Concept of Free Energy
Definition – Free energy is that portion of the energy of a system available to do work as the
system proceeds toward equilibrium under conditions of constant temperature and pressure and
volume
The change in free energy content (ΔG) depends on two components
ΔH (change in enthalpy, internal energy heat) and ΔS (change in entropy)
Chemical reaction performs work if it can be harnessed in utilizable form of energy. Amount of
work performed depends on the efficiency of the machinery.
Free energy change of a biological reactions is reported as the standard free energy change
(ΔG0’)
ΔG0’- is the value of ΔG for a reaction at standard conditions for biological reactions (pH 7,
1M, 25o
C, 1 atmosphere pressure)
Free energy change is used to predict the direction and equilibrium of chemical reactions
If ΔG is negative – net loss of energy (exergonic)
- reaction goes spontaneously
If ΔG is positive - net gain of energy (endergonic)
reaction does not go spontaneously
If ΔG is zero- reactants are in equilibrium
C - Oxidation-Reduction Reactions
The utilization of chemical energy in living system involves oxidation – reduction reactions. For
example, the energy of chemical bonds of carbohydrates, lipids and proteins is released and
captured in utilization form by processes involving oxidation- reductions.
I- Oxidation - removal of electron(s) from substance
- usually accompanied by a decrease in energy content of oxidized substance
II-Reduction - addition of electron(s) to a substance
usually accompanied by an increase in energy content of reduced substance
Oxidation reduction reactions are coupled processes
III- Reduction Potential (Oxidation-reduction potential, E'o )
-Concept
Definition - Measure of electron donating tendencies
Electrically measured in reference to a standard substance H2. Determined by measuring the
electromotive force generated by a sample half-cell with respect to standard reference halfcell
Anegative E’o = lower affinity for electrons
A positive E’o = higher affinity for electrons
In this example, 2H+
/H2 redox pair has negative E’o
- Electron donating tendency
where as ½ O2/H2O redox pair has positive E’o
- Electron accepting tendency
Hence, H2 - electron donor (oxidized)
O2 - electron acceptor (reduced)
Most molecules serve as both electron donors and electron acceptors of different times
depending upon what other substances they react with. In biological systems the primary
electron donors are fuel molecules such as carbohydrates, fats and proteins.
The oxidation of these substances transfers electrons to intermediate electron carriers such as
NAD+
, NADP+
and FAD to reduce them to their reduced state NADH, and FADH2.
Reduction potential of the NAD+
/ NADH pair is – 0.32Volt, placed high on the electron tower,
good electron donor and that of ½ O2 /H2O is +0.82volts.
In respiratory chain the electrons from NADH are transferred through a series of carriers
(organic or inorganic) until they are accepted by molecular oxygen (O2) releasing energy at
different levels. The free-energy change of an oxidation – reduction reaction can be calculated
from the difference in reduction potentials of the reactants using the formula:
Under cellular condition part of the free-energy of oxidation of reducing equivalents is conserved
in the form of high-energy phosphate compound, ATP. This occurs by the help of energy
conserving system in the inner mitochondrial membrane of eukaryotes or plasma membrane of
prokaryotes.
Fig 3.2. Change in free energy as a result of oxidation of NADH
Aerobic Energy-Generation
In aerobic organisms the complete breakdown of fuel molecules, carbohydrates, fats and
proteins takes place in mitochondria of eukaryotes and cytoplasmic membrane and cytoplasm
of aerobic prokaryotes.
The fuel molecules are metabolized to a common intermediate called aceyl CoA which is further
degraded by a common pathway called Kreb’s cycle.
This metabolic pathway in addition to providing energy provides building blocks required for
growth, reproduction, repair and maintenance of cellular viability.
Mitochondria
- it is an organelle where major amount of energy produced. Structurally it is
bounded by two separate membranes (outer mitochondrial membrane and inner mitochondrial
membrane)
Out membrane
- smooth and unfolded
- Freely permeable to most ions and polar molecules
(Contain porous channels)
Inner membrane
- folded into cristae-increased surface area
- Highly impermeable to most ions and polar molecules
Contain transporters which access polar and ionic molecules in and out
Cristae are characteristic of muscle and other metabolically active cell types
- Protein-rich membrane (about 75%)
Inter membrane space – space between outer and inner membranes
Matrix-the internal compartment containing soluble enzymes and mitochondrial genetic material
Oxidation of Pyruvate
Pyruvate is common intermediate of many catabolic reactions. It is still energy rich molecule. It
is a cross road molecule which can be converted into different intermediates depending on :-
type of cells-eukaryotes except RBC- acetyl CoA
- aerobic prokaryotes- acetyl CoA
- anaerobic prokaryotes- ethanol or lactate
- in RBC-always lactate
absence or presence of oxygen -lactate, ethanol or acetyl-CoA
- high ratio of NADH/NAD+ favors lactate formation in actively exercising muscle (oxygen limitation)
Oxidation of pyruvate into acetyl CoA-aerobic process (O2 terminal electron-acceptor) which
takes place in the mitochondrial matrix of eukaryotic cells Pyruvate is transported into
mitochondrial matrix by special transporter.Inside matrix pyruvate is oxidized into acetylCoA by
pyruvate dehydrogenase complex which is complex of E1, E2 and E3 enzymes.This enzyme
requires s five coenzymes- TPP, Lipoate, CoA, FAD and NAD+
Where:
E1 = pyruvate dehydrogenase,
E2 = dihydrolipoyl transacetylase,
E3 = dihydrolipoyl dehydrogenase
Regulation of Pyruvate Dehydrogenase
Product Inhibition by
- acetyCoA
- elevated levels of NADH
Covalent modification - dephosphorylated (active) - Increased ADP/ATP ratio
- Phosphorylated (inactive) - increased acetylCoA/CoA ratio
- Ncreased NADH/NAD+ ratio
KREB’S CYCLE
Also called-tricarboxylic acid cycle (TCA) or Citric acid cycle Final common pathway for
complete exudation of carbohydrates, fatty acids and many amino acids.Common pathway for
catabolism of acetyl COA ,a common intermediate of different catabolic pathways
Aerobic process (occurs in aerobic cells in presence of oxygen (O2). Reactions take place in
cytosol of prokaryotes and mitochondria matrix of eukaryotes
Fig 3.4 The citric acid cycle
Reactions of Kreb’s cycle
1. Condensation of acetyl COA with oxaloacetate by citrate synthase (condensing
enzyme) to form citrate.
Considerable free energy is lost as heat due to hydrolysis of thisester bond (drive the reaction
forward).
2. Isomerization of citrate to isocitrate by aconitase
Aconitase contains iron - sulfer (Fe:S) cluster that assists the enzymatic activity fluoroacetate
(potent rodenticide) inhibits aconitase with the ultimate effect of blocking Kreb’s cycle and
oxidative phosphorylation.
3. Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase
There are three types of isocitrate dehydrogenases
NAD+ - specific – only mitochondrial
NADP+ - specific – cytosolic and mitochondrial
Respiratory chain linked oxidation of isocitrate proceeds almost completely through NAD+ -
dependent enzyme
* Cytosolic isocitrate dehydragenase reaction generates NADPH and CO2
anabolic role
4. Oxidative decarboxylation of α- ketoglutarate by α - ketoglutarate dehydrogenase
complex
α-ketoglutarate is structurally and functionally similar to pyruvate dehydrogenase complex of
three enzymes (A’ B’ C’)
A’ (α - ketoglutarate dehydrogenase), B’ (transuccinylase), C’ (dihydrolipoyl dehydrogenase).
This enzyme has the same coenzyme requirement to that of pyruvate dehydrogenase complex.
The reaction releases considerable energy, part of which is used to form high-energy thioester
bond and NADH. Arsenite inhibits α-Ketoglutarate dehydrogenese complex blocking Kreb’s
cycle. α-Ketoglutarate accumulates upon poisoning of the enzyme. Arsenite is also inhibitor of
pyruvate dehydrogenase complex
5. Conversion of succinyl CoA into succinate by succinate thiokinase (succinyl CoA
synthetase)
GTP is formed by substrate – level phosphorylation
Fates of GTP - participate in mitochondrial protein synthesis
Converted into ATP.
6. Oxidation of succinate by succinate dehydrogenase
- Stereospecific for transfer of H-atoms of succinate (*)
- Succinate dehydrogenese – is integral part of inner mitochondrial membrane
- Contains Fe – S centers (non-heme iron protein) and FAD as prosthetic groups
Flavoprotein
- Inhibited by malonate which competes for the active site of the enzyme
-OOC-CH2-COO- (Malonate)
- Also inhibited by oxaloacetate resulting in succinate accumulation.
7. Hydration of Fumarate by Fumarase
- Fumarase is stereospecific for L-malate (catalyzes stereospecific trans addition of H and
OH).
8. Oxidation of L-malate by NAD–linked malate dehydrogenase
This reaction regenerates oxaloacetate, used in the first reaction.
N.B:
The cycle is aerobic process i.e. regeneration of oxidized coenzymes requires O2 as terminal
electron acceptor.
No net consumption or production of cycle intermediates
Oxaloacetate plays catalytic role in catabolism of acetyl CoA
Energy of acetyl CoA catabolism is partly conserved as reducing equivalents (NADH and
FADH2) and GTP
GTP is formed by substrate – level phosphorylation (synthesis of ATP related to oxidation of
substrates not related to electron transport)
Net consumption of two molecules of H2O
Enzymes – soluble and membrane attached (succinate dehydrogenase)
b-Functions of Kreb’s Cycle
Kreb’s cycle has catabolic and anabolic functions
Energy generation
– reducing equivalents,NADH and FADH2
– TP
Provide CO2 used for
– gluconeogenesis
– fatty acid synthesis
– urea synthesis
– ucleotide synthesis
Provide precursors for
– gluconeogenesis (all intermediates)
– amino acid synthesis (non-essial amino acids)
– heme synthesis (succinyl CoA)
– fatty acid synthesis (citrate)
Regulate other pathways – citrate (inhibit phosphofructokinase)
pyruvate carboxylation with formation of oxaloaccetate replenishes the cycle intermediates used
for biosynthesis
C - Regulation of Kreb’s Cycle
Primary function of the cycle is to provide energy, thus rate of the cycle is adjusted to meet an
animal cells ATP demand. Increased utilization of ATP increases the rate the cycle because of
availability of oxidized coenzymes necessary for the continuation of the cycle (NAD+, FAD) and
ADP which is needed for oxidative phosphorylation. High levels of ATP and NADH are inhibitory indicating high energy status of the cell. ATP inhibits both citrate synthase and isocitrate
dehydrogenase where as both are activated by high levels of ADP. NADH inhibits isocitrate
dehydrogenase and α-Ketoglutarate dehydrogenase. Complementary mechanisms of
controlling rate of acetyl CoA formation and rate of acetyl CoA degradation is also involved
III- Electron Transport system and Oxidative Phosphorylation
In aerobic organisms the major amount of ATP is synthesized by phosphorylation of ADP
related to electron transport, a process occurring on the inner mitochondrial membrane of
eukaryotes. It is a system composed of a chain of membrane associated electron carriers.
a- Components:
flavoproteins – FMN and FAD prosthetic groups. Accept H atoms but donate electrons
nonheme iron-sulfur proteins (Fe: S centers). Carry electrons not H – atoms. They are
component of complexes (I, II and III)
Coenzyme Q - Non protein component, Quinone derivative, lipid soluble. Common form in
mammals is Q10 Containing ten isoprene units. Accept H – atom but donate electrons. CoQ is
mobile carrier of electrons between flavoproteins and cytochromes. Accept electrons from
flavoproteins and donate to cytochromes. Transfer and accept two electrons at a time
Cytochromes – heme conjugated proteins
Heme = Fe2+/F3+ + porphyrin
Include classes of cytochromes designated a, b, and c. Iron at the center of cytochromes accept
and donates single electron
Cytochrome with relatively less positive reduction potential (i.e. cyt b) accepts electrons from
CoQH2 and transfers them to the next acceptor cytochrome with more positive reduction
potential the electron carriers except for CoQ are prosthetic groups of proteins
Components of electron transport system are arranged according to the increasing order
reduction potentials, a component with more negative reduction potential – at the top
component with more positive reduction potential at the bottom. The components of respiratory
chain are organized into four complexes and two mobile electron carriers
Complexes of respiratory chain are designated complex I, II, III and IV (integral parts of inner
mitochondrial membrane). CoQ and cytochrome C are mobile electron carriers which act as a
link between the complexes.
Complex Components
I (NADH dehydrogenase) FMN, Fe-S centers
II (Succinate dehydrogenese) FAD, Fe-S centers
III (Cytochrome reductase) cyt.b, cytc, Fe-S center
IV (Cytochrome oxidase) cyt.a, cyt.a3 ,copper (Cu1+/Cu2+)
Table 2. Components of electron transport chain
Complexes I, III and IC are proton pumps (trans-member and proteins) linked by CoQ and Cyt.c
(mobile electron carriers)
b - Oxidation of reducing equivalents (NADH & FADH2) and electron flow through
respiratory chain
Complex I
Electrons from NADH enter at complex I to be relayed to CoQ through series of carriers
Q also accepts a pair of electrons from FADH2 prosthetic group of complex II (succinate
dehydregenase), glycerol phosphate dehydrogenase (GPDH) and fatty acyl dehydrogenase
(FADH)
Complex IV
Every step of electron transport in the electron transport chain is accompanied by release of
energy
Useful energy is captured at three sites because large enough change in free energy (at least 9
- 10 Kcal/mole) occurs at three different sites when electron pairs flow down from NADH to O2.
The large enough drop in free energy is occurs when electrons are transferred: within the
NADH dehydrogenase complex, from NADH to CoQ, within cytochrome reductase from CoQ to
Cyt.c, within cytochrome oxidase from cytc to O2. Such drop in free-energy is more than enough
to sponsor the synthesis of three ATP from 3ADP and 3PI
Flow of a pair of electrons from FDH2 to O2 exhibits only two large drops in free-energy,
therefore sponsors the synthesis of only two ATP molecules. That is, the drop in free-energy as
electrons pass from FADH2 to CoQ is insufficient to sponsor ATP synthesis.
Oxidation of NADH releases more than enough free energy (-52.6kcal) needed for synthesis of
3ATP
Similarly oxidation of FADH2 releases more than enough free energy for synthesis of 2ATP.
C- Coupling of Electron Transport and ATP Synthesis (Oxidative Phosphorylation)
- Electron transport and oxidative phosphorylation are coupled processes
Suggested hypotheses for coupling mechanism
High-energy intermediate serves as precursor of ATP
Activated protein conformation drives the synthesis of ATP
Proton gradient across inner mitochondrial membrane couples electron transport and ATP
synthesis (chemiosmostic hypothesis of Peter Mitchell- postulated in 1961)
Mitchell’s chemiosmotic is the accepted theory
According to chemiosmotic theory, electron - transport and oxidative phosphorylation are
coupled by proton-gradient as follows. As electrons from NADH or FADH2 flow down the
respiratory chain to O2 free energy is released. The free energy released is captured at three
sites to pump protons against concentration gradient from matrix to inter membrane space
generating proton gradient across inner mitochondrial membrane. As a result of this pH gradient
is also formed, more positive (acidic) on the outer side more negative (basic) on the inner side
of mitochondria. In this process a proton motive force of 0.22Volts is created across inner
mitochondrial membrane. Now the kinetic energy of electrons is transformed into the proton –
motive force. This force drives later ATP synthesis
This happens when protons are back translocated into matrix through the channel portion of
ATP synthase (Fo) which is activated by electrochemical potential difference
across the membrane. Catalytic portion of ATP synthase (F1) synthesizes ATP from ADP and Pi
as protons are back translocated.
Fig 3.6. ATP synthesis coupled to electron transport
The precise number of protons pumped by each complex is not known with certainty
Current estimates
- 3 - 4 H + by complex I
- 4 H + by complex II
- 2 H + by complex IV.
Under cellular conditions, oxidation of:
NADH produces 3ATP from 3ADP and 3PI
FADH2 produces 2ATP from 2ADP and 2PI
Fates of mitochondrial ATP
used by mitochondria energy (ATP) requiring process
translocation to cytosol (where most of ATP is used for biosynthesis) by
ATP – ADP translocase (antiporter)
d- Respiratory Control
The most important factor determining the rate of oxidative phosphorylation is the level of ADP
which in turn is determined by the rate of ATP consumption cellular energy demand and
utilization determines the rate of ATP synthesis through its effect on the level of ADP which
controls the rates of oxidative phosphorylation and kreb’s cycle
ATP synthesis requires availability of ADP also regeneration of oxidized coenzymes (NAD+
and
FAD) required for Kreb’s cycle requires ADP that can be phosphorylated to ATP
e- Uncoupling of Electron Ttransport and Oxidative Phosphorylation
Uncouplers are substances which uncouple electron transport and oxidative phosphorylation
In the presence of uncouplers:
electron transport proceeds
proton translocation by proton pumps proceeds
oxygen consumption proceeds
aerobic oxidations proceed without control
Proton gradient is not formed since uncouplers carry protons back to matrix through IMM not
through ATP synthase (continuous dissipation of proton – gradient)
ATP synthase not activated
No ATP synthesis
Energy of oxidation lost as heat
Types of Uncouplers:
1. Chemical uncouplers – example 2, 4-dinitrophenol (2, 4-DNP) 2, 4, - DNP accepts proton and
carries it into matrix through IMM (membrane permeable)
2. Physiological uncoupler – thermogenin (protein)
H+
-channel in the IMM
Abundant in mitochondria of brown adipose tissue (low level of ATP synthase activity)
Responsible for diet induced thermo-genesis
Brown adipose tissue is absent or reduced in obese individuals
Present in newborns and cold adapted individuals
Thermogenin is opened by fatty acids liberated upon degradation of stored fat by activated
hormone sensitive lipase by norepinephrine released in response to drop in body temperature
due to cold environment.
Opening of thermogenin allows reentry of translated protons through IMM (no proton gradient
formed
No ATP synthesis
Energy of oxidation lost as heat
biological importance - maintain body temperature in newborns
- cold adaptation
f- Respiratory Poisons
Inhibit electron flow through respiratory chain
Inhibit proton translocation
Inhibit proton gradient formation
Inhibit O2 consumption
Inhibit ATP synthesis
Reducing equivalents remain reduced
Other aerobic oxidation’s inhibited
(Kreb’s cycle, pyruvate oxidation, fatty acid oxidation)