2. Phases of Metabolism
Primary metabolism:
Conversion of
macromolecules to simple
units.
Secondary metabolism:
Catabolism of small
components in the cell.
Catabolic products enter the
TCA cycle in the form of
Acetyl CoA molecule and
NADH and FADH2 are
generated.
Tertiary Metabolism:
Reduced
Coenzymes(NADH &
FADH2) enter the electron
transport chain and energy
is produced in the form of
ATP.
FADH2
3.
4. Reduced Coenzymes
Reduced co-enzymes are generated during oxidation
of substrates( glucose, fatty acids). Major portion of
reduced co-enzymes are generated in TCA cycle.
Each molecule of NADH and FADH2 carry 2 electrons
and transfer to Complex I and Complex II respectively
AH2 + NAD+ + 2e- A +NADH +H+
Succinate + FAD + 2e- Fumarate +FADH2
6. Fate of cytosolic reduced coenzymes
Cytosolic reduced coenzymes(NADH and FADH2) are
impermeable to inner mitochondrial membrane
and therefore need special transport system(shuttle)
Malate Aspartate shuttle
Glycerol-3-phosphate shuttle
These shuttle helps to transfer electrons from cytosolic
(NADH and FADH2) to Mitochondrial (NAD+ and
FAD+)
10. Redox potential and Redox couple
The redox potential is a quantitative measure of the tendency
of a redox couple to lose or gain electrons.
Positive redox potential: Has higher affinity for electrons than
hydrogen.
Negative redox potential: Has lower affinity for electrons than
hydrogen.
The electrons flow from electronegative potential (-0.32)
to electropositive potential (+ 0.82).
Redox Couple
12. High Energy Compounds
Substance which posses sufficient free energy to
liberate at least 7cal/mol.
On hydrolysis yields energy.
High energy in the bonds are represented by squiggle
bond(~).
Mostly consists of phosphate group.
Energy liberated is required for biological activities.
13.
14. ATP( Adenosine Tri -phosphate)
Universal currency of energy within the living cell.
ATP +H2O ADP + Pi + H (Go’ = -7.3 kcal/mol)
+
Energy in the ATP is used to
drive the biosynthetic reaction,
muscle contraction , cellular
motion.
ATP is synthesized at the rate
of 3 molecules per second , i.e.
1.5kg/day.
15. Substrate level phosphorylation
Direct transfer of phosphate group from substrate
to ADP or GDP to form ATP or GTP is called
substrate level phosphorylation
16.
17. Oxidative Phosphorylation
Oxidation of reduced coenzymes (NADH and
FADH2)in Electron transport chain leads to
phosphorylation of ADP molecule to form ATP is
called oxidative phosphorylation.
Oxidation is coupled with phosphorylation in Electron
transport chain
19. Organization of ETC
“ MITOCHONDRIA IS THE POWER HOUSE OF THE
CELL”
Complexes
of ETC
20. Organization of Electron Transport Chain
All the components of ETC are located in the inner
membrane of mitochondria.
There are five distinct multi-protein complexes; these
are named as complex-I, II, III IV and Complex
V(ATP synthase Complex). These are connected by
two mobile carriers, co-enzyme Q and cytochrome C.
21. Components and reactions of ETC
Consists of 4 enzyme complexes one ATP synthase
complexe and 2 mobile electron carriers.
Complex I to IV and one ATP synthase complex
Enzyme Complexes
Coenzyme Q Mobile Electron carriers
Cytochrome C
23. Co enzyme Q (Ubiquinone)
Ubiquinone are ubiquitous in
nature.
similar in structure and property
with Vitamin K.
hydrophobic and can diffuse across
the membrane and channel
electrons between carriers.
Ubiquinone can accept electrons as
well as protons but transfer only
electrons.
They accept electron from complex
1 and 2.
They can accept one e– and get
converted into semiquinone(QH
.) or
two e–s to from Ubiquinol(QH2).
24. Cytochromes
Cytochromes are the proteins with characteristic
absorption of visible lights due to the presence of
heme containing Fe as co-factor.
There are three different types of cytochrome a, b and
c.
Cytochrome- Fe2+ <————> Cytochrome- Fe3+ + e–
Cytochromes are arranged in the order cytochrome ‘b’,
cytochrome c1, cytochrome ‘c’ and cytochrome a/a3.
Cytochrome bC1 (Complex III) ( Cytochrome
reductase)
Cytochrome aa3 (Complex IV)(Cytochrome Oxidase)
25. COMPLEX I (NADH Dehydrogenase or NADH–Coenzyme
Q Oxidoreductase)
‘L’ shaped with its one arm
in the membrane and
another arm extending
towards the matrix.
NADH + H+ feeds two
electrons and 2 protons to
FMN
FMN turns to FMNH2
FMNH2 transfer one
electron at a time to Iron
sulphur centers(FeS) of
complex one
Finally 2 electrons are
passed to Coenzyme
Q(CoQ)
26. Complex II : Succinate dehydrogenase complex
located towards the matrix
side of the membrane.
Succinate is oxidized to
fumarate as it transfers two
e–s and two protons to FAD.
FAD is reduced to FADH2.
FADH2 transfers electrons
to FeS Centers and finally
electrons flows to
Coenzyme Q(CoQ)
27. Complex III: Cytochrome Reductase
Electrons are
channeled from
complex I and
complex II to
cytochrome bc1 via
coenzyme Q
One electron is
transferred from
complex III to
cytochrome C at a
time
29. Q Cycle
Steps that transfers 2 electrons from Coenzyme Q(QH2) to
Cytochrome C is called Q cycle
One molecule od QH2 binds to complex III and transfer
electron to Rieske centers and to Cyt C1 and finally to Cyt C
Another electron moves to Cyt bL and bH and transfer
electron to new molecule of CoQ and partially reduce it to Q.
(Semiquinone)
Again a new QH2 molecule donates electron to complex III.
Both the electrons follow two different path one to cyt C
and another to Semiquinone.
Semiquinone after receiving one more electron converts to
Ubiquinol and it is again used to transfer electrons
This way 2 QH2 is needed to transfer 2 electrons to Cyt C
30. Complex IV:Cytochrome Oxidase
Cyt C transfer electron to
complex IV
Complex IV consists of
iron containing heme-a and
heme-a3.
Along with iron atoms,
cytochrome oxidase also
consists of Cu A and Cu B.
Flow of electron in
complex III
Cytochrome c —> Cu A —–>
Heme a—–> heme a3—->Cu
B—> O2
31. What is the significance of transferring Electrons
The free energy released during the flow of electrons trough complex I, III and IV is
utilized by these complexes to pump out protons(H+) from mitochondrial matrix to
Intermembrane space
32. Theories of Oxidative phosphorylation
1. The chemical coupling hypothesis:
Put forth by Edward Salter
According to this hypothesis during the process of
electron transfer, a series of high energy
phosphorylated compounds are formed which is later
on utilized for ATP synthesis.
This reactions are considered analogous to substrate
level phosphorylation glycolysis and TCA cycle
But this hypothesis lacks experimental support
33. 2.Conformational Coupling Hypothesis:
which Paul Boyer formulated in 1964,
proposes that electron transport causes proteins of
the inner mitochondrial membrane to
assume“activated” or “energized” conformational
states. These proteins are somehow associated with
ATP synthase such that their relaxation back to the
deactivated conformation drives ATP synthesis.
As with the chemical coupling hypothesis,the
conformational coupling hypothesis has found little
experimental support.
34. 3. The chemiosmotic hypothesis.
proposed in 1961 by Peter Mitchell
the model most consistent with the experimental
evidence.
It postulates that the free energy of electron transport
is conserved by pumping H+ from the mitochondrial
matrix to the intermembrane space so as to create an
electrochemical H+ gradient across the inner
mitochondrial membrane.
The electrochemical potential of this gradient is
harnessed to synthesize ATP
35. Proton gradient helps ATP Synthesis
Electron flow is accompanied by proton transfer across
the membrane, producing both a chemical gradient
(∆pH) and an electrical gradient (∆⍦)
The inner mitochondrial membrane is impermeable to
protons; protons can reenter the matrix only through
proton-specific channels(Fo).The proton-motive
force that drives protons back into the matrix
provides the energy for ATP synthesis, catalyzed by the
F1 complex associated with Fo.
38. ATP Synthase Complex
ATP synthase, also called Complex V,
has
two distinct components:
1. Fo (o “oh”denoting oligomycin
sensitive)
2.F1, a peripheral membrane protein
Fo component:
Present in inner mitochondrial
membrane
It has four subunit attached to F1
subunit( c disk, a , b2 and δ )
It is water insoluble
Acts as proton channel from which
electrons flows
ATP SYNTHASE COMPLEX
39. F1 component:
It projects into mitochondrial matrix
It catalyzes ATP synthesis
It has 9 subunits{(3 alpha(α), 3 beta(β), 1 gamm(Ƴ), 1
sigma(σ), 1 epsilon(ε)}.
ADP and Pi bind to alpha subunit
ADP is phosphorylated to ATP in Beta subunit
40. Binding Change Mechanism
The binding change
mechanism proposed by Paul
Boyer (Nobel Prize, 1997)
explains the synthesis of ATP
by the proton gradient.
Flow of electron through Fo
leads to change is
conformation of F1 which
leads to ATP synthesis.
F1 has 3 chemically identical
but conformationally distinct
functional states.
O(open) state - Does not bind
substrate or products
L(Loose) state – Loose binding
of substrate and products
T(Tight) state – Tight binding of
substrate and products
41. Old and New Energetics concept for ATP synthesis
Old Energetics Concept:
According to the estimated free energy of synthesis, it was
presumed that around 3 protons are required per ATP
synthesized.
when one NADH transfers its electrons to oxygen, 10 protons
are pumped out. This would account for the synthesis of
approximately 3 ATP.
Similarly the oxidation of 1 FADH2 is accompanied by the
pumping of 6 protons, accounting for 2 molecules of ATP.
New Energetics Concept:
However, Peter Hinkle recently proved that actual energy
production is less, because there is always leakage of protons.
This results in harnessing of energy required for the production
of 2.5 ATP from NADH and 1.5 ATP from FADH2
45. Uncouplers
Substances that uncouples oxidation and
phosphorylation are termed as Uncouplers.
Oxidation and reduction is carried out in ETC
complexes but phosphorylation is disrupted.
Uncouplers makes inner mitochondrial permeable to
Proton( H+)
Proton gradient is disrupted and proton flows back
to mitochondrial matrix via inner mitochondrial
membrane
Now energy stored as electrochemical potential is
dissipated in the form of heat
46. Physiological Uncouplers
The uncoupling of oxidative phosphorylation is useful
biologically. In hibernating animals and in newborn
human infants, the liberation of heat energy is required to
maintain body temperature.
In Brown adipose tissue, thermogenesis is achieved by this
process.
Thermogenin, a protein present in the inner mitochondrial
membrane of adipocytes, provides an alternate pathway for
protons. It is one of the uncoupling proteins (UCP).
Thyroxine is also known to act as a physiological uncoupler.