Oxidative phosphorylation pathway

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Oxidative phosphorylation pathway

• Lecture 15
• Modified from internet resources, journals and
  books

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Introduction

• Oxidation of NADH with phosphorylation of ADP to
  form ATP ? processes supported by the
  mitochondrial electron transport assembly and ATP
  synthase (at inner mitochondrial membrane)
• electron transport assembly ? comprised of a
  series of protein complexes that catalyze
  sequential oxidation reduction reactions

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Principals of Reduction/Oxidation (Redox)
Reactions

• Redox reactions ? involve the transfer of
  electrons from one chemical species to another
• The oxidized plus the reduced form of each
  chemical species is referred to as an
  electrochemical half cell
• example of a coupled redox reaction is the
  oxidation of NADH by the electron transport
  chain
• NADH (1/2)O2 H -----gt NAD H2O

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Complexes of the Electron Transport Chain

• NADH is oxidized by a series of catalytic redox
  carriers that are integral proteins of the inner
  mitochondrial membrane
• free energy change in several of these steps is
  very exergonic
• Coupled to these oxidation reduction steps ? is a
  transport process in which protons (H) from the
  mitochondrial matrix are translocated to the
  space between the inner and outer mitochondrial
  membranes
• redistribution of protons ? leads to formation of
  a proton gradient across the mitochondrial
  membrane

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• result of these reactions ? redox energy of NADH
  is converted to the energy of the proton gradient
• This process ? facilitated by a proton carrier in
  the inner mitochondrial membrane ATP synthase ?
  this carrier is coupled to ATP synthesis

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Flow of electrons in the electron transport chain
of oxidative phosphorylation

• The electrons pass through the proteins of
  complex I, four protons (H) are pumped into the
  intramembrane space of the mitochondrion
• four protons are pumped into the intramembrane
  space as each electron pair flows through
  complexes III and as four electrons are used to
  reduce O2 to H2O in complex IV

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continued

• all of the components of the pathway (exception
  NADH, succinate, and CoQ) ? are integral proteins
  of the inner mitochondrial membrane
• the mitochondrial electron transport proteins are
  clustered into complexes Complexes I, II, III,
  and IV
• Electrons flow through the complexes ? Oxygen is
  the final electron acceptor, with water being the
  final product of oxygen reduction

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Oxidative Phosphorylation

• The free energy available as a consequence of
  transferring 2 electrons from NADH or succinate
  to molecular oxygen is -57 and -36 kcal/mol
• Oxidative phosphorylation traps this energy as
  the high-energy phosphate of ATP
• In order for oxidative phosphorylation to proceed
  ? two principal conditions must be met
• the inner mitochondrial membrane must be
  physically intact so that protons can only
  reenter the mitochondrion by a process coupled to
  ATP synthesis

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continued

• high concentration of protons must be developed
  on the outside of the inner membrane
• Electrons return to the mitochondrion through the
  integral membrane protein known as ATP synthase
  (or Complex V)
• In damaged mitochondria, permeable to protons ?
  the ATP synthase reaction is active in the
  reverse direction acting as a very efficient ATP
  hydrolase or ATPase

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Oxidative Phosphorylation

• For each pair of electrons originating from NADH
  ? 3 equivalents of ATP are synthesized
• proton gradient generated by electron transport
  contains sufficient energy to drive normal ATP
  synthesis

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Regulation of Oxidative Phosphorylation

• flow of electrons through the electron transport
  system is regulated by the magnitude of the PMF
  (proton motive force)
• Under resting conditions, with a high cell energy
  charge ? demand for new synthesis of ATP is
  limited
• energy demands are increased (vigorous muscle
  activity) ? cytosolic ADP rises and is exchanged
  with intramitochondrial ATP
• while the rate of electron transport is dependent
  on the PMF, the magnitude of the PMF at any
  moment simply reflects the energy charge of the
  cell

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Inhibitors of Oxidative Phosphorylation

• The pathway of electron flow through the electron
  transport assembly can be inhibited
• Some agents ? inhibitors of electron transport at
  specific sites in the electron transport assembly
• Rotenone ? Complex I
• Antimycin A ? Complex III
• Cyanide ? Complex IV
• Carbon Monoxide ? Complex IV

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Energy from Cytosolic NADH

• cytosolic NADH when oxidized via the electron
  transport system (in contrast to oxidation of
  mitochondrial NADH) ? gives rise to 2 equivalents
  of ATP if it is oxidized by the glycerol
  phosphate shuttle and 3 ATPs if it proceeds via
  the malate aspartate shuttle
• glycerol phosphate shuttle ? coupled to an inner
  mitochondrial membrane
• some tissues (heart, muscle) ? mitochondrial
  glycerol-3-phosphate dehydrogenase is present in
  very low amounts ? the malate aspartate shuttle
  is the dominant pathway for aerobic oxidation of
  cytosolic NADH
• the malate aspartate shuttle generates 3
  equivalents of ATP for every cytosolic NADH
  oxidized (in contrast to the glycerol phosphate
  shuttle)

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Brown Adipose Tissue and Heat Generation

• uncoupling of proton flow ? releases the energy
  of the electrochemical proton gradient as heat
• this process ? normal physiological function of
  brown adipose tissue
• Brown adipose tissue ? gets its color from the
  high density of mitochondria in the individual
  adipose cells
• Newborn babies contain brown fat in their neck
  and upper back ? serves the function of
  nonshivering thermogenesis
• muscle contractions that take place in the
  process of shivering ? generates ATP, produces
  heat
• Nonshivering themogenesis ? a hormonal stimulus
  for heat generation without the associate muscle
  contractions of shivering

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continued

• The process of thermogenesis in brown fat ?
  initiated by the release of free fatty acids from
  the triglycerides stored in the adipose cells
• The mitochondria in brown fat ? contain a protein
  called thermogenein
• Thermogenein ? acts as a channel in the inner
  mitochondrial membrane to control the
  permeability of the membrane to protons

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• norepinephrine is released in response to cold
  sensation ? binds to -adrenergic receptors on the
  surface of brown adipocytes triggering the
  activation of adenylate cyclase
• Activated adenylate cyclase ? leads to increased
  production of cAMP and the concomitant activation
  of cAMP-dependent protein kinase (PKA) ? result
  being phosphorylation and activation of
  hormone-sensitive lipase
• released free fatty acids bind to thermogenin ?
  triggering an uncoupling of the proton gradient
  and the release of the energy of the gradient as
  heat

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Other Biological Oxidations

• Oxidase complexes (e.g. cytochrome oxidase) ?
  transfer electrons directly from NADH and other
  substrates to oxygen, producing water
• Oxygenases ? widely localized in membranes of the
  endoplasmic reticulum ? catalyze the addition of
  molecular oxygen to organic molecules
• The chief components of oxygenase complexes ?
  include cytochrome b5, cytochrome P450 and
  cytochrome P450 reductase
• There are many P450 isozymes for example, up to
  50 different P450 gene products can be found in
  liver, where the bulk of drug metabolism occurs

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• Enzymatic reactions involving molecular oxygen ?
  usually produce water or organic oxygen in well
  regulated reactions having specific products
• under some metabolic conditions (e.g.,
  reperfusion of anaerobic tissues) ? unpaired
  electrons gain access to molecular oxygen in
  unregulated, non-enzymatic reactions
• The products free radicals (quite toxic)
• free radicals, especially hydroxy radical ?
  attack all cell components, including proteins,
  lipids and nucleic acids, potentially causing
  extensive cellular damage
• Tissues are replete with enzymes to protect
  against the random chemical reactions that these
  free radicals initiate

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• Superoxide dismutases (SODs) in animals ? contain
  either zinc (Zn2) and copper (Cu2), known as
  CuZnSOD, or manganese (Mn2) as in the case of
  the mitochondrial form
• SODs ? convert superoxide to peroxide and thereby
  minimizes production of hydroxy radical (the most
  potent of the oxygen free radicals)
• Peroxides produced by SOD ? also toxic --.
  detoxified by conversion to water via the enzyme
  peroxidase
• best known mammalian peroxidase is glutathione
  peroxidase, which contains the modified amino
  acid selenocysteine

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• Glutathione ? important in maintaining the normal
  reduction potential of cells and provides the
  reducing equivalents for glutathione peroxidase
  to convert hydrogen peroxide to water
• In red blood cells ? the lack of glutathione
  leads to extensive peroxide attack on the plasma
  membrane, producing fragile red blood cells that
  readily undergo hemolysis.
• Catalase (located in peroxisomes) ? provides a
  reductant route for the degradation of hydrogen
  peroxide
• Mammalian catalase ? has the highest turnover
  number of any documented enzyme