ETC

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Electron Transport and Oxidative Phosphorylation

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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• Electron Transport and Oxidative Phosphorylation
• Introduction
•  
• ?stage 3 of respiration
• ?NADH FADH oxidized, electrons are carried
  (ETS)? energy in form of ATP (Ox/Phos)
• ? aerobic acceptor oxygen

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• Mitochondrion --
• A. football shaped
• (1-2µ), 1-1000s in
• each cell
• B. electron transport
• and oxidative
• phosphorylation

Cytosol
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• C. Outer membrane- permeable to
• small molecules
• D. Inner membrane-
• ?electron transport
• enzymes embedded
• ? also ATP synthase
• ? Cristae increase area
• ? Impermeable to small molecules
• Integrity required for coupling ETS to
• ATP synthesis

Cytosol
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E. Matrix TCA enzymes, other enzymes also ATP,
ADP, NAD, NADH, Mg2, etc.
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? The Electron Transport System is the mechanism
the cell uses to convert the energy in NADH and
FADH2 into ATP. ? Electrons flow along an energy
gradient via carriers in one direction from a
higher reducing potential (greater tendency to
donate electrons) to a lower reducing potential
(greater tendency to accept electrons). ? The
ultimate acceptor is molecular oxygen.
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-- The overall voltage drop from NADH
E?? -(-0.32 V) to O Eº? 0.82 V is
?Eº? 1.14 V
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-- This corresponds to a large free energy change
of   ?G?? - nF?E?? -220 kJ/mole (n 2)   --
Since ATP requires 30.5 kJ/mole to form from ADP,
more than enough energy is available to
synthesize 3 ATPs from the oxidation of NADH.
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• NADH Dehydrogenase- Complex I
• NADH-CoQ oxidoreductase
• Contains FMN/FMNH2 and an Iron
• Sulfur Center as Electron Carriers
• NADH is substrate
• ?Coenzyme Q is second substrate

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Nicotinamide ?
NAD/NADH NADP/NADPH
?
Never covalently bound- freely diffusible
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Flavin mononucleotide FMN Flavin
adenine dinucleotide FAD Riboflavin ring
ribitol
isoalloxazine ring ribitol
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Coenzyme Q Ubiquinone ?a lipid in inner
membrane ? carries electrons ? polyisoprene tail
? moves freely within membrane
CoQ
CoQH2 (reduced form)
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For NADH, one of two entry points into the
electron transport chain
-- So the oxidation of one NADH results in the
reduction of one CoQ -- Another important
function of the enzyme will be mentioned later.
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• Succinate Dehydrogenase- Complex II
• ?SuccinateCoQ oxidoreductase
• ?Similar reaction can be written
• yielding CoQH2
• ?Second entry into electron transport
• ?Substrate is succinate
• Contains Iron Sulfur Center
• FAD is reduced, not FMN
• CoQH2 carries electrons to
• cytochrome b

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Cytochromes - proteins in ETS ?Carry
electrons ?Contain heme or heme-like group
? carries electrons only Fe(III) e- ? Fe(II)
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-- Cytochromes in respiration are on inner
mitochondrial membrane   ? cytochromes b, c1, c,
a, a3 , relay electrons,one at a time, in this
order ?
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? COMPLEX III b, an Fe-S and c1. ? Cytochrome
c is mobile. ? COMPLEX IV aa3 cytochrome
a-a3 cytochrome c oxidase -- large protein. --
both a and a3 contain heme A and Cu -- a3 Cu
binds to oxygen and donates electrons to
oxygen cytochrome a3 - only component of ETS that
can interact with O2
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Cytochrome c oxidase
Cu(II) ? Cu(I)
e- from cyt c to a
Heme A and Cu act together to transfer electrons
to oxygen
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Sequence of Respiratory Electron Carriers
Inhibitors in green
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How is amount ATP synthesized
measured? Quantify P/O ratio   Definition Pi
taken up in phosphorylating ADP per atom oxygen
(½O2), in other words per 2e-.
NADH 3 FADH2 2
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Experimental, we know As electrons are passed
through NADH oxidized by CoQ Cytochrome b
oxidized by cytochrome c1 Cytochrome a oxidized
by O2 Each yields enough energy to
synthesis about one ATP So oxidation of NADH
yields about 3 ATPs Oxidation of FADH2 gives
only 2 ATPs (succinate dehydrogenase others)
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What about energy and ATP stoichiometry? --
measured -- 220 kJ/mole from NADH oxidation --
Each ATP produced ADP Pi ? ATP ?G 30.5
kJ/mole   3(30.5)/220100 41 efficiency
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Oxidative Phosphorylation -- (ox-phos)
Definition Production of ATP using transfer of
electrons for energy coupled --for NADH,
we know
cyt b ?O2
NADH?FMN-FeS è CoQ è FeSècyt c è cyt aa3
?
cyt c1 ? ATP
ATP ATP Complex I Complex
III Complex IV   Note Several small energy
steps
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What are the requirements for coupling? --
Lehninger in the 50's and 60's ? Intact
mitochondria intact inner membrane,
respiratory chain ? Pi ? ADP ? NADH or other
reductant no other metabolites needed!
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Acceptor Control Suspend intact
mitochondria with NADH and Pi ?Add ADP
Requires ADP for oxygen uptake coupling
add ADP
O2 taken up
add ADP
time
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How is this coupling accomplished? -- It was
originally thought that ATP generation was
somehow directly done at Complexes I, III and
IV.   -- We now know that the coupling
is indirect in that a proton gradient
is generated across the inner mitochondrial
membrane which drives ATP synthesis.
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Matrix
ATP Synthetic Machinery   FoF1
ATP synthase Complex -- in inner mitochondrial mem
brane  
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-- knob-like projections on the matrix
side called F1 spheres.   -- responsible for
ATP production since when removed by
trypsin treatment, the resulting membranes
still transport electrons but do not make ATP.
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FoF1 ATP synthase -- ATP synthesized on matrix
side. -- electron transport complexes and FoF1
ATP synthase arranged on the inner membrane of
the mitochondrion facing in and lining the
membranes bordering the cristae.
 
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Chemiosmotic Theory --Peter Mitchell   -- A
proton gradient is generated using energy from
electron transport. --The vectorial transport of
protons (proton pumping) is done by Complexes I,
III, IV from the matrix to intermembrane space of
the mitochondrion.
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-- The protons have a thermodynamic tendency to
return to the matrix Proton-motive force The
proton move back into the matrix through
the FoF1ATP synthase driving ATP synthesis.
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The proton pumps are Complexes I, III and IV.
Protons return thru ATP synthase
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The return of protons downhill through Fo
rotates Fo relative to F1, driving
ATP synthesis. Note Subunit ? rotates through F1.
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ATP synthesis at F1 results from repetitive
comformational changes as ? rotates
? rotates 1/3 turn- energy for ATP release
animation
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• Experimental corroboration
• ? Uncoupling. The compound
• 2,4 dinitrophenol (DNP)
• allows proton
• through the membrane
• and uncouples.
• Blocking. The antibiotic oligomycin
• blocks the flow of H through the Fo,
• directly inhibiting ox-phos.

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Respiratory Control   -- Most
mitochondria are said to be tightly coupled. That
is there is no electron flow without
phosphorylation and no phosphorylation
without electron flow. -- Reduced substrate, ADP,
Pi and O2 are all necessary for oxidative
phosphorylation.
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For example, in the absence of ADP or O2 electron
flow stops, reduced substrate is not consumed and
no ATP is made acceptor control. Under certain
conditions, coupling can be lost. -- A toxic,
nonphysiological uncoupler, DNP, was described
previously.
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-- Brown adipose (fat) cells contain
natural uncouplers to warm animals - cold
adaptation and hibernation.
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Shuttling Reducing Equivalents from Cytosolic
NADH   -- Electrons from NADH are shuttled across
the mitochondrial membrane by carriers since NADH
cannot cross inner membrane. -- reoxidation of
cytosolic NADH leads to different energy yields
depending on mechanism the cell uses to
shuttle the reducing equivalents.
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-- The dihydoxyacetone phosphate shuttle yields 2
ATP/NADH   -- The malate shuttle yields 3 ATP/NADH
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mitochondrial malate dehydrogenase
cytosolic malate dehydrogenase
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Review of the Energy Yield from Glycolysis,
Pyruvate Dehydrogenase and the TCA
Cycle  Glycolysis glucose ?2pyruvate
2NADH2ATP 6-8 ATPs  Pyruvate
Dehydrogenase pyruvate ? acetyl CoA NADH
6 ATPs TCA cycle acetyl CoA?
2CO23NADHFADH2GTP 2x12ATPs   OVERALL yield
from glucose 36-38 ATPs