Electron Transport System

1
Oxidative PhosphorylationStructure and function
of ATP synthase, mitochondrial transport systems,
and inhibitors of Ox Phos
Bioc 460 Spring 2008 - Lecture 30 (Miesfeld)
The ATP synthase complex is the molecular motor
of life
Uncoupling proteins generate metabolic heat to
protect vital organs during animal hibernation
Dinitrophenol uncouples proton motive force and
ATP synthesis
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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.
• Mitochondrial shuttle systems are required to
  move metabolites across the impermeable inner
  mitochondrial membrane.
• Numerous inhibitors have been identified that
  interfere with ATP synthesis in mitochondria.
• The uncoupling protein UCP-1 converts redox
  energy into metabolic heat.

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• 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.

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Structure and Function ofthe ATP Synthase Complex

• 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.

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Three functional unitsof ATP Synthase

• The rotor turns 120º for every H that crosses
  the membrane using the molecular carousel
  called the c ring.
• The catalytic head piece contains the enzyme
  active site in each of the three ? subunits.
• The stator consists of the a subunit imbedded in
  the membrane which contains two half channels for
  protons to enter and exit the F0 component, and a
  stabilizing arm.

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Proton movement through the ATP synthase complex
forces conformational changes in the catalytic
head piece in response to rotor rotation
?
?top
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http//www.cnr.berkeley.edu/hongwang/Project/ATP_
synthase/MPEG_movies/F1_side_sp_2.mpeg
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Proton movement through the ATP synthase complex
forces conformational changes in the catalytic
head piece in response to rotor rotation
?
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?
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?top
?
http//www.cnr.berkeley.edu/hongwang/Project/ATP_
synthase/MPEG_movies/F1_top_sp_2.mpeg
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Proton flow through F0alters the conformation of
F1 subunits

• 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.
• These studies showed that the dissociation
  constant (Kd) decreased by a million-fold in the
  presence of proton-motive force.
• Paul Boyer proposed the binding change mechanism
  of ATP synthesis to explain how conformational
  changes in ß subunits control ATP production.

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The binding change mechanism

• The ? subunit directly contacts all three ?
  subunits, however, each of these interactions are
  distinct giving rise to three different ß subunit
  conformations.
• The ATP binding affinities of the three beta
  subunit conformations are defined as T, tight
  L, loose and O, open.
• As protons flow through F0, the ? subunit rotates
  such that with each 120º rotation, the ß subunits
  sequentially undergo a conformational change from
  O --gt L --gt T --gt O --gt L --gt etc.
• The binding change mechanism model predicts that
  one full rotation of the ? subunit should
  generate 3 ATP.

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Follow the the conformational changes in the ?1
subunit which will be O - L - T.
?
Looking down onto the catalytic head piece from
the viewpoint of the mitochondrial matrix side.
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From this viewpoint the ? subunit rotates
counter-clockwise.
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ATP is formed in the ?1 subunit but it is not
released in the T state release of ATP is the
key step.
Three more H pass through the c ring channel and
the ? subunit rotates another 120º.
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ATP is released from the ?1 subunit when it is in
the O conformation. The ? subunit sequence is
O - L - T - O.
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The numbers dont quite add up, but close enough

• We will use 3 H/ATP because it is a close
  approximation and it fits with the observation
  that 10 H are translocated across the inner
  mitochondrial membrane for each NADH that is
  oxidized.
• The observed ATP currency exchange ratio of 2.5
  ATP/NADH is consistent with this because one full
  360º rotation of the ? subunit should produce 3
  ATP for 9 H translocated.
• 10 H translocated/NADH oxidized/3ATP
  synthesized.

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• 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.

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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.
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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.

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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.
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Energy released by ATP hydrolysis was the driving
force for ? rotation, not a proton gradient
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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 didnt 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.
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How does H movement through the c ring lead to ?
subunit rotation and subsequent conformational
changes?

• 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.

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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.

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Two Translocase Proteins

• 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.
• Phosphate Translocase
• translocates one Pi and one H into the matrix by
  an electroneutral import mechanism.

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The Phosphate translocase functions as a channel
The phosphate translocase functions as a
symporter because both molecules are translocated
in the same direction. This is an electroneutral
translocation since the two charges cancel each
other out.
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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.

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Most widely used shuttle is the malate-aspartate
shuttle

• The key enzymes in this shuttle pathway are
  cytosolic malate dehydrogenase and mitochondrial
  malate dehydrogenase.

Cytolosolic malate dehydrogenase
Mitochondrial malate dehydrogenase
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The primary NADH shuttle in brain and muscle
cells is the glycerol-3-phosphate shuttle

• The electron pair extracted from cytosolic NADH
  enters the electron transport chain at the point
  of Q rather than complex I.

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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.
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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.

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Succinate increases rates of Ox Phos and O2
consumption in isolated mitochondria, whereas,
cyanide, CN-, which inhibits ETS, inhibits Ox
Phos and O2 consumption - what the...?
31
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 short-circuits the proton
circuit.
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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.
33
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?
34
Summary of known ETS and Ox Phos inhibitors
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The UCP1 uncoupling protein, also called
thermogenin, controls thermogenesis in newborn
and hibernating animals