Reading: Berg,
Tymoczko and Stryer 5th edition, Chapter 18 pp. 507-523
The last two lectures focused
on events that take place in mitochondria. To clarify what we have been talking about,
here is a decent picture of a mitochondrion. The outer membrane is permeable to small molecules including
nucleotides, because it has the protein porin. The inner membrane is where all the electron transport
complexes are found. The internal
space is the matrix. Approximately
800 proteins that are coded by nuclear genes and made on cytoplasmic ribosomes
have to be imported across this double membrane system and then folded or
assembled in the mitochondrion.
There are a number of essential genes that are specific for the assembly
of electron transport complexes, but they do not exist in the final complex
structure. This would include the
gene(s) for iron sulfur cluster
assembly and scaffold proteins.
In April of 1997, a report
appeared (PNAS 94, 4526-4531 1997) that mutations in two different subunits of
cytochrome c oxidase COX1 and COX2 correlate with late onset Alzheimer's
disease. These are mitochondrially
encoded subunits. A defect in
cytochrome oxidase can reduce the level of ATP in a cell and produce unwanted
toxic oxygen metabolites like superoxide anion and hydroxyl ion. Brain tissue is especially dependent on
energy and is vulnerable to a mitochondrial defect.
OXIDATIVE PHOSPHORYLATION
Respiration is very tightly
coupled to synthesis of ATP in a process called oxidative phosphorylation. The synthesis of ATP uses the energy
stored in the proton and membrane potential gradient to drive a molecular motor
called the ATP synthase. This is
another protein complex located in the inner mitochondrial membrane, sometimes
called complex V, but more often called the F0F1 ATPase, named for the reverse
reaction that is easier to measure.
This protein has a lollipop structure in electron microscopic images.
The lollipop part is the F1 portion and it can be removed from the membranes by
treatment with urea. The F1 part
contains the catalytic subunits.
It has been crystallized and the structure solved. See fig. 18.27 p. 509). The FO part is membrane bound (the 0 in
FO stands for oligomycin,
an inhibitor that binds to this
part of the protein). It contains
the proton channel. Part of FO has been crystallized from the yeast ATP
synthase, and it contains the stalk and 10 c subunits, but subunits a and b are
missing. Science 286 1700-1705 Nov. 26 1999. A model of the whole structure is given in Fig. 18.27 p.
509.
When the F1 part of the ATPase
is removed from the membranes, the electron transport chain can still work and
oxygen can be reduced to water.
The oxidative phosphorylation (synthesis of ATP) cannot work, and the
ATP synthesis is said to be uncoupled from respiration. There are several ways that
mitochondria can become uncoupled.
The easiest and the most troublesome to people who isolate mitochondria,
is that their membranes can be leaky.
Not with big holes, but it does not take much to let protons through and
destroy the proton gradient.
Another way is the addition of a chemical uncoupler. These small molecules actually serve as
proton shuttles that carry a proton across the membrane down the gradient. They are protonated on the cytosolic
side where the pH is lower (H+ concentration is higher) Then they diffuse
across the membrane as uncharged species and release the proton to the matrix,
where the pH is higher. At this
point the uncoupler is charged and cannot go back across the membrane for
another proton, so these are one way proton carriers that accumulate in the
matrix. Chemical uncouplers
include CCCP, 2,4-dinitrophenol, FCCP (fig. 18.44 p. 519)
If the F1 part of the ATP
synthase is added back to the membranes where it was originally, the two parts
reassemble and ATP synthesis can once again be coupled to electron transport
and respiration. This is called a
reconstitution experiment.
Oxidative phosphorylation is an
unusual process, that depends on the integrity of the inner mitochondrial
membrane. Peter Mitchell, who
first proposed this mechanism, is highly regarded by bioenergeticists because
he had the insight to come up with this model which is called the chemiosmotic
hypothesis. He also proposed the Q
cycle as a means for pumping protons.
He predicted that the inner membrane would be impermeable to protons on
theoretical grounds and it is.
This permits the controlled leak of protons down the gradient through
the ATP synthase to make ATP. How
this is done is a very intensely studied area, and quite a bit is known. Because ATP synthesis is done
indirectly, oxidative phosphorylation is distinct from ATP synthesis seen in
glycolysis or GTP synthesis seen in the TCA cycle at succinyl CoA synthetase. Those direct syntheses of a high energy
bond are called substrate level phosphorylations to distinguish them from
oxidative phosphorylation.
One famous reconstitution
experiment provided evidence that the chemiosmotic hypothesis was correct. In this experiment performed by Efriam
Racker and Walther Stoeckenius, bacteriorhodopsin was reconstituted into
phospholipid vesicles with the FOF1 ATPase. When light was applied, the bacteriorhodopsin pumped protons
into the vesicles, since it is a light driven proton pump. Fortunately for the
experiment, the orientation of the pump could be prevented from being random
and there was net pumping into the vesicles not out from them. With this proton gradient, the ATP
synthase was able to make ATP.
This was an elegant experiment that showed there was no high energy
intermediate, or no specific interaction with the electron transport
complexes. Just a simple proton
gradient could power ATP synthesis. (see fig. 18.26 p. 508)
THE STRUCTURE OF ATP SYNTHASE
The whole molecule has a
dumbbell appearance. (fig. 18.27
p. 509). The membrane part and the
F1 part are connected by a 50 angstrom stalk. The number of subunits in the FO part varies depending on
the source, but there are only 3 FO subunits in E coli. They are called a, b and c and they
exist in the ratio 1:2:10-12. The
c subunit is very interesting, since it is small and exists in so many
copies. It has only two
transmembrane segments and one of these has a critical aspartate residue in the
middle of the helix. This
aspartate is required for the proton channel. A chemical called dicyclohexyl carbodiimide reacts with this
side chain and kills the enzyme.
In fact it is often called the DCCD binding protein. In an interesting experiment Robert
Fillingame mutated the E. coli subunit c aspartate residue to eliminate the
carboxyl group. He then selected
for regain of function mutants that could make ATP again. What he found was the negative charge
from the carboxyl reappeared in the protein structure, but it came back as a
glutamate on the other transmembrane helix at about the same depth into the
membrane. This carboxyl is
protonated at some point during the transport of protons through the channel
and this is required to make the protein work.
In yeast the three subunits
that are equivalent to the three E. coli FO subunits are all encoded in the
mitochondrial genome. This is just
another example of the mitochondrion keeping control of these very critical
proteins and not giving them up to the nuclear genome. Doug Wallace, an expert in
mitochondrial diseases, wrote in Scientific American that the symbiont (the
mitochondrion) ensures its own survival by keeping its fingers on the jugular
vein of cellular energy flow.
The FO part of the ATPase includes
about half of the long stalk. The
other half comes from the F1 particle.
In the stalk region, there is a protein called the oligomycin sensitivity conferring protein or
OSCP. OSCP is required for
sensitivity to oligomycin, an antibiotic inhibitor of the ATPase. However, oligomycin binds to the FO
part of the protein, not to the OSCP.
The other portions of the stalk are just being characterized.
The F1 particle has been
crystallized and the structure is known.
This work earned John Walker the Nobel Prize in Chemistry in 1997. The subunit composition of F1 is alpha
3, beta 3, gamma, delta, epsilon.
Alpha and beta alternate like 6 slices of an orange to make a ball. Only the beta subunits are active
catalytic subunits. The alpha
subunits have binding sites for nucleotide, but they are not catalytic. In the crystal structure, delta and
epsilon are not visible, but gamma has two long helices that run through the
center of the ball made by alpha and beta. This long helical gamma subunit sticks out the bottom of the
ball formed by alpha and beta to form part of the stalk. It is known that the gamma subunit
rotates relative to the F1 ball during ATP synthesis, so that gamma is like a
molecular axle and the central hole in F1 is like a molecular bearing. It is very important to this model that
the gamma subunit is not symmetrical.
It has significant differences in its interactions with the three
catalytic beta subunits, and this causes distortions or conformational changes
in the beta subunits. This is important as we will see below.
THE BINDING CHANGE MECHANISM
The model of how the ATP
synthase works is called the binding change mechanism. (figure 18.32 p.
511)This mechanism has three main tenets or postulates. Paul Boyer received the Nobel Prize in
Chemistry in 1997 for devising this model and the experiments to confirm it.
1. The first postulate is that
the energy of the proton gradient is not used to form ATP but to release ATP
from a very tight binding site where it forms spontaneously. The release of this tightly bound ATP
requires the energy of the proton gradient converted into the mechanical energy
of protein conformational changes probably brought about by interactions with
the gamma subunit and cooperative changes with the other alpha and beta
subunits.
2. The second postulate says
that the three catalytic sites are each in a unique conformation and the
conformations are interconvertible.
They represent three different stages of the catalytic cycle. One site L is loose, with ADP and Pi
bound. ATP forms without added
energy input. Afterwards the contacts with the newly formed ATP become very
strong. This tight conformation T
then needs the energy from the proton gradient to release the ATP. The third state is the open or O state
before nucleotide is bound.
3. The third postulate is that conformational changes at the
three sites are driven by rotation of the asymmetric gamma subunit relative to
the F1 ball.
EVIDENCE FOR THE THREE
POSTULATES.
The first tenet was shown to be
correct by O18 water exchange experiments. If the ATP synthase is incubated with ADP, Pi and O18 water,
Paul Boyer showed that O18 gets into free phosphate. The only way this can happen is by forming ATP and then
rehydrolyzing it. The O18
incorporation occurred without a proton gradient, therefore, ATP was formed
without energy input from the gradient.
ATP was not released unless the gradient was applied, so the energy is
needed to release the ATP not to form the ATP.
The crystal structure
immediately supported the second tenet, because the three catalytic sites were
each in a different conformation.
The third tenet, that the
protein rotates has now been proved by Richard Cross and others. He has made a mutant with cysteine
added into the beta subunits very near a naturally occurring cysteine in the
gamma subunit. If the cysteine
mutation is added to the beta subunit without oxidation, nothing happens and
ATP is made. If both cysteines are
present together and the disulfide is formed by oxidation, then no ATP can be
made. The protein is locked and
cannot turn. (Proc. Natl. Acad.
Sci USA 92, 10964-10968 1995).
This experiment was done with soluble F1. A similar experiment was done with membrane bound FOF1
ATPase to show the relevance to the membrane bound intact ATP synthase
(Biochim. Biophys Acta 1275, 96-100 1996).
There is an argument against
the rotation interpretation that says the conformational changes in alpha and
beta are large enough that a disulfide might prevent them even if the protein does
not rotate. In this kind of model
the ball sits in one place and beats like a heart.
To try and prove that the
protein actually rotates, a more sophisticated experiment has been
designed. The disulfide linked
ATPase has been dissociated and reconstituted with beta subunits that have the
cysteine mutation and epitope tags.
The F1 protein has been reconstituted with the FO part. The disulfide was reduced and the
protein was allowed to make ATP.
After a brief time the disulfide was reformed by oxidation and the
epitope tags
were used to detect the
location of beta-gamma cross linked subunits. If no rotation had occurred then the epitope tag should not
be covalently attached to gamma. The
original untagged subunit should reform the cross link. However if the F1 portion rotates, then
some of the epitope tagged beta should now be in a position to cross link with
gamma. This will be detectable on
SDS gels by an increase in the size of the epitope tagged protein. And that is what was seen. (Proc. Natl.
Acad. Sci USA 94, 10583-10587 1997). The same type of experiment was done with
cysteines added between epsilon and beta.
These proved that epsilon is part of the rotor, just like gamma. (J Biol
Chem. 1998 Nov 27;273, 31765-9).
Another crosslinking experiment from Fillingame's lab showed that
epsilon is in contact with the c subunit (J Biol Chem 1999 Jun
11;274(24):17011-6)
So there is evidence for
rotation. Richard Cross is now
doing another experiment to actually measure the rate of the rotation. He has engineered a tryptophan into the
protein near the site where the disulfide was. On the other subunit he has added a label that will quench
the tryptophan fluorescence when it is close. In this experiment, the fluorescence of the tryptophan
should vary with the rotation of the F1 part. The period of the fluctuation in fluorescence should be
equal to the period of rotation.
This experiment is being done now, but the results are not in yet. One problem with an experiment like
this is synchronization. It will
be necessary for all the ATPases to start at the same time, or there will only
be an average fluorescence. This
can be achieved by using caged ATP that can be released by a pulse of light, so
that all the ATPases will see the ATP at the same time.
In March of 1997, another very
simple and elegant experiment showed the rotation of the gamma subunit
directly. Hiroyuki Noji and
collaborators engineered ten histidines onto the N-terminal of the the ATPase beta
subunit and expressed these his-tagged proteins in bacteria. The F1 part of the ATPase was purified
and attached to a glass slide that was coated with nickel. The ATPase beta subunits in the F1 complex
bound to the slide through their his tags and immobilzed the F1 particle upside
down with the gamma subunit facing up.
The gamma subunit was mutated so it had only one cys residue near its
end. The cys was used as a site
for biotinylation and attachment of streptavidin. Finally, a biotinylated flourescent actin filament was added
to the complex and it bound to the streptavidin. Fluorescence of the actin
filament tagged to the gamma subunit was observed in a flourescence
microscope. When ATP was added to
the slide, the filaments spun around.
(figure 18.33 p. 511) It moved at about 4 cycles per second, slower than
expected for the free enzyme, (about 20 cycles per second) but it has a big
drag on it from the actin filament.
(Nature 386, 217-219 1997 news and views article, and 299-302 research
article.) A more recent experiment repeats this strategy with intact F1FO and
couples the actin fiber to the c subunits. The result is the same, showing that the c subunits rotate.
Science 286, 1722-1724 1999.
RECENT FINDINGS ABOUT THE
STRUCTURE OF THE ATP SYNTHASE
Most of the data we have talked
about concerns the F1 part of the ATPase.
What about the rest of the complex. Some additional experiments have been done in 1997 and 1998
that clarify the mechanism of the whole complex. Once again, disulfide bonds have been used to show
interaction of subunits and rotation or lack of rotation between subunits. Two of the subunits of the F1 particle
that we have not discussed are the delta and epsilon subunits. The question is do these subunits
rotate with gamma or stay fixed with alpha and beta in the ball structure. by introducing more site directed
cysteines at critical places, the motion or lack of motion of these subunits
has been probed. Experiments with
delta crosslinked to alpha show that no effect is found when the crosslink is
formed by oxidation (J. Biol. Chem. 272, 16652-16656 1997). Therefore delta is fixed relative to
alpha. It is argued that delta is
part of the stator or fixed part of the motor. For gamma to turn relative to the alpha and beta ball, some
part of the structure must connect the ball to the FO membrane structure,
otherwise the ball would spin too and no ATP would be made. Similar experiments with epsilon show
that linking epsilon to alpha kills the activity, so epsilon moves with gamma
in the interior of the ball structure.(J. Biol. Chem. 272, 19621-19624 1997; J.
Biol. Chem. 1998 Nov 27;273, 31765-9.)
The FO part of the enzyme is
also critical to function. Since
there are 10-12 c subunits in FO and only one a and two b subunits, it is
important to know which of these move and which don't. Experiments like the ones described
above using cys linkages have shown that subunit b binds to delta and is part
of the stator, while subunits c are rigidly fixed to gamma and are part of the
rotor. The single a subunit is also
part of the stator. Therefore, the
ball is fixed to the membrane subunit a through delta and b. This makes up the stator, while gamma
and epsilon are coupled with the 10-12 c subunits to make the rotor.
In November of 2000, two groups
reported the x-ray crystal structure of the stalk subunitsgamma, delta and
epsilon from bovine heart and the equivalent subunits from E. coli. (Nature Structural Biology 7, 1002
(commentary by Fillingame) 1051 (E. coli) 1055 (bovine heart). The structure identified an ARG (R75)
that probably interacts with an acidic sequence DELSEED in the beta subunit
with five aspartate and glutamate residues. This interaction may provide a catch mechanism to stabilize
the protein at 120o intervals as it turns. A foot for the stalk was also described that would bind to
the ring of c subunits and join them to the stalk, since they are both part of
the rotor. The only part of the
ATPase missing in the structures now is the a and b subunits that connect the
membrane part of the stator to the ball of F1. (please note that bovine delta is not the same as delta in
E. coli that anchors the b subunits of the stator to the alpha-beta ball. The bovine delta subunit is equivalent
to the E. coli epsilon subunit).
CONSIDERING THE ELECTRON TRANSPORT
CHAIN AND ATPase AS A WHOLE INPUTS AND OUTPUTS AND THE P/O RATIO
There are some aspects of the
electron transport chain and FOF1 ATPase that need to be looked at from the
perspective of a system. So far we
have examined each part as a unique structure. One property that depends on the whole chain is the P/O
ratio, or how many ATP molecules are produced per oxygen consumed. The traditional way to score this is
number of ATP produced per pair of electrons sent down the chain. Since it takes 4 electrons to reduce
molecular oxygen to two waters, the P/O ratio is really ATP produced/ atom of
oxygen. This number is dependent
on how many protons are pumped per site in the chain and how many are consumed
to make one ATP. The best
estimates are 4 protons per electron pair at complex I, 4 protons per electron
pair at complex III (2 for each turn of the Q cycle) and 2 protons per electron
pair at cytochrome oxidase (This amounts to 4 protons for complete reduction of
O2 to water).
The estimate for protons
consumed per ATP synthesized is 4.
There is an interesting theory about the ATPase that might explain this
number. It is called the elevator
model. Remember that the FO part
had 10-12 c subunits, each with a single DCCD binding aspartate. Single mutations in subunit a stop
transport, so subunit a probably forms part of the proton channel. The interesting fact is that mutation
of even 1 of the 10-12 subunits of c will also stop transport. This suggests that the a subunit
rotates relative to the c subunits to form a complete proton pathway. The elevator model suggests that protons
start their passage through the membrane in the a subunit. At some point they cannot continue
unless they move onto one of the c subunits which then carries the proton as
the ATPase rotor moves 1/12th (or 1/10th) of a full circle. This opens up the proton path in
subunit a once again for another proton to leave its c subunit and exit. By this model, the rotor would have to
turn 1/12th (or 1/10th) of a circle for every proton, or 10-12 protons could
pass through per revolution. We
already know that three ATPs are
made per revolution of the F1
relative to gamma, so that gives 3/12 = 1 ATP per 4 protons. If the number of c subunits were
variable, then there might be a variable stoichiometry. If there were 10 c subunits the math
would be 3.3 protons per ATP, It does not have to be an integer number if the
number of c subunits is variable.
This elevator model is a
speculative model that is not supported with hard evidence like the disulfide
experiments.
Back to the P/O ratio. For electron transport from NADH to O2,
there are 10 protons pumped at 4 protons/ATP = 2.5 ATP/NADH. For transport starting with FADH2, only
6 protons are pumped at 4/ATP = 1.5 ATP/FADH2. There is a way to add electrons directly to cytochrome c and
bypass complex III. Ascorbic acid
can reduce a compound called tetramethyl-para-phenylenediamine (TMPD) which can
reduce cytochrome c. If this
method is used, 2 protons are pumped per pair of electrons (2 cytochrome cs)
for a ratio of 2/4 = 0.5 ATP/pair of electrons.
By using different substrates
and inhibitors, it is possible to tell where a complex lies in the electron
transport pathway. (see fig. 18.43
p. 519) In practice, NADH and FADH2 are not added in experiments. Succinate can be added that enters the
TCA cycle at complex II and makes FADH2.
This bypasses complex I. beta-hydroxybutyrate is oxidized by
beta-hydroxybutyrate dehydrogenase to form NADH that feeds into the pathway at
complex I. You should know where
these substrates enter the chain.
If I told you a new complex had
been isolated from chili peppers that accepted electrons from some exotic
compound X in chilis, you should be able to interpret the results of adding in
inhibitors and substrates along with P/O ratios to place the complex in the
electron transport chain. If
compound X is added and you measure a P/O ratio of 1.5 that is completely
blocked by antimycin, but not affected by rotenone, you should be able to tell
that this complex is like complex II and it feeds in electrons upstream of
complex III, but does not interact with complex I. If you add compound X and the P/O ratio is 2.5 and blocked
by all known inhibitors, then it must be upstream of complex I and probably is
a compound X dehydrogenase that produces NADH.
The inputs and outputs of
glucose oxidation (Table 18.4 p.
518) depend on the method used to transport reducing equivalents from the
cytosol into the mitochondrion. We
already talked about the glycerophosphate shuttle, with two forms of
glycerophosphate dehydrogenase, one membrane bound and the other
cytosolic. These enzymes feed
electrons from NADH into the electron transport chain at ubiquinone, so some of
the available energy was lost.
There is another way to get NADH reducing equivalents across the inner
membrane that keeps the electrons entering the path at the level of NADH. This is called the malate-aspartate
shuttle.
This shuttle is illustrated in
fig 18.38 p. 515. To get the NADH reducing equivalents across the membrane,
they are first transferred to oxaloacetate to form malate by the malate
dehydrogenase in the cytosol.
Malate moves into the mitochondrion on a carrier that exchanges malate
for alpha-ketoglutarate. Once
inside, the mitochondrial malate dehydrogenase oxidizes the malate back to
oxaloacetate and NADH is formed.
This gets the NADH inside the mitochondrion, but it does not complete
the shuttle. So far oxaloacetate
has moved in and alpha-ketoglutarate has moved out. We need to restore these to the original side of the
membrane where they started. This
is done by aspartate amino transferase that converts oxaloacetate in the matrix
to aspartate and glutamate to alpha-ketoglutarate. A second carrier now exchanges the aspartate for
glutamate. Now everything is
correct on the matrix side. On the
cytosolic side aspartate transaminates alpha-ketoglutarate to glutamate and
reforms oxaloacetate. All is back
to normal now except that NADH has moved inside the mitochondrion.
Notice this is an example of
the same enzymes existing in two different compartments. This is fairly common.
This malate-aspartate shuttle
illustrates another common feature of mitochondria. These are the carriers. In yeast, there are 35 genes for mitochondrial carriers,
some of them are redundant, but there seem to be at least 28 different
functions performed by these carriers.
In humans there is sequence evidence for at least 46 different genes.
Two very important carriers in
mitochondria are the phosphate carrier that brings in phosphate for ATP
synthesis in exchange for hydroxide ion, and the ADP/ATP carrier that exchanges
ADP from the cytosol for ATP freshly made in the matrix. In a resting human, the ATP turnover
amounts to about 80 pounds of ATP per day, and almost all of this goes through
the ADP/ATP carrier twice per cycle, once going in and once going out. So approximately your body weight in
adenine nucleotide passes through this protein every day. The direction of transport is affected
by the electrical membrane potential which is inside negative. ADP and ATP move through this carrier
without complexed magnesium. The
movement is electrogenic, because ATP is -4 and ADP is -3. This favors movement of ATP out and ADP
in (the most negative species goes to the positive side of the membrane).
Another member of this family
is the uncoupler protein, also called thermogenin because it generates
heat. This protein transports
fatty acid anions across the inner membrane from the matrix in exchange for a
counter ion like chloride or hydroxyl ion. Once the fatty acids are released on the outer positive side
of the membrane they become protonated and flip flop back across the membrane
as non-charged species. On the
matrix side they deprotonate and become charged again. This is like an uncoupler, except the
fatty acids do not have to accumulate at the matrix side since the uncoupler protein
can transport them out again. This
process uncouples oxidative phosphorylation and wastes the proton gradient as heat. It is advantageous to be able to
generate heat in special situations and this protein only occurs in brown fat
adipose tissue where it is hormonally regulated. It is activated by free fatty acids that are produced by
lipolysis in response to hormones.
Ubiquinone is a required cofactor of uncoupling protein. The protein is
important in newborn mammals and hibernating animals when they need to wake
up. It is a new evolutionary
invention, because it only exists in mammals. In March of 1997 a new UCP2 protein was found in humans and
everyone got very excited, because it was in many tissues, not just brown fat. Adult humans have no brown fat, so UCP1
was pretty useless to adults. UCP2
and now UCP3 can be turned on in adults as a way to regulate weight by wasting
energy through uncoupling oxidative phosphorylation. The search is now on to find drugs that will activate UCP2
or UCP3.
One final consideration about
mitochondria and respiration/oxidative phosphorylation is what are the
consequences of a tightly coupled system.
Electron flow is linked to ATP synthesis. If electrons do not flow no ATP can be made. That is obvious from all we have
seen so far. But the opposite is also true if no ATP
can be made then electrons cannot flow because of the tight coupling. This is not so obvious. If substrate is present, like succinate,
but no ADP or Pi is present, the ATPase cannot make ATP, but one would still expect
that electrons could flow down the chain to water. However they do not.
Why? The reason they don't
is the proton gradient. When electrons
flow, protons are pumped against an existing proton gradient. Once this gradient is large enough, the
energy released by electron movements to higher reduction potential is just equal
to the force applied by the gradient and no more protons can be pumped. If the gradient is relaxed by synthesis
of ATP, then more electrons can flow.
If the gradient is relaxed by an uncoupler then the electrons can
flow. Otherwise a steady state is
reached and electron flow ceases.
This is illustrated in the
following oxygen electrode traces.
When mitochondria are incubated with succinate as an electron donor, and
ADP and Pi are present, we have rapid oxygen consumption. This is called state 3
respiration. When the ADP runs
out, the slope drops back to a much lower level called state 4
respiration. (see fig. 18.42 p.
518) This is not a zero slope, because there is always some turnover of ATP in
the matrix and the membranes are always a little bit leaky to protons. The ratio of state 3 to state 4
respiration is called the respiratory control ratio and it should be about 5-6
in good preps of isolated mitochondria.
Addition of an uncoupler produces the same state 3 level of respiration. The mitochondria show a different
appearance in electron microscope images when they are in state 3 respiration versus
state 4. The actively respiring
mitochondria have a large periplasmic space and the matrix appears
condensed. In state 4 they look
more typical with very little periplasmic space.