2. 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
3. Mitochondrion --
A. football shaped
(1-2μ), 1-1000s in
each cell
B. electron transport
and oxidative
phosphorylation
Cytosol
4. 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
6. 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.
7. -- The overall voltage drop from
NADH
E = -(-0.32 V)
to O Eº = +0.82 V
is Eº = 1.14 V
8. -- This corresponds to a large free
energy change of
G = - nFE = -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.
9. 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
14. 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.
15. Succinate Dehydrogenase- Complex II
Succinate:CoQ 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
16. Cytochromes - proteins in ETS
Carry electrons
Contain heme
or heme-like group
carries electrons only:
Fe(III) + e- Fe(II)
17. -- Cytochromes in respiration are on
inner mitochondrial membrane
cytochromes b, c1, c, a, a3 ,
relay electrons,one at a time,
in this order
18. COMPLEX III = b, an Fe-S and c1.
Cytochrome c is mobile.
COMPLEX IV = a+a3 =
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
19. Cytochrome c oxidase
Heme A and Cu act together to
transfer electrons to oxygen
Cu(II) Cu(I)
e- from cyt c to a
22. 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
23. 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)
24. 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)/220]×100 = 41% efficiency
25. Oxidative Phosphorylation
-- (ox-phos)
Definition: Production of ATP using
transfer of electrons for energy
= coupled
--for NADH, we know
cyt b O2
NADHFMN-FeS CoQ FeScyt c cyt aa3
cyt c1
ATP ATP ATP
Complex I Complex III Complex IV
Note: Several small energy steps
26. 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!
27. Acceptor Control
Suspend intact mitochondria with
NADH and Pi
Add ADP
add ADP
O2
taken
up
time
add ADP
Requires
ADP for
oxygen
uptake
= coupling
28. 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.
30. -- 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.
31. 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.
*********************************************************
32. 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.
33. -- 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.
34. The proton pumps are Complexes I,
III and IV.
Protons return thru ATP synthase
35. The return of protons “downhill”
through Fo rotates Fo
relative to F1,
driving ATP
synthesis.
Note: Subunit
rotates
through F1.
36. ATP synthesis at F1 results from
repetitive comformational changes
as rotates
rotates 1/3 turn- energy
for ATP release animation
37. 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.
38. 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.
39. 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.
40. -- Brown adipose
(fat) cells
contain natural
uncouplers to
warm animals -
cold adaptation
and hibernation.
41. 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.
42. -- The dihydoxyacetone phosphate
shuttle yields 2 ATP/NADH
-- The malate shuttle yields
3 ATP/NADH
