Coupling

1
Lecture 4

• Coupling

2
How Fuel made into ATP
WORK
ATP
ADP
H
ATP
ADP
Fuels
H
NAD
NADH
H
H
CO2
H2O
H/e
H/e
O2
H
H
3
Terms Principles

• Oxidative Phosphorylation
• Use of the proton gradient to make ATP
• Electron Transport
• Use of NADH to make proton gradient
• COUPLING
• Interdependence of Oxidative Phosphorylation and
  Electron Transport
• One generally cannot occur without the other
• RATE OF FUEL OXIDATON IS COUPLED TO RATE OF ATP
  CONSUMPTION
• Rate of ATP generation is very finely linked to
  the rate of ATP usage
• Cant burn fuels without doing work!

4
Important Rules I

• ATP is made when H come through the F0F1ATPase
• Note that ATP is made from ADP and P
• BUT H will ONLY come in through the F0F1ATPase
  IF ADP is available and is being made into ATP
• The movement of protons and the synthesis of ATP
  at the F0F1ATPase are all part of the same
  reaction and cant occur independently
• The proton gradient is made as electrons/hydrogens
  move down the electron transport chain from NADH
  to oxygen
• Note that the H that are pumped out come from
  the matrix and not necessarily the Hs on NADH
• Indeed, the one hydride on NADH causes the
  pumping of 10 H as it goes down the chain
• BUT electrons/hydrogens can ONLY move down the
  electron transport chain IF protons are
  simultaneously pumped out
• The movement of H/e- and the proton movement from
  the matrix to the cytoplasm are part of the same
  reaction and cant occur independently

5
Important Rules II

• The bigger the pressure of protons (ie, the
  bigger the size of the proton gradient), the
  harder it is to pump protons out of the matrix.
• So the bigger the gradient, the slower the
  movement of H/e down the chain from NADH to
  oxygen
• The carriers in the process are in limited supply
• NAD our initial carrier of Hs and electrons
  rips Hs/electrons out of fuels and necessary for
  continued fuel oxidation
• ADP our carrier of phosphate made into ATP
• Electron and Hydrogen carriers in the electron
  transport chain will alternately collect and
  pass-on the H/e to each other
• CoA a carrier involved in fatty acid oxidation
  and the full oxidation of glucose

6
No Work

• Imagine a cell is not doing any work
• Obviously this never actually happens, but its a
  good example of one extreme!
• If we do no work, we dont make ADP
• Remember ATP is quite stable and it will only
  break down if an enzyme tells it to
• If ATP is not used then no ADP is regenerated
• All the adenine nucleotides in the form of ATP
• ADP not available for new ATP synthesis
• H cannot pass through F0F1ATPase
• Proton gradient stays high
• Its not being dissipated
• H cannot be pumped out since the H gradient is
  so big
• H/e- wont be able to move down the chain
• Because the back pressure is so large
• If H/e- cannot move down the chain, NADH cannot
  pass its H/e to the ETC complexes
• So no regeneration of NAD all NAD stays as NADH
• And no oxygen consumption
• Since there are limited amount of NAD, if no NAD
  is regenerated, then no carriers will be
  available to pick up H from fuels ? so there will
  be no fuel oxidation

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Doing Work

• Now ATP will start to be used
• ADP gets made
• Now proton gradient can be dissipated
• Proton pressure relieved
• Pumping starts again
• Hs move down the chain to oxygen
• Oxygen consumption increases
• NADH now re-oxidised to NAD
• Availability of NAD allows fuel oxidation to
  occur again
• Rate of fuel oxidation is matched to the rate at
  which the ATP is used
• Rate of oxygen consumption a good indicator of
  the rate of fuel oxidation and energy expenditure

8
Uncoupling

• Imagine if the protons didnt have to go through
  the F0 channel to get back into the matrix
• If there was a leak/hole in the membrane
• Or if something carried the H across
• A short circuit!
• No driving force for ATP synthesis
• No back-pressure to stop H pumping
• No restriction on H/e movement down the transport
  chain to oxygen
• Instant regeneration of NAD from NADH
• Massive fuel oxidation rate
• Massive oxygen consumption
• But no ATP synthesis ?
• So ATP levels and cell death
• Unless it can be controlled!!!!!!

9
Dinitrophenol (DNP)

• Hydrophobic - can move freely across membrane
• H on OH group can come on/off easily
• Weak acid
• When H comes off, -ve charge can be delocalized
• Through nitro group
• Dissipates the H gradient
• Allows uncoupling of fuel oxidation from
  oxidative phosphorylation
• Energy in fuels all lost as heat
• Non-specific - affects every tissue

10
DNP mechanism

• In matrix, DNP loses H
• Because the pH in there is relatively high as
  many protons have be pumped out
• Outside mitochondria, DNP picks up H
• Because the pH in the cytoplasm is relatively low
• The DNP can shuttle across the membrane quite
  rapidly
• After H comes off from DNP in mitochondria, the
  negatively charged DNP leaves mitochondria and
  goes back out to pick up another H
• First documented as affecting munitions workers
• Massive weight loss and heat production
• Later used as weight loss agent

11
Natural Uncoupler - Thermogenin

• A.k.a. UCP-1 (Uncoupling protein-1)
• Found only in brown adipose tissue
• Its function is to generate heat
• Especially in small mammals and hibernating
  animals
• High in neonates, lost as we grow up
• But perhaps more of it than we think!
• Under hormonal control noradrenalin binds
  ?3-receptors on brown adipocytes to switch on
  thermogenin