Oxidative phosphorylation

1
Oxidative phosphorylation
2
Oxidative phosphorylation

• Oxidative phosphorylation
• Process by which ATP formed as electrons
  transferred from NADH and FADH2 to O2 by series
  of electron carriers
• Carried out by enzymes and electron carriers
  located within inner mitochondrial membrane
• Electron transport chain/respiratory chain

3
Oxidative phosphorylation

• Electrons transferred from NADH and FADH to O2
  via series of electron carriers
• Transfer of electrons to three carriers
• NADH dehydrogenase
• Cytochrome C reductase
• Cytochrome C oxidase
• releases sufficient energy to pump protons across
  inner-membrane into inter-membrane space
• NADH enters chain at NADH dehydrogenase
• 2.5 - 3 ATP
• FADH2 enters chain at Coenzyme Q
• 1.5 - 2 ATP

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.526.
4
Oxidative phosphorylation

• Cytochrome C oxidase combines electrons with
  oxygen and 2H to form water
• Oxygen final electron acceptor

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.526.
5
Oxidative phosphorylation

• Chemiosmotic hypothesis
• Electron transport linked to ATP synthesis
• Protons trapped in intermembrane space form
  electrochemical gradient
• Protons flow down gradient through ATP synthase
  complex
• phosphorylates ADP and Pi to form ATP (precise
  mechanism not known)

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.526.
6
NADH shuttles

• NADH produced in cytosol during glycolysis
• Mitochondrial membranes impermeable to NADH
• Reducing equivalents shuttled into mitochondria
  to ETC
• Two shuttles operate
• Glycerol phosphate shuttle
• Malate-aspartate shuttle

From Summerlin LR (1981) Chemistry for the Life
Sciences. New York Random House p 543.
7
Glycerol phosphate shuttle

• Operates to minor extent in variety of tissues,
  but very important in fast-twitch muscle fibres
• Transfers reducing equivalents held by cytosolic
  NADH to FAD in ETC
• Yields 1.5 - 2 ATP

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.519.
8
Malate-aspartate shuttle

• Dominant shuttle in liver, heart, and slow-twitch
  muscle fibres
• Transfers reducing equivalents held by cytosolic
  NADH to NAD in ETC
• Yields 2.5 - 3 ATP

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.519.
9
Mitochondrial ATP transport

• Most ATP synthesised in mitochondria but
  hydrolysed in cytosol
• ADP-ATP translocase and Pi-OH- translocase
  located in inner mitochondrial membrane
• Antiporter systems

From Houston ME (2001) Biochemistry Primer for
Exercise Science. Champaign Human Kinetics. p70
10
Mitochondrial ATP transport

• Charge difference between ATP4- and ADP3-
  provides driving force for translocation
• ATP moves from more -ve matrix to more ve
  intermembrane space
• ADP moves in opposite direction
• Reduces charge gradient across inner membrane by
  1
• Does not affect proton concentration gradient, so
  will not affect ATP production

From Houston ME (2001) Biochemistry Primer for
Exercise Science. Champaign Human Kinetics. p70
11
Regulation of oxidative phosphorylation

• ADP, Pi, NADH, and O2 are all substrates for
  oxidative phosphorylation
• All must be available for process to proceed

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.526.
12
Regulation of oxidative phosphorylation at rest

• At rest
• O2 readily available
• Sufficient NADH and Pi usually present
• ADP concentration low
• ADP limiting rate of Oxidative Phosphorylation
• Addition of ADP in well oxygenated isolated
  mitochondria will increase oxygen consumption
  (indirect measure of oxidative phosphorylation)

13
Regulation of oxidative phosphorylation during
exercise

• During exercise changes in individual substrate
  concentrations do not correspond to changes in
  oxidative phosphorylation
• Regulation based on combination of factors
• Cytoplasmic phosphorylation potential ATP /
  ADP x Pi
• Mitochondrial redox state NADH / NAD
• Cellular O2 content

14
Cytoplasmic phosphorylation potential

• Cytoplasmic phosphorylation potential ATP /
  ADP x Pi decreases with increasing exercise
  intensity
• ATP concentration decreases little
• Increase in ADP parallels decrease in ATP
• Pi increases a lot due to hydrolysis of PCr to
  buffer decrease in ATP
• Cytoplasmic phosphorylation potential varies more
  than any of the individual constituents

15
Mitochondrial redox state

• Mitochondrial redox state NADH / NAD changes
  when rate of electron transfer from NADH is not
  matched by rate of formation of NADH by
  dehydrogenase enzymes
• increases with increasing exercise intensity

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Mitochondrial oxygen availability

• Availability of O2 in mitochondria depends on gas
  exchange, blood flow and diffusion
• Oxygen availability usually only limiting during
  isometric exercise or at altitude
• Long transportation route for O2 primarily
  responsible for slow onset of VO2 kinetics
  relative to step increase in ATP hydrolysis (i.e.
  relative to O2 utilisation in mitochondria)

From Houston ME (2001) Biochemistry Primer for
Exercise Science. Champaign Human Kinetics. p76
17
Regulation of oxidative phosphorylation during
exercise

• Step increase in exercise intensity causes
  decrease in ATP / ADP x Pi
• Results in increased ADP Pi transport into
  mitochondria and increases electron transport
  from NADH to O2
• This also affects regulation of KC
• As NADH oxidized to NAD, inhibitory effect of
  NADH on KC control enzymes reduced
• Increased Ca2 also increases activity of PDH and
  KC control enzymes
• Net result is that KC speeds up

18
Regulation of oxidative phosphorylation during
exercise

• Mitochondrial oxygen tension will decline
• mismatch between O2 utilization by Cytochrome C
  oxidase and oxygen delivery to muscle fibre
• Oxidative phosphorylation will be maintained by
  decrease in cytosolic phosphorylation potential
  and increase in mitochondrial redox state despite
  declining oxygen availability.
• Multisubstrate reactions

19
Regulation of oxidative phosphorylation during
exercise

• Any mismatch between ATP demand and ATP supplied
  by oxidative phosphorylation must be provided by
  PCr and glycolysis
• Increases in cytoplasmic phosphorylation
  potential (such as during moderate - heavy
  exercise) stimulate PFK and accellerate
  glycolysis