4018766.ppt biological oxidation and ETC

biochem

1. Chemiosmotic theory of oxidative
phosphorylation. Inhibitors and
uncouplers of oxidative
phosphorylation.

2. A PROTON GRADIENT POWERS
THE SYNTHESIS OF ATP
The transport of electrons from NADH or FADH
2
to O
2via the electron-transport chain is exergonic
process:
NADH + ½O
2+ H
+
H
2O + NAD
+
FADH
2+½O
2H
2O + FAD
+
G
o
’ = -52.6 kcal/mol for NADH
-36.3 kcal/mol for FADH
2
How this process is coupled to the synthesis of ATP
(endergonic process)?
ADP + P
iATP + H
2O G
o
’=+7.3 kcal/mol

3. •Proposed by Peter Mitchellin the
1960’s (Nobel Prize, 1978)
•Chemiosmotic theory: electron
transport and ATP synthesis
are coupled by a proton
gradient across the inner
mitochondrial membrane
Mitchell’s postulates for chemiosmotic theory
1.Intact inner mitochondrial membraneis required
2.Electron transport through the ETC generates a proton
gradient
3. ATP synthasecatalyzes the phosphorylation of ADP in a
reaction driven by movement of H
+
across the inner
membrane into the matrix
The Chemiosmotic Theory

4. As electrons flowthrough complexes of ETC, protons are
translocatedfrom matrix into the intermembrane space.
The free energy stored in the proton concentration gradientis
tapped as protons reenter the matrix via ATP synthase.
As result ATP is formed from ADP and P
i.
++ + +++
----
Overview of oxidative phosphorylation

5. An artificial system demonstrating the basic
principle of the chemiosmotic hypothesis
Synthetic vesicles
contains
bacteriorhodopsin and
mitochondrial ATP
synthase.
Bacteriorhodopsin -
protein that pumps
protons when illuminated.
When the vesicle is
exposed to light, ATP is
formed.

6. ATP Synthase
Two units, F
oand F
1(“knob-and-
stalk”; “ball on a stick”)
F
1contains the catalytic subunits
where ADP and P
iare brought
together for combination.
F
0spans the membraneandserves as
a proton channel.
Energy released by collapse of proton
gradient is transmitted to the ATP
synthesis.

7. •F
1contains 5 types of
polypeptide chains -
a
3b
3gde
•F
o-a
1b
2c
10-14
(csubunitsform
cylindrical, membrane-
bound base)
•F
oand F
1are
connected by agestalk
and by exterior column
(a
1b
2 andd)
•The proton channel –
between c ringand a
subunit.

8. •there are 3 active sites,
one in eachbsubunit
•c-e-gunit forms a “rotor”
•a-b-d-a
3b
3unit is the
“stator”
•passage of protons
through the F
ochannel
causes the rotor to spin
•rotation of thegsubunit
inside thea
3b
3hexamer
causes domain
movements in theb-
subunits, opening and
closing the active sites

9. Each bsubunit
contains the catalytic
site.
At any given time,
each site is in
different
conformation: open
(O), loose (L) or
tight (T).
O conformationbinds
ADP and P
i
The affinity for ATP
of T conformationis
so high that it
converts ADP and P
i
into ATP.

10. 1. ADP and P
ibind to an open site
2. Passage of protons causes each of three sites to change
conformation.
3. The open conformation(containing the newly bound ADP and P
i)
becomes a loose site. The loose sitefilled with ADP and Pi
becomes a tight site. The ATP containing tight sitebecomes an
open site.
4. ATP released from open site, ADP and P
iform ATP in the tight
site
Binding-Change Mechanism of ATP Synthase

11. Experimental observation of ATP synthase
rotation
•Fluorescent protein
arm (actin) attached
togsubunits
•a
3b
3subunitsbound
to a glass plate
•Arm seen rotating
when ATP added
(observed by
microscopy)

12. MOVEMENT ACROSS THE
MITOCHONDRIAL MEMBRANES
Electrons from Cytosolic NADH Enter
Mitochondria by Shuttles
NADHis
generated in the
cytosolin
glycolysis.
The inner mitochondrial membrane is impermeable to
NADH and NAD
+
.
Electronsfrom NADH,but not NADH itself, are carried
across the mitochondrial membrane.
Two shuttles move electrons: glycerol 3-phosphate
shuttleandmalate-aspartateshuttle

13. Glycerol 3-phosphate shuttle
Active in
skeletal
musclesand
brain.
Electrons
enter the
electron-
transport
chain via
complex II.
Therefore
only 1.5
molecules of
ATPare
produced.

14. Active in heartand liver.2.5 molecules of ATPare produced.
Malate-aspartate shuttle

15. •ATP must be transported to the cytosol, and ADP and P
imust
enter the matrix
•ADP/ATP carrier, adenine nucleotide translocase, exchanges
mitochondrial ATP
4-
for cytosolic ADP
3-
•The exchange causes a net loss of -1 in the matrix(draws some
energy from the H
+
gradient)
•Phosphate (H
2PO
4
-
) is transported into matrix in symport with
H
+
.Phosphate carrierdraws on pH.
•Both transporters consume proton-motive force
Active Transport of ATP, ADP and P
iAcross the
Inner Mitochondrial Membrane

16. Mechanism of ATP and ADP Transport
ATP-ADP translocaseis abundantin the inner mitochondrial
membrane (about 14% of the protein)
The entry of ADP into the matrix is coupled to the exit of ATP.

17. Mitochondrial Transporters
ATP-ADP translocase–antiport of ATP and ADP
Phosphate carrier–antiport of H
2PO
4
-
and OH
-
(symport of H
2PO
4
-
and H
+
)
Dicarboxylate carrier–antiport of malate, succinate, or fumarate and H
2PO
4
-
Tricarboxylate carrier–antiport of citrate and H
+
and malate
Pyruvate carrier–antiport of pyruvate and OH
-
(symport of pyruvate and H
+
)

18. REGULATION OF OXIDATIVE
PHOSPHORYLATION
Coupling of Electron Transport with ATP Synthesis
Electron transport is tightly coupled to phosphorylation.
ATP can not be synthesized by oxidative phosphorylation
unless there is energy from electron transport.
Electrons do not flow through the electron-transport chain
to O
2unless ADP is phosphorylated to ATP.
Important substrates: NADH, O
2, ADP
Intramitochondrial ratio ATP/ADPis a control mechanism
High ratio inhibits oxidative phosphorylation as ATP
allosterically binds to a subunit of Complex IV

19. The most important factor in determining the rate of
oxidative phosphorylation is the level of ADP.
The regulation of the rate of oxidative
phosphorylation by the ADP level is calledrespiratory
control
Respiratory control

20. Uncoupling of Electron Transport with ATP Synthesis
Uncoupling of oxidative phosphorylation generates heat to maintain
body temperature in hibernating animals, in newborns,and in mammals
adapted to cold.
Brown adipose tissuesis specialized for thermogenesis.
Inner mitochondrial membrane contains uncoupling protein (UCP),or
thermogenin.
UCP forms a pathway for the flow of protons from the cytosol to the
matrix.

21. •Uncouplers are lipid-soluble aromatic weak acids
•Uncouplers deplete proton gradientby transporting
protons across the membrane
Uncouplers
2,4-Dinitrophenol: an uncoupler
•Because the negative charge is delocalized over the ring,
both the acid and base forms of DNP are hydrophobic
enough to dissolve in the membrane.

22. Specific inhibitors of electron
transport are invaluable in revealing
the sequence of electron carriers.
Rotenoneand amytalblock electron
transfer in Complex I.
Antimycin Ainterferes with electron
flow thhrough Complex III.
Cyanide, azide,and carbon monoxide
block electron flow in Complex IV.
ATP synthaseis inhibitedby
oligomycinwhich prevent the influx of
protons through ATP synthase.
Specific inhibitors of electron
transport chain and ATP-synthase

23. Translocation of 3H
+
required by ATP synthase
for eachATP produced
1 H
+
needed for transport of P
i.
Net:4 H
+
transported for each ATP
synthesized
ATP Yield
Ten protonsare pumped out of the matrix during
the two electrons flowing from NADH to O
2
(Complex I, III and IV).
Six protonsare pumped out of the matrix during
the two electrons flowing from FADH
2to O
2
(Complex III and IV).
3
4
2
4

25. Glucose
Pyruvate
Acetyl Co AFatty Acids
Amino Acids
Citric acid
cycle supplies
NADH and
FADH
2to the
electron
transport
chain

26. Reduced coenzymes NADHand FADH
2are
formed in matrix from:
(1) Oxidative decarboxilation of pyruvate to
acetyl CoA
(2) Aerobic oxidation of acetyl CoA by the
citric acid cycle
(3) Oxidation of fatty acids and amino acids
The NADHand FADH
2are energy-rich
moleculesbecause each contains a pair of
electronshaving a high transfer potential.

27. The reduced and oxidized forms of NAD

28. The reduced and oxidized forms of FAD

29. Electronsof NADH or FADH
2are used to
reduce molecular oxygen to water.
A large amount of free energy is liberated.
The electrons from NADH and FADH
2are not
transported directly to O
2but are transferred
through series of electron carriersthat undergo
reversible reduction and oxidation.

30. The flow of electrons through carriers leads to
the pumping of protons out of the mitochondrial
matrix.
The resulting
distribution of
protons
generates a pH
gradientand a
transmembrane
electrical
potentialthat
creates a
protonmotive
force.

31. ATP is synthesized when protons flow back to the
mitochondrial matrix through an enzyme complex
ATP synthase.
The oxidation of fuels and the phosphorylation of
ADP are coupled by a proton gradient across the
inner mitochondrial membrane.
Oxidative
phosphorylationis
the process in which
ATP is formed as a
result of the
transfer of electrons
from NADH or
FADH
2to O
2by a
series of electron
carriers.

32. OXIDATIVE PHOSPHORYLATION IN
EUKARYOTES TAKES PLACE IN MITOCHONDRIA
Two membranes:
outer membrane
inner membrane(folded into
cristae)
Two compartments:
(1) the intermembrane space
(2) the matrix
•Inner mitochondrial membrane:
Electron transport chain
ATP synthase
•Mitochondrial matrix:
Pyruvate dehydrogenase complex
Citric acid cycle
Fatty acid oxidation
Location of mitochondrial complexes
The outer membrane
is permeableto small
molecules and ions
because it contains
pore-forming protein
(porin).
The inner membrane
is impermeableto ions
and polar molecules.
Contains transporters
(translocases).

33. THE ELECTRON TRANSPORT CHAIN
Series of enzyme complexes (electron carriers)
embedded in the inner mitochondrial membrane,
which oxidize NADH
2and FADH
2and transport
electrons to oxygen is calledrespiratory
electron-transport chain(ETC).
The sequence of electron carriers in ETC
cyt b
NADH FMN Fe-S Co-Q Fe-S cyt c
1cyt c cyt a cyt a
3O
2
succinate FAD Fe-S

34. High-Energy Electrons: Redox Potentials
and Free-Energy Changes
Inoxidativephosphorylation,theelectron
transferpotentialofNADHorFADH
2
is
convertedintothephosphoryltransfer
potentialofATP.
PhosphoryltransferpotentialisG°'(energy
releasedduringthehydrolysisofactivatedphos-
phatecompound).G°'forATP=-7.3kcalmol
-1
ElectrontransferpotentialisexpressedasE'
o
,
the(alsocalledredoxpotential,reduction
potential,oroxidation-reductionpotential).

35. E'
o
(reduction potential)is a measure of how easily a
compound can be reduced (how easily it can accept
electron).
All compounds are compared to reduction potential of
hydrogenwich is 0.0 V.
The larger the value of E'
o
of a carrier in ETC the better
it functions as an electron acceptor (oxidizing factor).
Electrons flow through the ETC components spontaneously
in the direction of increasing reduction potentials.
E'
o
of NADH= -0.32volts (strong reducing agent)
E'
o
of O
2= +0.82volts (strong oxidizing agent)
cyt b
NADH FMN Fe-S Co-Q Fe-S cyt c
1cyt c cyt a cyt a
3O
2
succinate FAD Fe-S

37. Important characteristic of ETC is the amount of
energyreleased upon electron transfer from one
carrier to another.
This energy can be calculated using the formula:
G
o
’=-nFE’
o
n –number of electrons transferred from one carrier
to another;
F –the Faraday constant (23.06 kcal/volt mol);
E’
o–the difference in reduction potential between
two carriers.
When two electrons pass from NADH to O
2 :
G
o
’=-2*96,5*(+0,82-(-0,32)) = -52.6 kcal/mol