Electron transport

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• Electron transport

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Chemiosmotic Theory

• Electron Transport Electrons carried by reduced
  coenzymes are passed through a chain of proteins
  and coenzymes to drive the generation of a proton
  gradient across the inner mitochondrial membrane
• Oxidative Phosphorylation The proton gradient
  runs downhill to drive the synthesis of ATP
• Electron transport is coupled with oxidative
  phosphorylation
• It all happens in or at the inner mitochondrial
  membrane

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Outer Membrane Freely permeable to small
molecules and ions. Contains porins with 10,000
dalton limit Inner membrane Protein rich (41
proteinlipid). Impermeable. Contains ETR, ATP
synthase, transporters.
Cristae Highly folded inner membrane structure.
Increase surface area. Matrix- cytosol of the
mitochondria. Protein rich (500 mg/ml) Contains
TCA cycle enzymes, pyruvate dehydrogenase, fatty
and amino acid oxidation pathway, DNA,
ribosomes Intermembrane Space composition
similar to cytosol
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Reduction Potentials

• High Eo' indicates a strong tendency to be
  reduced
• Crucial equation ?Go' -nF ?Eo'
• ?Eo' Eo'(acceptor) - Eo'(donor)
• NADH ½ O2 H ? NAD H H2O
• NAD H 2e-? NADH Eo -0.32
• ½ O2 2e- 2H ? H2O Eo 0.816
• ?Go -nF(Eo'(O2) - Eo'(NADH))
• ?Go -nF(0.82 (-0.32)) -nF(1.14)
• -2(96.5 kJ mol-1V-1)(1.136) -220 kJ mol-1

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Electron Transport

• Four protein complexes in the inner mitochondrial
  membrane
• A lipid soluble coenzyme (UQ, CoQ) and a water
  soluble protein (cyt c) shuttle between protein
  complexes
• Electrons generally fall in energy through the
  chain - from complexes I and II to complex IV

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Standard reduction potentials of the major
respiratory electron carriers.
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Complex I

• NADH-CoQ Reductase
• Electron transfer from NADH to CoQ
• More than 30 protein subunits - mass of 850 kD
• 1st step is 2 e- transfer from NADH to FMN
• FMNH2 converts 2 e- to 1 e- transfer
• Four H transported out per 2 e-

         
FMN
         
Fe2S
         
FMNH2
Fe3S
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Complex II

• Succinate-CoQ Reductase
• aka succinate dehydrogenase (from TCA cycle!)
• four subunits
• Two largest subunits contain 2 Fe-S proteins
• Other subunits involved in binding succinate
  dehydrogenase to membrane and passing e- to
  Ubiquinone
• FAD accepts 2 e- and then passes 1 e- at a time
  to Fe-S protein
• No protons pumped from this step

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Q-Cycle

• Transfer from the 2 e- carrier ubiquinone (QH2)
  to Complex III must occur 1 e- at a time.
• Works by two single electron transfer steps
  taking advantage of the stable semiquinone
  intermediate
• Also allows for the pumping of 4 protons out of
  mitochondria at Complex III
• Myxothiazol (antifungal agent) inhibits electron
  transfer from UQH2 and Complex III.

UQ
UQ.-
UQH2
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Complex III

• CoQ-Cytochrome c Reductase
• CoQ passes electrons to cyt c (and pumps H) in a
  unique redox cycle known as the Q cycle
• Cytochromes, like Fe in Fe-S clusters, are one-
  electron transfer agents
• cyt c is a water-soluble electron carrier
• 4 protons pumped out of mitochondria (2 from UQH2)

         
         
         
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Complex IV

• Cytochrome c Oxidase
• Electrons from cyt c are used in a four-electron
  reduction of O2 to produce 2H2O
• Oxygen is thus the terminal acceptor of electrons
  in the electron transport pathway - the end!
• Cytochrome c oxidase utilizes 2 hemes (a and a3)
  and 2 copper sites
• Complex IV also transports H (2 protons)

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Inhibitors of Oxidative Phosphorylation

• Rotenone inhibits Complex I - and helps natives
  of the Amazon rain forest catch fish!
• Cyanide, azide and CO inhibit Complex IV, binding
  tightly to the ferric form (Fe3) of a3
• Oligomycin and DCCD are ATP synthase inhibitors

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Shuttling Electron Carriers into the Mitochondrion

• The inner mitochondrial membrane is impermeable
  to NADH.
• Electrons carried by NADH that are created in the
  cytoplasm (such as in glycolysis) must be
  shuttled into the mitochondrial matrix before
  they can enter the ETS

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Glycerol phosphate shuttle
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malate/aspartate shuttle system
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Electron transport is coupled to oxidative
phosphorylation
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Uncouplers

• Uncouplers disrupt the tight coupling between
  electron transport and oxidative phosphorylation
  by dissipating the proton gradient
• Uncouplers are hydrophobic molecules with a
  dissociable proton
• They shuttle back and forth across the membrane,
  carrying protons to dissipate the gradient
• w/o oxidative-phosphorylation energy lost as heat
• Dinitrophenol once used as diet drug, people ran
  107oF temperatures

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• Oxidative phosphorylation

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Proton Motive Force (Dp)

• PMF is the energy of the proton concentration
  gradient
• The chemical (DpH pHin pHout) potential and
  the electrical potential(DY Yin Yout)
  contribute to PMF
• DG nfDY and DG 2.303nRT DpH
• DG for transporting 1 H from inner membrane
  space to matrix DG nfDY 2.303nRTDpH
• Dp Dp DG/nF
• Dp Dy (0.059)DpH

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Proton Motive Force (Dp)

• What contributes more to PMF, DY or DpH?
• In liver DY-0.17V and DpH0.5
• Dp Dy (0.059)DpH -0.17-(0.059)(0.5V)
• Dp -0.20 V
• DY/Dp(-0.17V/-0.20V) X 100 85
• 85 of the free energy is derived form DY

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Proton Motive Force (Dp)

• How much free energy generated from one proton?
• DG nFDP (1)(96.48kJ/Vmole)(-0.2V) -19
  kJ/mole
• To make 1 ATP need 30 kJ/mole.
• Need to translocate more than one proton to make
  one ATP
• ETC translocates 10 protons per NADH

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ATP Synthase

• Proton diffusion through the protein drives ATP
  synthesis!
• Two parts F1 and F0

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Racker Stoeckenius confirmed Mitchells
hypothesis using vesicles containing the ATP
synthase and bacteriorhodopsin
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Binding Change Mechanism

• ADP Pi lt-gt ATP H2O
• In catalytic site Keq 1
• ATP formation is easy step
• But once ATP is formed, it binds very tightly to
  catalytic site (binding constant 10-12M)
• Proton induced conformation change weakens
  affinity of active site for ATP (binding constant
  10-5)

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Binding Change Mechanism

• Different conformation at 3 catalytic sites
• Conformation changes due to proton influx
• ADP Pi bind to open-site in exchange for ATP
• Proton driven conformational change (loose site)
  causes substrates to bind more tightly
• ATP is formed in tight-site.
• Requires influx of three protons to get one ATP

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ATPase is a Rotating Motor

• Bound a,b,g subunits to glass slide
• Attached a fluroescent actin chain to g subunit.
• Hydrolysis of ATP to ADP Pi cause filament to
  rotate 120o per ATP.

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How does proton flow cause rotation?
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Active Transport of ATP, ADP and Pi Across
Mitochondrial Inner Membrane

• ATP is synthesized in the matrix
• Need to export for use in other cell compartments
• ADP and Pi must be imported into the matrix from
  the cytosol so more ATP can be made.
• Require the use of transporters

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Transport of ATP, ADP and Pi

• Adenine nucleotide translocator ADP/ATP
  antiport.
• Exchange of ATP for ADP causes a change in DY due
  to net export of 1 charge
• Some of the energy generated from the proton
  gradient (PMF) is used here
• Pi is imported into the matrix with a proton
  using a symport.
• Because negative charge on the phosphate is
  canceled by positive charge on proton no effect
  on DY, but effects DpH and therefore PMF.

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Transport of ATP, ADP and Pi

• NRG required to export 1 ATP and import 1 ADP and
  1 Pi NRG generated from influx of one proton.
• Influx of three protons required by ATPase to
  form 1 ATP molecule.
• Need the influx of a total of 4 protons for each
  ATP made.

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P/O Ratio

• The ratio of ATPs formed per oxygens reduced
• e- transport chain yields 10 H pumped out per
  electron pair from NADH to oxygen
• 4 H flow back into matrix per ATP to cytosol
• 10/4 2.5 for electrons entering as NADH
• For electrons entering as succinate (FADH2),
  about 6 H pumped per electron pair to oxygen
• 6/4 1.5 for electrons entering as succinate

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Regulation of Oxidative Phosphorylation

• ADP is required for respiration (oxygen
  consumption through ETC) to occur.
• At low ADP levels oxidative phosphorylation low.
• ADP levels reflect rate of ATP consumption and
  energy state of the cell.
• Intramolecular ATP/ADP ratios also impt.
• At high ATP/ADP, ATP acts as an allosteric
  inhibitor for Complex IV (cytochrome oxidase)
• Inhibition is reversed by increasing ADP levels.

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Uncouplers

• Uncouplers disrupt the tight coupling between
  electron transport and oxidative phosphorylation
  by dissipating the proton gradient
• Uncouplers are hydrophobic molecules with a
  dissociable proton
• They shuttle back and forth across the membrane,
  carrying protons to dissipate the gradient
• w/o oxidative-phosphorylation energy lost as heat
• Dinitrophenol once used as diet drug, people ran
  107oF temperatures

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Physiological Uncoupling

• Uncoupling of ETC and Ox-phos occurs in animals
  as a means to produce heat nonshivering
  thermogenesis.
• Impt. In hibernating mammals, neborn animals and
  mammals adapted to cold
• Occurs in brown adipose tissues (rich in
  mitochondria)
• Uncoupling protein (UCP) channel to allow
  influx of protons to matrix (dissipates proton
  gradient)

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Uncoupling in Plants

• Plants generate heat during fruit ripening and to
  emit odors to attach pollinators.
• Plants can by pass normal ATP generating ETC
• Alternative ETC in plants does not pump protons,
  just transfers electron.
• All plant have this pathway, actual physiological
  reason not known