Electron Transport Chain #3

ATP Synthesis
Redox Potential Changes
 Paul D. Boyer and John E. Walker

Nobel Prize in Chemistry, 1997

" for elucidation of the enzymatic

mechanism underlying the
synthesis of ATP"
The Mammalian Respiratory Chain
The ATP Synthase
Asp61 (E.coli) Glu59 (mitochondria)
Asp61 (E.coli) Glu59 (mitochondria)
Proton Motion Across the Membrane Drives Rotation
of the C Ring
Proton Motion Across the Membrane Drives Rotation of the C
Ring
Role of Arg176 (mito) or
Arg220 (E.coli)
g subunit of the
ATPase
ATP Synthase Nucleotide-Binding Sites Are Not Equivalent
• In the active site Keq for the reaction ADP + Pi ↔ ATP + H2O is
near 1.0.

• Thus ATP formation at the active site is readily accomplished.

However, the ATP produced is in a thermodynamic pit; that is,
ATP has such a high binding energy that release is prevented.

• A conformational change driven by proton influx wakens the

binding of ATP sufficiently to allow the product to be released
from the enzyme.

• The dissociation constant for ATP changes from about 10 -12 M

to 10-5 M when the conformation of one of the three b
subunits in the ATP synthase changes.
Binding-change mechanism of the
ATP synthase
 Mechanism of ATP synthase.
ATP is shown in red, ADP and phosphate in
pink, and the rotating γ subunit in black
Rotation of the ATP synthase
 The P/O Ratio
• Before the acceptance of the chemiosmotic
hypothesis for oxidative phosphorylation, the
overall reaction equation was

• xADP + xPi + ½ O2 + H+ + NADH → xATP + H2O +

NAD+

• with the value x (called the P/O ratio) always an

integer.
 The P/O Ratio
• However with the acceptance of the chemiosmotic theory
there was no theoretical requirement for the P/O to be an
integer.

• The question became how many protons are pumped

outwards from NADH to O2 and how many must return to
drive ATP synthesis.

• The measurement of proton fluxes is very complicated as the

buffering capacity of the mitochondria has to be taken into
account, the use of the proton gradient for other purposes
and the leakage of protons across the inner membrane.
 The P/O Ratio
• The consensus values are that 10 protons are pumped out
per NADH and 6 for succinate.

• The number of protons believed to be required to drive

ATP synthesis is 4 of which one is used to transport Pi, ATP
and ADP across the inner membrane.

• Therefore the P/O ratio is 2.5 for NADH and 1.5 for
succinate. However the final values are still in doubt and
will not be fully determined until the full details of the
actions of the respiratory chain are determined.
Interaction between three transporters of
the inner membrane
Malate-Aspartate Shuttle
Glycerol 3-Phosphate Shuttle
Glycerol 3-Phosphate Shuttle
• Although this pathway
results in 2 less ATP per
glucose than the malate
–aspartate shuttle its
main advantage is the
speed of usage of the
cytosolic NADH.

• Found extensively in
very active muscles. E.g.
insect flight muscle
Redox Potential Changes
 Brown Adipose Tissue (BAT)
• BAT is the seat of non-shivering
thermogenesis, the ability of hibernators, cold
adapted rodents and newborn mammals in
general to increase their respiration and
generate heat without shivering

• In extreme cases the whole body respiration

can increase up to tenfold as a result of the
enormous respiration of this tissue
Uncoupling Protein and Thermogenesis
Regulation of Thermogenesis
• Free fatty acids (FFA)
liberated from TAGs
accumulates and binds
to UCP activating its
proton conductance.

• On the termination of
lipolysis residual fatty
acids are oxidized and
UCP reverts to its
inactive state
The Basal Proton Leak

• A proton leak is
found in
mitochondria that
do not express
UCP.
• This basal leak is
responsible for
state 4 respiration
 The Basal Proton Leak
• This leak accounts for a high proportion of the
basal metabolic rate in tissue such as skeletal
muscle.

• The molecular basis of the leak is still unclear

but it cannot be correlated with the presence
of a specific protein and does not correlate
with the lipid composition of the bilayer.
 The Basal Proton Leak
• It is possible that proton leak across the
membrane between protein and lipid.

• The basal leak displays a non-ohmic

current/voltage relationship.

• Proton conductance is highest at very high Dp

and decreases more that proportionately with
Dp
 The Basal Proton Leak
• The non-ohmic relationship may suggest that
the leak evolved to limit the maximal value of
Dp attainable by mitochondria.

• Since the production of ROS by mitochondria

is highly dependent on Dp, proton leaks may
serve the process of restricting oxidative
damage