Mitochondria and chloroplasts

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SBS922 Membrane Biochemistry
Mitochondria and chloroplasts
John F. Allen School of Biological and Chemical
Sciences, Queen Mary, University of London
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http//jfa.bio.qmul.ac.uk/lectures/
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School of Biological and Chemical Sciences
Seminars 2006-07 WEDNESDAYS AT 12 NOON IN LECTURE
THEATRE G23, FOGG BUILDING, SCHOOL OF BIOLOGICAL
AND CHEMICAL SCIENCES
6 December 2006 PROFESSOR SO IWATA David Blow Chair of Biophysics and Director of Centre for Structural Biology Division of Molecular Biosciences, Imperial College London, and Diamond Light Source, Rutherford Appleton Laboratory, Chilton Structural studies on membrane proteins
28 February 2007 Professor COLIN ROBINSON Department of Biological Sciences, University of Warwick, Coventry Pathways for the targeting of proteins across chloroplast and bacterial membranes
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The membrane energised state We have seen how
observed characteristics of oxidative
phosphorylation led to conclusion that there was
a membrane energised state linking electron
transfer in mitochondria to ATP synthesis and
other membrane-linked energy-dependent functions
such as active transport of solutes.
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There was convincing evidence by 1960 that the
transfer of energy via this membrane energised
state between the respiratory electron transfer
chain, the synthesis of ATP and the various
solute transfer systems is both efficient and
fully reversible.
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Membrane Energised State (cont.) It was occurring
via a common (to all these activities) and stable
non-phosphorylated energised state. This
energised state was dissipated by uncoupling
agents (uncouplers), which led to a permanent
increase in the rate of electron transfer. The
energised state was not dissipated by
phosphorylation inhibitors, which caused a
decrease in the rate of electron transfer which
could be overcome by uncouplers.
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Thus respiration will drive ATP synthesis, ATP
hydrolysis will drive reversed electron transfer,
and both respiration and ATP hydrolysis will
drive solute transport. Similarly certain solute
gradients of the correct magnitude and direction
will power both ATP synthesis and reversed
electron transfer. It was clear therefore that
the energised state occupies a central position
in the mechanism of membrane-associated energy
transduction.
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This energised state was essentially linking
together two types of protein complex in coupling
membranes. a) electron transfer complexes b) ATP
synthase Three types of coupling membranes are
the IMM, thylakoid membrane and plasma membrane
of prokaryotes, and all used same energised
state. Search for the nature of the energised
state became one of central problems in
Biochemistry. Only precedent was the
phophorylated intermediate in substrate-level
phosphorylation, but energised state of coupling
membranes was non-phosphorylated, and an intact
membrane was required to couple electron transfer
to ATP synthesis.
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CHEMIOSMOTIC HYPOTHESIS   Proposed by Peter
Mitchell 1961 and further elaborated
1966   Competing hypotheses 1) Chemical
hypothesis (i.e. like substrate-level
phosphorylation) 2) Conformational hypothesis 3)
Localised proton hypothesis (variation of
chemiosmotic)   Peter Mitchell awarded Nobel
Prize in Chemistry in 1978.
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Mitchell proposed that electron transfer directly
produced an electrochemical gradient of protons
across the coupling membrane that was
subsequently used to drive ATP synthesis. The
theory was subsequently adapted and expanded,
principally by Mitchell and his colleague
Jennifer Moyle to account for other
membrane-linked energy dependent functions such
as the active transport of solutes across the
membrane. The chemiosmotic hypothesis is named
because it is postulated to involve both a)
chemical reactions, the transfer of chemical
groups (electrons,protons and O22-) within the
membrane b) osmotic reactions, the transport of a
solute (protons) across the membrane
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Transmembrane Proton gradient Energy transduction
occurs via a proton circuit which circulates
through the insulating coupling membrane and the
two adjacent bulk phases ( the matrix and the
cytosol/ intermembrane space in
mitochondria). Since each of the two bulk phases
is in equilibrium, energy storage is
transmembrane rather than intramembrane
(intramembrane proton gradients were the basis of
the localised proton hypothesis).
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This energy storage takes the form of a
delocalised electrochemical potential difference
of protons (??H), otherwise known as the
protonmotive force (p.m.f. or ?p). It is an
electrochemical gradient because it is composed
of both   A chemical potential difference ? pH
( pHout-pHin )   An electrical potential
difference or membrane potential ??  These
two contribute to ?p according to the following
relationship  ?p ?? - Z ? pH where Z
2.303RT F  R gas constant,
T absolute temperature in Kelvin, F Faraday  Z
constant approximately equal to 60 at 25oC and
serves to convert pH into electrical units, mV.
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ESSENTIAL REQUIREMENTS OF THE CHEMIOSMOTIC
HYPOTHESIS   MITHCHELL PROPOSED THREE ESSENTIAL
REQUIREMENTS THAT HAD TO BE VERIFIED
EXPERIMENTALLY BEFORE THE CHEMIOSMOTIC
HYPOTHESIS COULD BE ACCEPTED AS PROVEN.
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Mitchells three essential requirements 1) That
the respiratory chain redox system (electron
transfer) translocates protons across the
membrane in one direction (anisotropic,direction-o
riented) as electrons flow down the chain.  2)
The coupling membrane should be impermeable to
protons and other ions except via specific
exchange-diffusion systems which are involved in
active solute transport  3) That the ATP synthase
can transport protons across the membrane in one
direction down the concentration (? pH ) and
charge (?? ) gradient , using the energy for ATP
synthesis. Alternatively it should be able to use
the energy from ATP hydrolysis to pump protons in
the opposite direction (active transport against
the concentration and charge gradient).
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Only central part of Mitchells hypothesis
accepted, that an electrochemical gradient of
protons was both necessary and sufficient for ATP
synthesis linked to electron transfer. Specific
mechanisms he proposed for proton pumping were
only partially correct, and his chemiosmotic
mechanism for ATP synthase was wrong (this
actually involves conformational change in ATP
synthase caused by p.m.f., and was partly
elucidated as a result of part of the structure
of the ATP synthase being solved by John Walker
at Cambridge, who also received the Nobel Prize).

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Was convincing evidence by 1960 that the transfer
of energy via this membrane energised state
between the respiratory electron transfer chain,
the synthesis of ATP and the various solute
transfer systems is both efficient and fully
reversible.
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Transmembrane Proton gradient Energy transduction
occurs via a proton circuit which circulates
through the insulating coupling membrane and the
two adjacent bulk phases ( the matrix and the
cytosol/ intermembrane space in
mitochondria). Since each of the two bulk phases
is in equilibrium, energy storage is
transmembrane rather than intramembrane
(intramembrane proton gradients were the basis of
the localised proton hypothesis).
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This energy storage takes the form of a
delocalised electrochemical potential difference
of protons (??H), otherwise known as the
protonmotive force (p.m.f. or ?p). It is an
electrochemical gradient because it is composed
of both   A chemical potential difference ? pH
( pHout-pHin )   An electrical potential
difference or membrane potential ??  These
two contribute to ?p according to the following
relationship  ?p ?? - Z ? pH where Z
2.303RT F  R gas constant,
T absolute temperature in Kelvin, F Faraday  Z
constant approximately equal to 60 at 25oC and
serves to convert pH into electrical units, mV.
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Mitchells three essential requirements 1) That
the respiratory chain redox system (electron
transfer) translocates protons across the
membrane in one direction (anisotropic,direction-o
riented) as electrons flow down the chain.  2)
The coupling membrane should be impermeable to
protons and other ions except via specific
exchange-diffusion systems which are involved in
active solute transport  3) That the ATP synthase
can transport protons across the membrane in one
direction down the concentration (? pH ) and
charge (?? ) gradient , using the energy for ATP
synthesis. Alternatively it should be able to use
the energy from ATP hydrolysis to pump protons in
the opposite direction (active transport against
the concentration and charge gradient).
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ATP synthase can transport protons across the
membrane in one direction down the concentration
(? pH ) and charge (?? ) gradient , using the
energy for ATP synthesis.
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Alternatively the ATP synthase should be able to
use the energy from ATP hydrolysis to pump
protons in the opposite direction (active
transport against the concentration and charge
gradient).
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Only central part of Mitchells hypothesis
accepted, that an electrochemical gradient of
protons was both neccesary and sufficient for ATP
synthesis linked to electron transfer. Specific
mechanisms he proposed for proton pumping were
only partially correct, and his chemiosmotic
mechanism for ATP synthase was wrong (this
actually involves conformational change in ATP
synthase caused by p.m.f., and was partly
elucidated as a result of part of the structure
of the ATP synthase being solved by John Walker
at Cambridge, who also received the Nobel Prize).

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• EVIDENCE FOR FIRST REQUIREMENT
• That the respiratory chain redox system (electron
  transfer) translocates protons across the
  membrane in one direction (anisotropic,direction-o
  riented) as electrons flow down the chain. i.e
  "An anisotropic proton-translocating respiratory
  electron transfer chain"
• YOU CAN ONLY MEASURE THE INITIAL EJECTION OF
  PROTONS FROM MITOCHONDRIA AS ELECTRON TRANSFER
  STARTS, BEFORE THE RE-ENTRY OF PROTONS HAS BECOME
  ESTABLISHED. BY DEFINITION DURING STEADY STATE
  ELECTRON TRANSFER THE RATE AT WHICH PROTONS ARE
  PUMPED OUT OF MITOCHONDRIA EQUALS THE RATE OF
  THEIR RE-ENTRY.

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Precautions )incubate mitochondria anaerobically
so no electron transfer )in lightly-buffered
medium so pH/H changes can be observed )add
oligomycin to inhibit proton re-entry via the ATP
synthase )add valinomycin ( a potassium ionophore
) and a high concentration of KCl to abolish ??
and thus maximise ?pH.
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)add a small non-saturating pulse of oxygen to
start electron transfer and measure the extent of
proton extrusion into the medium surrounding the
mitochondria.
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RESULTS
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FACTORS CONTRIBUTING TO DECAY OF ?pH
Protons decay back in to mitochondria across IMM
because of 1 inherent proton permeability of IMM
note that FCCP accelerates the decay 2 action
of endogeneous Na/H antiport 3 electroneutral
PO4 entry
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EVIDENCE FOR SECOND REQUIREMENT   2) The coupling
membrane should be impermeable to protons and
other ions except via specific exchange-diffusion
systems which are involved in active solute
transport.   It can be deduced that the inner
mitochondrial membrane has a low effective proton
conductance by studying the action of uncouplers.
A majority of uncouplers act by increasing the
effective proton conductance of the coupling
membrane, dissipating the proton motive force and
thus breaking the link between electron transfer
and ATP synthesis. It is reasonable to assume
that the coupling membrane has a low proton
conductance in their absence.
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MECHANISM OF ACTION OF UNCOUPLERS (i.e.
FCCP) Uncouplers are lipophilic
(membrane-permeable) weak acids that can cross
the membrane in either the protonated or
deprotonated form. So they act as proton
translocators, catalysing a proton uniport across
the coupling membrane.
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Driven by ? pH
Driven by ??
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Subsequently Mithchell and Moyle measured the
effective proton conductance of the inner
mitochondrial membrane. This effective proton
conductance is one million times less than that
of the surrounding aqueous phases. i.e. CmH lt
or 0.5µmho cm-2 Or 0.2 nmol H min-1 mg
protein-1 mV ?p-1
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EVIDENCE FOR THIRD REQUIREMENT   3) That the ATP
synthase can transport protons across the
membrane in one direction down the concentration
(? pH ) and charge (?? ) gradient , using the
energy for ATP synthesis. Alternatively it should
be able to use the energy from ATP hydrolysis to
pump protons in the opposite direction (active
transport against the concentration and charge
gradient) i.e. "A reversible proton-translocating
ATP synthase"
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EVIDENCE FOR THIRD REQUIREMENT (CONT.) a)Mitchell
and Moyle showed that if you injected a small
amount of ATP into a suspension of anaerobic ( so
no electron transfer) mitochondria there was an
expulsion of protons as ATP was hydrolysed.
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b)In mitochondria need to impose a artificial ??
across the inner mitochondrial membrane, as
active solute transport uses up ?pH, so
mitochondria work on ?? for ATP synthesis.
Finally achieved in early 1970's.
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EVIDENCE FOR THIRD REQUIREMENT (CONT.) First
demonstration of artificial ?pH driving ATP
synthesis was in 1966 by Jagendorf and Uribe,
using thylakoid membranes of chloroplasts which
don't have solute transport and normally work on
high ?pH and low ??. see handout
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EVIDENCE FOR THIRD REQUIREMENT (CONT.) First
demonstration of artificial ?pH driving ATP
synthesis was in 1966 by Jagendorf and Uribe,
using thylakoid membranes of chloroplasts which
don't have solute transport and normally work on
high ?pH and low ??. see handout
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OTHER EVIDENCE FOR MITCHELLS CHEMIOSMOTIC
HYPOTHESIS
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