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
Mitochondrion
Cytosol
A. football shaped
(1-2), 1-1000s in
each cell

B. electron
transport
and oxidative
phosphorylation
C. Outer membrane- permeable to
Cytosol
small molecules
D. Inner membrane-
electron transport
enzymes embedded;
also ATP synthase

Cristae increase area

Impermeable to small molecules
Integrity required for coupling ET
to ATP synthesis
E. Matrix

TCA enzymes,
other
enzymes;

also ATP, ADP,

NAD+, NADH,
Mg2+, etc.
 Biological redox = Two half-reactions
A:H A
Reductant Oxidant + e-

B B:H
Oxidant + e- Reductant
(acceptor) (donor)
Standard reduction potential, E
-- measure of the tendency of
oxidant to gain electrons, to
become reduced, a potential
 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.
-- The overall voltage drop from
NADH
E = -(-0.32 V)
to O E = +0.82 V
is E = 1.14 V
 -- 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
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

read about
flavoproteins, pp 519-521.
 Nicotinamide

NAD+/NADH
NADP+/NADPH

Never covalently bound- freely diffusib
Flavin mononucleotide isoalloxazine
= FMN ring
Flavin adenine
dinucleotide = FAD
ribitol
Riboflavin = ring
+ ribitol
Coenzyme Q = Ubiquinone

a lipid in inner membrane

carries electrons
polyisoprene tail
moves freely within membrane

2H+
+2e
Coenzyme Q
Coenzyme Q

CoQ CoQH2 (reduced form)

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.
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
Cytochromes - proteins in
ETS
Carry electrons
Contain heme
or heme-like group
carries electrons only:
Fe(III) + e- Fe(II)
-- Cytochromes in respiration are on
inner mitochondrial membrane

cytochromes b, c1, c, a, a3 ,
relay electrons,one at a time,
in this order
 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 c oxidase

e- from cyt c to a Cu(II)

Cu(I)
Heme A and Cu act together to
transfer electrons to oxygen
Sequence of
Respiratory
Electron
Carriers

Inhibitors
in green
How is amount ATP synthesized
measured?

Quantify P/O ratio

Definition: # Pi taken up in
phosphorylating ADP per atom
oxygen (O2), in other words
per 2e-.
NADH 3
FADH2 2
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
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

 Oxidative Phosphorylation
-- (ox-phos)
Definition: Production of ATP
using
transfer of electrons for energy
= coupled
--for NADH, we know
cyt b
O2
NADH
NADH FMN-FeS CoQ FeS
FeS cyt c cyt
aa3
cyt c1
ATP ATP
What are the requirements
for coupling?
-- Lehninger in the 50's and 60's
=
simplist reconstitution
Intact mitochondria
= intact inner membrane,
respiratory chain
Pi
ADP
NADH or other reductant
 Acceptor Control
Suspend intact mitochondria with
NADH and Pi
Add ADP
Requires
ADP for
oxygen
O2 uptake
take add ADP = coupling
n up
add ADP
time
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.
ATP Synthetic Matrix
Machinery

= FoF1 ATP
synthase
Complex
-- in inner
mitochondrial
membrane

-- 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.
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
******************************************************

Chemiosmotic Theory --Peter

Mitchell

-- A proton gradient is
generated using energy
from electron transport.
--The vectorial transport
of protons (proton
pumping)
pumping is done by
Complexes I, III, IV from the matrix
into the intermembrane space of
the mitochondrion.
-- 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.
The proton pumps are Complexes I,
III and IV.

Protons return thru ATP synthase

The return of protons downhill
through Fo rotates Fo
relative to F1,
driving ATP
synthesis.
Note: Subunit
rotates
through F1.
ATP synthesis at F1 results from
repetitive comformational change
as rotates

rotates 1/3 turn-

energy for ATP release
animation
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.
 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.
For example, in the absence of AD
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.
-- Brown
adipose (fat)
cells
contain natural
uncouplers to
warm animals -
cold adaptation
and
hibernation.
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 dihydoxyacetone phosphate
shuttle yields 2 ATP/NADH

-- The malate shuttle yields

3 ATP/NADH
cytosolic malate mitochondrial malate
dehydrogenase dehydrogenase
Review of the Energy Yield from
Glycolysis, Pyruvate Dehydrogenas
and the TCA Cycle
Glycolysis:
glucose 2pyruvate + 2NADH+2ATP 6-8 AT
Pyruvate Dehydrogenase:
pyruvate acetyl CoA + NADH 6 AT
TCA cycle:
acetyl CoA 2CO2+3NADH+FADH2+GTP 2x12

OVERALL yield from glucose 36-38 ATP