SBS922 Membrane Biochemistry
Mitochondria and chloroplasts
John F. Allen
School of Biological and Chemical Sciences, Queen Mary, University of
London
1
http://jfa.bio.qmul.ac.uk/lectures/
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 Structural studies on membrane
David Blow Chair of Biophysics proteins
and Director of Centre for
Structural Biology Division of
Molecular Biosciences, Imperial
College London, and Diamond
Light Source, Rutherford
Appleton Laboratory, Chilton
28 February 2007 Professor COLIN ROBINSON Pathways for the targeting of
Department of Biological proteins across chloroplast and
Sciences, University of Warwick, bacterial membranes
Coventry
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.
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.
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.
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.
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.
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
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).
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.
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.
Mitchell’s three essential requirements
1) That the respiratory chain redox system (electron transfer)
translocates protons across the membrane in one direction
(anisotropic,direction-oriented) 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).
Only central part of Mitchell’s 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).
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.
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).
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.
Mitchell’s three essential requirements
1) That the respiratory chain redox system (electron transfer)
translocates protons across the membrane in one direction
(anisotropic,direction-oriented) 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).
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 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).
Only central part of Mitchell’s 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).
EVIDENCE FOR FIRST REQUIREMENT
1. That the respiratory chain redox system (electron
transfer) translocates protons across the membrane in
one direction (anisotropic,direction-oriented) 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.
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.
)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.
RESULTS
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
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.
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.
Driven by pH
Driven by
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+ < or = 0.5µmho cm-2
Or 0.2 nmol H+ min-1 mg protein-1 mV p-1
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"
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.
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.
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]
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]
OTHER EVIDENCE FOR MITCHELL’S CHEMIOSMOTIC
HYPOTHESIS