Chemiosmotic Theory

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Chemiosmotic Theory

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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• Theories of oxidative phosphorylation
• Chemiosmotic theory
• Boyers binding change mechanism

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• The Chemiosmotic Theory of oxidative
  phosphorylation, for which Peter Mitchell
  received the Nobel prize
• Coupling of ATP synthesis to respiration is
  indirect, via a H electrochemical gradient.

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Chemiosmotic theory proposed by Peter Mitchell
The transport of protons from matrix to
intermembrane space is accompanied by the
generation of a proton gradient across the
membrane.
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• Protons (H) accumulate intermembrane space
  creating an electrochemical potential difference,
  proton gradient or electrochemical gradient.
• This proton motive force (PMF) drives the
  synthesis of ATP by ATP synthase complex.

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CHEMIOSMOTIC THEORY
Peter mitchel
IMM- Inner mitochondrial membrane
IMS- Inter membrane space
OMM- outer mitochondrial membrane
H
H
H
H
4H
H
4H
H
2H
III
2e-
H
2e-
H
I
H
Iv
H
4H
H
H
H
2H
4H
H
H
H
ADPPi
H
4H
H
H
4H
H
H
V
H
H
H
ATP
H
H
H
H
H
H
H
H
H
H
MATRIX
H
H
H
H
H
H
H
H
H
H
IMM
H
H
H
H
H
Complex I, III and IV are proton pumps
H
H
IMS
H
OMM
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• Proton gradient / electrochemical gradient
• Proton motive force

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Generation of ATP

• Proton dependant ATP synthese
• Uses proton gradient to make ATP
• Protons pumped through channel on enzyme
• From intermembrane space into matrix
• 4 H / ATP
• Called chemiosmotic theory

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Totals

• NADH
• 10 H X 1 ATP 2.5 (3) ATP
• 4 H
• FADH2
• 6 H X 1 ATP 1.5 (2) ATP
• 4 H

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Boyer s binding change mechanism ATP synthase
is a protein assembly in the inner mitochondrial
membrane.
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ATP synthase has two units F1 - projects into
matrix -has 3 a , 3 ß , gamma , delta,
epsilon chains -catalyses ATP
synthesis Peripheral catalytic sites are present
on beta subunits. Fo - embedded in membrane
- acts as channel for transport of H
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4
H H H H
H H H H
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• Mechanism of ATP synthesis (Boyers Hypothesis)
• Boyers binding change hypothesis
• Synthesis of ATP occurs on the surface of F1.
• Binding change mechanism states that 3 beta
• subunits change CONFORMATIONS during
• catalysis with only one beta subunit acting as
  
• Catalytic site.

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• ß subunits occur in 3 forms
• O form (Open form). It has low affinity for
  substrates ADP Pi
• L form (loose form). Can bind substrates ADP
  and Pi but catalytically it is inactive.
• T form (Tight form). Binds substrates ADP Pi
  tightly and catalyses ATP synthesis.

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• When protons pass through the disk of C subunits
  of F0 unit it causes rotation of ? sub unit.
• The ß subunits which are fixed to the membrane
  donot rotate.
• ADP Pi are taken up sequentially by the
  ßsubunits which undergo conformational changes
  and form ATP.

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Gamma subunit is in the form of axle . It rotates
when protons flow. ATP synthase is smallest known
MOLECULAR MOTOR in the living cells.
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ETC - inhibitors Complex I site I of ATP
synthesis inhibitors Rotenone, Peircidin, Amytal,
Barbiturates ComplexII Carboxin,Thenoyltrifluroac
etone,malonate Complex III site II of ATP
synthesis inhibitors Antimycin, Myxothiazol ,
stigmatellin Complex IV site III of ATP
synthesis inhibitors Cyanide, azide , carbon
monoxide
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• Complex I inhibitors (Site I inhibitors)
• Rotenone, insecticide, also used as fish poison.
  Binds to complex I and prevents the reduction of
  Ubiquinone.
• Piercidin, Amytal (sedative), Barbiturates
  inhibit by preventing the transfer of electrons
  from iron sulfur center of complex I to
  Ubiquinone.

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Complex II inhibitors Malonate acts as a
competitive inhibitor with the substrate
succinate
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• Complex III inhibitors (Site II Inhibitors)
• Antimycin inhibit electron transfer from cytb
  to C1.
• Myxothiazol and stigmatellin, antibiotics inhibit
  electron transfer from Cytb to C1.

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• Complex IV (site III inhibitors)
• Cyanide and azide bind tightly to oxidized form
  of heme a3 ( of complex iv ) preventing electron
  flow.
• Cyanide is potent and rapidly acting poison.
• Cyanide prevents binding of oxygen to Cytochrome
  oxidase ( aa3 ).
• Mitochondrial respiration and energy production
  stops cell death occurs rapidly.

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• Carbonmonoxide binds to the reduced form of
• heme a3(Fe2) competitively with oxygen and
  prevents
• electron transfer to oxygen.

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• Uncouplers of oxidative phosphorylation
• Uncouplers will allow oxidation to proceed but
  energy instead of being trapped as ATP is
  dissipated as heat.
• They are hydrophobic weak acids.
• They are protonated in the intermembrane space
  where a higher concentration of protons exists.

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• These protonated uncouplers due to their
  lipophilic nature rapidly diffuse across the
  membrane into matrix where they are deprotonated
  since matrix has a lower concentration of
  protons.
• Thus, the proton gradient is dissipated.

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• 2-4 dinitrophenol a classical uncoupler
  electrons from NADH to oxygen proceeds normally
  but ATP not formed as proton motive force across
  inner mitochondrial membrane is dissipated .

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• 2. Penta chloro phenol
• 3. Dinitro cresol
• 4.Bilirubin
• 5.Thyroxine-Physiological uncoupler
• 6.Valinomycin
• 7.Nigericin
• Note They are Lipophilic

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Intermembrane space
matrix
H H
H
H
H H H H
H H
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• Physiological Uncouplers
• 1.Excessive thyroid hormones
• 2. Unconjugated hyper bilirubinaemia
• 3. In high doses aspirin uncouple oxidative
  phospharylation which explains fever that
  accompanies toxic over dosage of these drugs.

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• Uncoupling proteins
• UCPs occur in the inner mitochondrial membrane of
  mammals, including humans.
• UCPs create a proton leak, that is they allow
  protons to re-enter the mitochondrial matrix
  without energy being captured as ATP.
• Energy is released as heat, and the process is
  called nonshivering thermogenesis.

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• UCP1, also called thermogenin, is responsible for
  the activation of fatty acid oxidation and heat
  production in the brown adipocytes of mammals.
• Brown fat , unlike the more abundant white fat,
  uses almost 90 of its respiratory energy for
  thermogenesis in response to cold, at birth,etc.

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• Inhibitors of Oxidative phosphorylation
• Oligomycin acts through one of the proteins
  present in F0 - F1 stalk .
• Oligomycin blocks the synthesis of ATP by
  preventing the movement of protons through ATP
  synthase.

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Respiratory control

• The regulation of the rate of oxidative
  phosphorylation by ADP level is called
  respiratory control.
• The ADP level increases when ATP is consumed and
  so oxidation is coupled to the utilization of
  ATP.
• Under physiological conditions, electron
  transport is tightly coupled to oxidative
  phosphorylation.

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• Electrons do not usually flow through the
  electron transport chain to O2 unless ADP is
  simultaneously phosphorylated to ATP.
• In the presence of excess substrate and Oxygen,
  respiration continues until all ADP is converted
  to ATP.
• After that the respiration rate or utilization of
  oxygen decreases
• In the presence of adequate oxygen and
  substrate, ADP becomes rate limiting it exerts a
  control over the entire oxidative phosphorylation
  process

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• The rate of respiration of mitochondria
  (Oxidative phosphorylation) can be controlled by
  ADP.
• Oxidation cannot proceed via ETC without
  simultaneous phosphorylation of ADP.
• Chance Williams defined 5 conditions that can
  control rate of respiration.

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• Generally most cells in the resting state are in
  state 4 , and respiration is controlled by the
  availability of ADP.
• The availability of inorganic phosphate could
  also influence the respiration.
• As respiration increases (Exercise) cell
  approaches state 3 ( ETC working to its full
  capacity ) or state 5 ( Availability of O2 is a
  limiting factor ).
• ADP / ATP transporter may also be a rate
  limiting factor

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• PO ratio (ADP O ratio)
• PO ratio is defined as number of phosphates
  incorporated into ATP to 1 atom of oxygen
  utilized during the transfer of 2 electrons
  through ETC.
• For NADH PO ratio is 3 i.e 3 ATPs are produced
  (2.5)
• For FADH2 PO ratio is 2 i.e 2 ATPs are
  produced(1.5)

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