Oxidative Phosphorylation:
Structure and function of ATP synthase, mitochondrial
transport systems, and inhibitors of Ox Phos
Bioc 460 Spring 2008 - Lecture 30 (Miesfeld)
Dinitrophenol uncouples proton
motive force and ATP synthesis
The ATP synthase
complex is the
molecular motor of life
Uncoupling proteins
generate metabolic heat to
protect vital organs during
animal hibernation
Key Concepts in Oxidative Phosphorylation
•
The ATP synthase complex is a molecular motor that undergoes protein
conformational changes in response to proton motive force across the inner
mitochondrial membrane. For each proton that flows through the ATP synthase
complex, the motor rotates 120º; 3 H+ are required for each ATP synthesized.
•
Mitochondrial shuttle systems are required to move metabolites across the
impermeable inner mitochondrial membrane. These shuttles move redox
energy from the cytosol to the mitochondrial matrix using carrier molecules such
as malate and glycerol-3-P, whereas, ATP translocase exchanges ATP for ADP.
•
Numerous inhibitors have been identified that interfere with ATP synthesis in
mitochondria. Some of these inhibit the electron transport system (rotenone,
cyanide), or the ATP synthase complex (oligomycin, DCCD), while others
function as chemical uncouplers that permit protons to cross the membrane
without passing through the ATP synthase complex (DNP, FCCP).
•
The uncoupling protein UCP-1 iconverts redox energy into metabolic heat.
UCP-1 is expressed in brown adipose tissue of newborns and hibernating bears.
The mitochondrial ATP
synthase complex uses
the proton-motive force
generated via the electron
transport system to
synthesize ATP through
protein conformational
changes in a process
called oxidative
phosphorylation.
In addition to generating
ATP during aerobic
respiration, a similar ATP
synthase complex
synthesizes ATP in
response to proton motive
generated by light-driven
photosynthetic processes
in plant chloroplasts.
Structure and Function of
the ATP Synthase Complex
•
ATP synthase complex represents
the molecular motor of life on planet
earth - natures own rotary engine.
•
Mitochondrial ATP synthase complex
consists of two large structural
components called F1 which
encodes the catalytic activity, and
F0 which functions as the proton
channel crossing the inner
mitochondrial membrane.
Three functional units
of ATP Synthase
1. The rotor turns 120º for
every H+ that crosses the
membrane using the
molecular “carousel” called
the c ring.
2. The catalytic head piece
contains the enzyme active
site in each of the three 
subunits.
3. The stator consists of the
α subunit imbedded in the
membrane which contains
two half channels for
protons to enter and exit
the F0 component, and a
stabilizing arm.
Proton movement through the ATP synthase
complex forces conformational changes in the
catalytic head piece in response to rotor rotation

top
QuickTime™ and a
Video decompressor
are needed to see this picture.

bottom
http://www.cnr.berkeley.edu/~hongwang/Project/ATP_synthase/MPEG_movies/F1_side_sp_2.mpeg
Proton movement through the ATP synthase
complex forces conformational changes in the
catalytic head piece in response to rotor rotation

top
top

QuickTime™ and a
Video decompressor
are needed to see this picture.


top
http://www.cnr.berkeley.edu/~hongwang/Project/ATP_synthase/MPEG_movies/F1_top_sp_2.mpeg
Proton flow through F0
alters the conformation of F1 subunits
The realization that the catalytic activity of the three  subunits was
regulated by conformational changes induced by the rotating  subunit
provided the key to understanding the enzyme mechanism of the F1F0
ATP synthase complex. Nucleotide binding studies revealed that it was
the affinity of the  subunit for ATP, not the rate of ATP synthesis (or
ATP hydrolysis in isolated F1 fragments), that was altered by proton flow
through the F0 component.
This conclusion came from studies showing that in the presence of protonmotive force, the dissociation constant (Kd) decreased by a millionfold. Based on these results, and on what was known about the subunit
composition of the F1 component, Paul Boyer at UCLA proposed the
binding change mechanism of ATP synthesis to explain how
conformational changes in β subunits control ATP production
The binding change mechanism
1. The  subunit directly contacts all three  subunits, however, each of
these interactions are distinct giving rise to three different β subunit
conformations.
2. The ATP binding affinities of the three beta subunit conformations are
defined as: T, tight; L, loose; and O, open; in which ADP and Pi bind to
the O and L conformations, and ATP binds tightly to the T conformation
but is released from the enzyme when the subunit is in the O
conformation.
3. As protons flow through F0 the subunit rotates in a counter-clockwise
circle (looking at F1 from the matrix side) such that with each 120º
rotation the β subunits sequentially undergo a conformational change
from O --> L --> T --> O --> L --> etc.
4. The binding change mechanism model predicts that one full rotation of
the  subunit should generate 3 ATP since each  subunit will have
cycled once through the T state.
Looking down
onto the
catalytic head
piece from
the viewpoint
of the
mitochondrial
matrix side.

Follow the the
conformational
changes in the
1 subunit which
will be O - L - T.
O
L
From this
viewpoint
the 
subunit
rotates
counterclockwise.
ATP is formed
in the 1
subunit but it is
not released in
the T state;
release of ATP
is the key step.
L
T
Three more H+
pass through
the c ring
channel and the
 subunit
rotates another
120º.
T
O
ATP is
released from
the 1
subunit when
it is in the O
conformation.
The 
subunit
sequence is
O - L - T - O.
The numbers don’t quite add up, but close enough
The ratio of 3 H+/ATP generated (3H+/120º rotation of the  subunit), is
not yet certain because there are unanswered questions regarding the
molecular mechanism of the proton-driven rotor.
Nevertheless, we will use 3 H+/ATP for now because it is a close
approximation and it fits pretty well with the observation that 10 H+ are
translocated across the inner mitochondrial membrane for each NADH
that is oxidized (~1 full 360º rotation of the  subunit).
The observed ATP currency exchange ratio of ~2.5 ATP/NADH is
consistent with this because one full rotation of the  subunit should
produce 3 ATP for 9 H+ translocated. So call it ~10 H+/NADH/~3ATP.
Boyer's model predicts that ATP hydrolysis by the F1 headpiece
should reverse the direction of the subunit rotor.
To test this idea, Masamitsu Yoshida and Kasuhiko Kinosita of Tokyo
Institute of Technology used recombinant DNA methods to modify the
, , and  subunits of the E. coli F1 component in order to build a
synthetic molecular motor.
When they viewed the motor from the c ring side (inter-membrane
space side), it was found to rotate counter clockwise for ATP
hydrolysis. Normally for ATP synthesis, the  subunit rotates
clockwise when viewed from the inter-membrane space.
Inter-membrane space side
ATP synthesis
Clockwise
ATP hydrolysis
Counter clockwise
Biochemical Application of the Oxidative Phosphorylation
The F1 component of the ATP synthase
complex can be used as a "nanomotor" to
drive ATP synthesis by attaching a magnetic
bead to the  subunit and forcing
clockwise rotation (viewed from the
bottom) using electromagnets.
Clockwise, counterclockwise, matrix side, inter-mitochondrial
membrane side - what is the take-home message?
The structure-function relationships in the ATP synthase complex that
catalyze ATP synthesis as a result of proton-motive force, are the same
ones that catalyze ATP hydrolysis.
Note that in the Yoshida experiment, energy released by ATP hydrolysis
was the driving force for  rotation, no proton gradient was required. In
this case, ATP binding to the O conformation, and subsequent ATP
hydrolysis, caused  conformational changes that pushed against the 
subunit to cause the sequence of events to be O - T - L - O - T etc.
Typical exam question on ATP motor rotation
The ATP synthase catalytic head piece rotates counterclockwise as viewed
from the matrix side of the inner mitochondrial membrane during ATP
synthesis.
What direction does it rotate during ATP hydrolysis when viewed from the
inter-membrane space?
The opposite side of the membrane would be clockwise, but since it is also the
opposite function (hydrolysis), the answer is counterclockwise.
You didn’t have to know which direction it rotates a priori, I gave that information
in the question. However, you did have to know that if you switch the orientation
and/or the function, the rotation is reversed - this the key concept.
How does H+ movement through the c ring lead to  subunit
rotation and subsequent conformational changes?
A proposed model for the F0 "rotary engine" is shown below based on
structural analysis of the yeast mitochondrial c subunit ring that was found
to contain 10 identical subunits. In response to proton motive force, a H+
will enter the half channel in the a subunit where it then comes in contact
with a negatively charged aspartate residue in the nearby c subunit.
Transport Systems In The Mitochondria
Key element of the Chemiosmotic Theory:
The inner mitochondrial membrane must be impermeable to ions in order
to establish the proton gradient.
Biomolecules required for the electron transport system and oxidative
phosphorylation must be transported, or "shuttled," back and forth across
the inner mitochondrial membrane by specialized proteins
For Pi and ADP/ATP, this is accomplished by two translocase proteins
located in the inner mitochondrial membrane.
Two Translocase Proteins
1. ATP/ADP Translocase
– also called the adenine nucleotide translocase.
– functions to export one ATP for every ADP that is imported.
– an antiporter because it translocates molecules in opposite
directions across the membrane.
– for every ADP molecule that is imported from the cytosol, an ATP
molecule is exported from the matrix.
2. Phosphate Translocase
– translocates one Pi and one H+ into the matrix by an electroneutral
import mechanism.
The Phosphate translocase functions as a channel
When the negatively
charged Pi ion (H2PO4-)
accompanies the
positively charged H+
across the inner
mitochondrial
membrane in response
to the proton gradient, it
is acting as a symporter
because both molecules
are translocated in the
same direction.
This is an electroneutral
translocation since the
two charges (H2PO4and H+) cancel each
other out.
Cytosolic NADH transfers electrons to the matrix
via shuttle systems
• Numerous dehydrogenase reactions in the cytosol generate NADH,
one of which is the glycolytic enzyme glyceraldehyde-3-phosphate
dehydrogenase.
• However, cytosolic NADH cannot cross the inner mitochondrial
membrane, instead the cell uses an indirect mechanism that only
transfers the electron pair (2 e-), or two reducing equivalents, from
the cytosol to the matrix using two different "shuttle" systems.
Most widely used shuttle is the malate-aspartate shuttle
Found to operate in liver, kidney, and heart cells, the malate-aspartate
shuttle functions as a reversible pathway. The key enzymes in this shuttle
pathway are cytosolic malate dehydrogenase and mitochondrial
malate dehydrogenase.
Cytolosolic malate dehydrogenase
Mitochondrial malate dehydrogenase
This is the
enzyme that
replaces
cytosolic
NAD+ during
aerobic
respiration.
The primary NADH shuttle in brain and
muscle cells is the glycerol-3-phosphate shuttle
Differs from the malateaspartate shuttle: the
electron pair extracted
from cytosolic NADH
enters the electron
transport chain at the
point of Q rather than
complex I.
The result of this is that
cytosolic NADH using this
shuttle system can only
produce 1.5 ATP/NADH
rather than 2.5 ATP
because of the loss of 4
H+ that are normally
pumped across the
membrane by complex I.
The net yield of ATP from glucose oxidation
in liver and muscle cells
Let's add everything up to see how one mole of glucose can be used to
generate 32 ATP in liver cells via the malate-aspartate shuttle, or 30 ATP in
muscle cells which use the glycerol-3-phosphate shuttle.
The ETS and Ox Phos are functionally linked
The role of the electrochemical proton gradient in linking substrate
oxidation to ATP synthesis can be demonstrated by experiments using
isolated mitochondria that are suspended in buffer containing O2,
but lacking ADP + Pi and also lacking an oxidizable substrate such as
succinate which has 2 e- to donate to the FAD in complex II of ETS.
• When ADP + Pi are added, O2 consumption, and ATP synthesis
increase only slightly over time because ETS runs out of substrate.
• When succinate is also added, both the rates of O2 consumption and
ATP synthesis increase dramatically until substrates become limiting.
• Both O2 consumption and ATP synthesis are blocked when cyanide
(CN-) is added to the suspension since proton translocation by the ETS
stops, resulting in a shut down of the ATP synthase complex because
the proton gradient is dissipated.
The ETS and Ox Phos are functionally linked
(ETS activity)
Succinate increases rates of Ox Phos and O2 consumption
(ETS activity) in isolated mitochondria, whereas, cyanide, CN-,
which inhibits ETS, inhibits Ox Phos and O2 consumption what the...?
Dinitrophenol (DNP) dissipates the proton gradient
by carrying H+ across the inner mitochondrial membrane
through simple diffussion-mediated transport
The result is that
carbohydrate
and lipid stores
are depleted in
an attempt to
make up for the
low energy charge
in cells resulting
from decreased
ATP synthesis;
DNP shortcircuits the
proton circuit.
Dinitrophenol is a hydrophobic
molecule that remains in the
mitochondrial membrane as a
chemical uncoupler for a long
time - a very dangerous way to
burn fat.
J Anal Toxicol. 2006 Apr;30(3):219-22.
(ETS activity)
Oligomycin inhibits proton flow through the Fo subunit of
ATP synthase and blocks ATP synthesis, but oligomycin
also blocks O2 consumption - what the…?
Addition of DNP to oligomycin-inhibited mitochondria
leads to increased rates of O2 consumption, but no change
in rates of ATP synthesis - what the, what the, what the…?
Summary of known ETS and Ox Phos inhibitors
You should be able to answer questions about changes in the rates of
succinate oxidation, O2 consumption, and ATP synthesis in mitochondrial
suspensions if provided information about any of these ETS, Ox Phos, or
translocase inhibitors.
The UCP1 uncoupling protein, also called thermogenin,
controls thermogenesis in newborn and hibernating animals
Cell-specific expression of
the UCP1 protein leads to
heat production under
aerobic conditions by short
circuiting the proton gradient
across the mitochondrial
inner membrane.
The UCP1 protein is
expressed at high levels in
special fat cells called brown
adipose tissue which contain
fatty acids for the production
of acetyl CoA to drive NADH
production by the citrate
cycle, and large numbers of
mitochondria to increase the
output of heat by the electron
transport system.