacteria to
enter)
• potentially continuous luminescence

Pyrosoma
 Bacterial photophores- 3 genera

• Photobacterium (symbiotic relationship)

• Achromabacteria (2 types of squid use bacteria, the
rest (17) have make their own luminescence)
• Beneckea (not associated with symbiotic relationship)

Squid Euprymna- squid hatches w/out bacteria; w/in hours it is infected w/natural
populations of bacteria
Tunicate- Pyrosoma- bacterial symbiont (intracellular)
 

Examples of fish that have bacterial photophores:

• Anglerfish (ceratioids)
• Pinecone fish (Monocentrids)
• Lantern eyes/flashlightfish (Anomalopids)
• Ponyfishes/slipmouths (Leiognathids)
• Ichthyococcus
Intrinsic photophores:
1. Widely distributed, ex. Cookie cutter shark
2. Numerous photophores 1000’s
3. Make own luminescence
4. Control output of light (on and off)
Control of Bioluminescence:
They can control biolum intensity by controlling blood
supply to light organ (i.e., control the amt of O2 -- O2
decreases light intensity decreases)

Light control using a shield

• Lid
• Vascular control
• Rotation of organ
• Reproductive advantage
• Countershading
• Escape and avoid predation
• Species recognition
• Feeding
• In evolution
Some mesopelagic copepod
species are red/black in color.
Why?
Malacosteus, possess a cheek photophore
that emits a red light, which allows it to detect
red animals.
squids- looking for mates.
Some predators can lure prey by mimicking
signals of prey. Other predators dangle a lure to
attract prey.
mid-water squid releases a bioluminescent cloud
to startle and confuse predators.
Photoblepharon- blink and run method.
Ctenophore
pterapods
Firefly squid

Deep sea squid

  
Photophores on ventral
surface
                                                                         

              

Deep sea gulper

Deep sea viper fish
angler fish