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MATTHEW VANDER HEIDEN: Last time, we
discussed standard reduction potential, this E0 prime value,
that really is a way to quantitate
the propensity of a molecule to accept or donate electrons.
And this is useful to think about how energy is transduced
in biological systems.
Because remember, as I have now tried to stress many times,
how energy is moved through biology
is largely about oxidation and reduction reactions
involving carbon and ultimately oxygen.
And so remember that oxidation of carbon is favorable.
That is removing electrons from the carbon
and giving them to oxygen, exactly what we
described for burning wood or burning gasoline.
And so that's why coming from the most reduced
carbon like fatty acids or other reduced carbon
like carbohydrates, not as reduced
as fatty acids, that oxidizing that carbon releases energy.
And of course, it means that storing carbon
as reduced carbon either as carbohydrates or fatty acids
is a way to store energy for later.
And so moving from left to right, equilibrium strongly
favors moving to the right.
That means that delta G is less than 0
for the oxidation of carbon.
Or we can describe this as moving from a smaller
standard reduction potential to a larger standard reduction
potential because, remember, the relationship between the change
in standard reduction potential is related
to the equilibrium constant or delta G0 prime,
which is also related to the equilibrium
constant by the above formula.
And so we closed last time by saying that with this formula
we know that, if we move from a smaller standard reduction
potential-- that is donate electrons from a smaller
standard reduction potential to a larger standard reduction
potential-- that that number, that difference,
will then be positive, which is the opposite of delta G
because of that formula.
And so a positive change in standard reduction potential,
moving from lower to higher, equilibrium
is going to favor that direction--
should make sense from everything we discussed before.
And of course, that change tells you
the propensity of electron transfer
that ultimately is going to be favorable at equilibrium.
However, exactly as we discussed for delta G,
whether an exact reaction is favorable
is still going to be dependent on delta G.
And so the change in standard reduction potential
tells you about the equilibrium constant,
and so which direction is going to be
net favorable at equilibrium.
However, what actually happens is still
going to depend on conditions for all the reasons
we talked about previously.
Still, this change in standard reduction potential
is still a useful concept to think about
because it tells us about how energy transfer can
occur across redox pairs.
And that will be useful in thinking
about this process of oxidative phosphorylation
and how cells really couple electron transfer to oxygen
as a way to do lots of useful things
in cells, including making ATP.
Now, one point I want to come back to again,
which we discussed at length when we talked about changes
in free energy and how that worked for pathways,
is that, remember, we discussed that, if we start with wood
or oil and all in one step burn it to CO2,
it releases a certain amount of energy.
Or we can break that into individual steps,
as we did in glycolysis.
The same amount of energy is released.
But by breaking into individual steps,
we can now capture that energy as intermediates
such that we can, say, favorably synthesize ATP
at a high ATP/ADP ratio in cells.
The exact same thing is true about any of these electron
transfers.
There is the change in standard reduction potential.
Or the change in free energy as one moves from electron
transfer from one acceptor to one donor
is the same if you go in one step
or in many different steps.
And so by doing these stepwise electron transfers
in exactly the same way we discussed about for glycolysis,
we can capture that energy release in smaller packets.
And this is a way to basically better harness this energy
release to do useful things for cells.
Ultimately, you'll see it's how you make ATP,
but it can do other work as well,
generate heat, move ions, et cetera.
And so this is really what I want to discuss today.
But it's important to remember these concepts when we think
through how these systems work.
And so we've now spent several lectures
discussing the chemical details of how we can oxidize.
First, we discussed carbohydrates.
Then we discussed fatty acids.
And what we saw is that many of the electrons that
are lost from the carbon oxidation
are used to generate molecules such as NADH or FADH2
and effectively charge up a ratio, if you will, of
reduce oxidized cofactors.
And these molecules then donate those electrons, ultimately,
to oxygen. And it's that, how you couple those electron
transfers from these carriers to oxygen,
ultimately is what oxidative phosphorylation is
about as a way to allow cells to convert this energy released
into a biologically useful form.
Now, effectively, the way biology
does this is by using that favorable electron transfer
to oxygen to charge a battery.
So really oxidative phosphorylation, if you will,
is about a biological battery.
And it's really the same concepts
that you may have learned in other courses
about how chemical batteries work.
And so let's just think about that.
What is a battery?
Well, a battery is two different solutions of ions usually
that is separated into two compartments.
It has a unit of volts.
That's exactly what the units are
for this change in standard reduction potential is volts.
By using that voltage to run a current between the two
compartments, you can do work.
And if you short circuit a battery, of course,
you can get some heat.
And so how are these really made?
Well, you basically just need to get that voltage.
You need to have something that insulates between those two
compartments.
And you can then control electron flow
from one side of the circuit to the other.
And it's exactly the same issue that happens in biology,
except biology needs some insulator
between two compartments.
How do we create different compartments in cells?
Well, we create them with membranes.
And so an important function of membranes in cells,
other than things like separating cellular contents
from the outside world or breaking up
different organelles, is that cells actually
require a membrane.
They're essential for life because they
are what allows an insulation between two aqueous
compartments in a way that you can build
a biological battery that is really central to how life uses
energy as a way to do work.
And so life creates a voltage, which I'm going to call delta
psi, delta pH-- we'll come back to that in a little bit--
to basically couple favorable electron transfer from, say,
an electron carrier like NAD/NADH to oxygen,
so reoxidizing NADH to reduce oxygen to water.
That is moving from a lower to a higher standard reduction
potential that's favorable.
Delta G0 prime is negative.
Net transfer is favorable.
This can be coupled to doing some work that otherwise would
be unfavorable, in this case pumping protons
from one side of the membrane to another.
By pumping protons to one side of a membrane or the other,
you create a voltage membrane potential, often
abbreviated delta psi.
It's also a pH gradient, so delta H. The protons
have lower pH on one side of the membrane than the other.
And it's this charging of a battery at a membrane that
is what is captured by oxidative phosphorylation, what's
typically referred to as that process, as a way to make ATP.
But of course, this is a useful thing.
It's a battery.
And so it can also do other work for cells.
Now, I just want to say and really
stress that this is really critical, this process.
And it's really central to how energy works for cells.
You will see that, effectively, this
is the key steps in photosynthesis,
do exactly the same thing.
It's how mitochondria work.
It's also how bacteria work.
And really prokaryotes end up doing this at their plasma
membranes, whereas mitochondria and chloroplasts end up
doing this at intracellular membranes in eukaryotes.
But ultimately, it's the same idea
of charging this biological battery that really allows
cells to convert, do energy conversions
in a way that's useful for them to allow otherwise
unfavorable things to happen in cells.
Now, this works largely because you
have this insulating membrane system, which is,
of course, made of lipids.
Lipids are what separates the two compartments.
Remember, we discussed earlier that these lipid bilayers
are useful because they create a hydrophobic interface
between two aqueous compartments.
It's also useful because lipids are
poor conductors of electrons.
And so this really ends up being the insulating system
that makes this work.
Now, in the previous lecture, we discussed
a lot about the polar and non-polar properties
of phospholipids really allow them to assemble
into these membrane barriers.
And I just want to do a slight diversion
to revisit this in the context of how
this works for bioenergetics.
So remember, you have this lipid bilayer where
you separate two aqueous compartments
with this hydrophobic membrane.
In this membrane are these phospholipids,
where you have these polar head groups with two lipid tails.
You can look back to a former lecture where I drew
and we discussed what those phospholipids
are in great detail.
But it's really having that polar, non-polar interface
that allows you to form these bilayers that's
creating this separation between the aqueous compartments,
but also this insulator that allows a battery to be built
across the membrane.
Now, there are several consequences, though,
to this system that is worth thinking about at least
in terms of how lipids and membranes work.
Now, a consequence of this is that, if we're
going to create this system, now we
create a system where we have issues with transport
across from one compartment to another.
Now, this, of course, is an opportunity
for a layer of regulation because it also
allows then these conditions where
you could have different chemical conditions,
say, different ATP/ADP ratios, NAD/NADH ratios
in the different compartments, which, of course,
for all the thermodynamic reasons we've already discussed
can make things more or less favorable to occur
in different compartments in cells.
But it also means metabolism then
has to have ways to transport stuff across these membranes.
And we've discussed this a couple of times.
A few examples-- I talked briefly
about the pyruvate carrier.
That is, if glycolysis is happening
in this compartment in the cytosol
and we need to get the pyruvate inside the mitochondria
across a membrane for the pyruvate dehydrogenase
reaction, now you needed this pyruvate carrier molecule.
And it turns out that that transport process
is an opportunity for regulation,
but it still needs to occur.
We discussed the carnitine shuttle, right?
Fatty acid, we generate fatty acyl-CoAs
from breakdown of lipids in one side
in the cytosolic compartment.
We need to get those fatty acyl-CoAs into the mitochondria
where they can be oxidized by fatty acid oxidation.
And that required that carnitine shuttle system.
Now, ultimately, what mediates this transport is, of course,
proteins that can sit within this membrane bilayer.
And you can imagine you can have proteins
that span the membrane bilayer, such
that there's hydrophilic surfaces that
are on either face of the membrane and hydrophobic
residues in the middle with various ways
to make channels and other transport mechanisms to get
things from one side of the membrane to the other,
all right?
Or you could have complexes that float freely within the lipid
bilayer, such that there's a hydrophilic surface that
faces one compartment and a hydrophobic surface that faces
the inside of the membrane.
Now, having these complexes will become very important
for ways in which one can regulate processes that
occur either within the membrane or by moving things
across membranes, like generating moving protons
and generating a voltage, or even
move large things like proteins across the membrane.
But it has additional consequences because it now
means that the properties of the membrane
also matter somewhat as well.
And this brings us back to the other discussion
we had in the last lecture.
And that is how the various properties of the fatty acids
that exist within this hydrophobic membrane area also,
now, can really matter because they're
going to affect things about how leaky this
is as an insulator between the two compartments,
as well as how fluid the membrane is,
such that things like these proteins
can actually move within this hydrophobic barrier
between the two compartments.
In other words, the fatty acid composition, as well as
the lipid species themselves, that
is which phospholipid, other polar hydrophobic lipid
species analogous to phospholipids that form up
membranes, can change properties such as how leaky
the membrane is or how fluid the membrane is.
You can imagine, if you have a really rigid membrane surface,
you're not going to get any flow of materials.
It's not going to be very leaky, which might be great
if you want to build an insulator between the two
compartments.
But it's going to be very difficult to regulate transport
across that compartment.
And so nature has come up with a bunch of different solutions
for how to play with fatty acid and other lipid composition
to change the properties of membranes.
We don't have time to get into that in great detail
in this course, but I want you to appreciate
that these things exist.
But one thing I want to discuss is that often,
when you see phospholipids drawn like this,
they're drawn in this way where the polar head
group is a circle.
And then they'll draw the two lipids tails often
with a kink in one of the lipid tails.
That's because many times phospholipids, of the two lipid
tails, one is saturated.
And one has a monounsaturation in it.
And I just want to go back to the models here.
Remember, the monounsaturated, the double bond,
ends up being this cis double bond.
And so here's two fatty acids.
If you put these here in with the head groups on one side--
so this would be esterified to the glycerol, the phospholipid,
you end up getting this kink in the membrane, which
is much different than two straight chains.
You can imagine that within the membrane
itself this ends up basically not letting those lipid tails
pack quite so tightly in the membrane, which certainly
contributes to an ability to have space
to put other stuff in the membrane to allow
other processes to happen.
Now, recall that there's other ways
we can modulate the properties of fatty acids,
introduce more double bonds, chain length, et cetera.
And different organisms use different strategies
to deal with this.
And so plants, remember, have all kinds
of polyunsaturated fatty acids, just like we talked about
with plant oils being liquid at room temperature.
Having more polyunsaturated fatty acids in their lipid
tails is one way plants use to keep
their membranes more fluid.
Animals, remember, have more saturated fatty acids.
All right, those saturated fatty acids--
less double bonds, going to be higher melting
temperature, more likely to be solid at room temperature.
So that's not going to be very good for membrane fluidity.
And it turns out animals use a different strategy
to keep membrane fluidity that's not
polyunsaturated fatty acids.
And that is that they incorporate this molecule shown
here on the slide, which is cholesterol,
which you're well aware of.
And so animals use you sterols, like cholesterol,
as a way to keep their membranes more fluid.
And so that cholesterol that is present in animal products
is not there to give you heart attacks.
It's really there as a way animals use
to keep their membranes fluid.
And this is really, at heart, why
plants are cholesterol free foods and animals have
cholesterol.
And it's because animals use cholesterol as a way
to keep their membranes fluid, whereas plants
use a different strategy, polyunsaturated fatty acids.
All right, now, ultimately setting up
the system this way is important because it allows
for these flexible membranes that
can enable ion and other transport between compartments,
as well as hold a charge so that you can form this battery which
allows oxidative phosphorylation or other processes to occur
where you can charge this membrane
and ultimately use that voltage to do work,
like make ATP, all right?
Now, how this membrane charging occurs,
again, is really taking advantage of favorable electron
transfer across a series of redox pairs,
ultimately, to oxygen or another good electron acceptor.
And it's really that favorable electron transfer
that's being coupled to this pumping of proton ions
to charge a battery.
That's the oxidation part.
And then that battery doing work such as driving favorable ADP
to ATP conversion, the phosphorylation
part of oxidative phosphorylation,
is the type of work that you can then
do once you generate this chemical battery, although not
the only work you can do with that battery.
And it's why this is so much more
flexible than just making ATP.
All right, so ultimately this process
by coupling transfer of electrons
from favorable oxidation of carbon
to charge up these other electron carriers that
ultimately transfer to oxygen is a major way
that life uses nutrient oxidation to convert it
to biologically useful energy.
And this is where we get to this process of what is typically
referred to as oxidative phosphorylation, which
is a concept or a process that was often abbreviated oxphos.
And it was discovered by or first described by a gentleman
by the name of Peter Mitchell.
And like many novel or revolutionary ideas,
when Peter Mitchell first described or proposed
the processes I'm about to tell you about,
his ideas were initially dismissed.
It was thought at the time, when people were thinking about how
do you couple metabolism to, say, charge up your ATP/ADP
ratio, that that process would work
via the oxidative phosphorylation type reactions
that we otherwise described, things
like what happens at the GAPDH step or the succinic thiokinase
step in the TCA cycle.
Those are two examples where we could directly
couple the oxidation of carbon to a phosphorylation event that
ultimately allowed us to favorably produce
ATP to maintain an ATP/ADP ratio that's high
and is present in cells.
However, what Peter Mitchell described
was really this battery phenomena
that we're talking about.
And it turned out to be largely right
and represents something that's actually
quite unique about biological processes
even if you think about other biological processes that
occur.
And that is what he proposed was the oxidation
and phosphorylation events were actually separate processes.
That is nature does not have to have these two things touch
in order for them to work.
That is that you use oxidation to create
this battery as one event.
So oxidation generates this potential across a membrane.
And then that potential across a membrane
is a totally separate event, can drive phosphorylation, ATP,
or do other work, ultimately making
this a more flexible system for biology.
And so turns out, as I said before, in a prokaryotic cell--
so this here is some kind of prokaryote, OK?
Prokaryotes have a membrane that separates them
from the outside world.
This cell membrane effectively couples that oxidation step
to the pumping of protons.
That creates a battery at the cell membrane
of the prokaryote.
And separate in space, that can then
be used to drive ATP synthesis or do
other work that's useful to maintain that prokaryotic cell.
Now, in eukaryotes, this process occurs at membranes
within the mitochondria.
So this here is a mitochondria, OK?
So just to remind you of some very basic cell biology--
and so, remember, mitochondria have two membranes.
So there's this outer mitochondrial membrane,
outer mitochondrial membrane.
And then there's this inner mitochondrial membrane,
so two membranes in the mitochondria.
Remember, there's this matrix space.
The cytosol would be out here.
And then there's this space between the two membranes
called the intermembrane space, intermembrane space
between the two membranes.
If you'll recall, many of these oxidation reactions, the TCA
cycle, fatty acid oxidation, pyruvate dehydrogenase step,
all of those were happening in the matrix of the mitochondria.
Remember, glycolysis was in the cytosol,
but all these other processes were
happening in the matrix of the mitochondria.
And really what's going on is that this battery is
being charged across this inner mitochondrial membrane, OK?
So protons are being pumped into the inner mitochondrial
membrane into the intermembrane space of the mitochondria
and then used to synthesize ATP in the matrix
of the mitochondria.
Now, part of this came, many people know-- remember,
mitochondria have their own DNA, et cetera.
And so really where people thought,
think eukaryotic cells came from is basically
the mitochondria is an enslaved bacteria or an obligate
captured symbiont, if you will, where one cell engulfed
a different prokaryotic cell.
And if you track this, think of the inner mitochondrial
membrane as a enslaved prokaryotic cell,
you can see that you're generating
this membrane potential across the inner mitochondrial
membrane, which is really equivalent
to the bacterial membrane of the enslaved prokaryotic cell.
In both cases, it's creating this battery
at a membrane, the inner mitochondrial membrane
or the bacterial membrane of a prokaryote, that is then
being used to separately in space
use that gradient to do work, such as synthesize ATP.
So if I draw this more explicitly as this here
being the membrane--
so the inner mitochondrial membrane
of a mitochondria or the bacterial membrane
of a prokaryotic cell, such that this
is outside the bacteria, that's inside the bacteria.
This is the intermembrane space often referred to
as the cytosol side.
Of course, there's another outer mitochondrial membrane here
that, for the sake of this class,
you can just think of as being generally permeable.
This would be the matrix inside of the inner mitochondrial
membrane.
And so all this NADH is being generated
from nutrient oxidation TCA cycle,
fatty acid oxidation, pyruvate dehydrogenase,
whatever, within the matrix or within the bacteria.
Those electrons are ultimately being transferred from NADH,
reoxidizing it to NAD, to reduce oxygen to water.
This is favorable.
This is, therefore, coupled to pumping protons.
That creates a membrane potential and gradient
across this membrane, which can be
used to do work such as driving the synthesis of ATP
for other work.
So this all has to happen at the same membrane,
but this process does not have to touch that process.
It's coupled because you create this battery,
this membrane potential and pH gradient, that
can be used elsewhere along the membrane
to do some kind of work.
Now, I draw this is delta psi, delta pH.
You can imagine, if you're moving protons,
you're going to create a voltage.
You're also going to create a pH gradient.
It turns out I don't have time to discuss
this further in the class.
A lot is written about how to think
about this membrane potential as either a true voltage
or as a pH gradient.
It turns out that they're useful to think
about in different ways for different processes.
But if you're interested in this,
there's certainly lots written about this on various papers
and texts on bioenergetics.
And I would encourage you to read more about that.
OK.
All right, what I want to do next
then is discuss each of these processes
in turn, that is how does this work
and how does that work, to really understand
what's going on in mitochondria and bacteria
to carry out this process of oxidative phosphorylation.
So first, let's talk here.
First, how does one use favorable electron transfer
to make this membrane potential?
Obviously, if we're going to pump protons
against a gradient, such that we have different concentration
of protons on either side of the membrane,
that's going to require energy input.
And that energy is going to come from favorable electron
transfer.
And really this process is taking electrons from, say,
a cofactor like NADH, and ultimately transferring
those electrons to oxygen to get water.
The standard reduction potential of NAD/NADH
is less than the standard reduction
potential of oxygen and water.
That means that equilibrium is going to favor electron
transport in this direction.
To say it another way, relative to oxygen-water,
NAD/NADH is a better electron donor.
Oxygen-water is a better electron acceptor pair.
And so by this electron transfer being favorable,
it can be used then to release energy
coupled to other processes, such as moving protons
and make that otherwise unfavorable process favorable.
Now, you'll note that, in doing this,
we now regenerate the oxidized cofactor NAD+.
And so that solves our electron balance problem for glycolysis,
solves any electron balance problem
that would exist for fatty acid oxidation or the TCA cycle
in the mitochondria.
Because ultimately, those electrons
are being given to oxygen. And so oxygen,
being this final electron acceptor,
being stoichiometrically produced to generate water,
is effectively water being the alternative waste product
to, say, lactate or ethanol that we
saw in fermentation, all right?
Now, as we started the lecture with and discussed
in glycolysis, if we do this in one step, it's favorable.
But it's also favorable if we do it in lots of little steps.
By doing it and lots of little steps,
just like we did in glycolysis, we
can break up that energy release in a way
to allow us to do more work, right?
So in glycolysis, we could turn carbohydrate into CO2--
one step, burning wood, release a lot of light and heat
all in one step.
That can be used to do work.
But it was more efficient to break it up into little steps
where we could then capture intermediates
that made ATP synthase favorable despite high ATP/ADP ratio.
Same idea here, we could transfer this all in one step.
But instead, you break it up into a bunch
of individual steps, which can then be coupled individually
to pumping protons across the membrane
to generate more efficiently this delta PSI delta pH
to charge this battery.
And so this occurs across a series of electron carriers,
multi-protein complexes, electron carriers,
called the electron transport chain.
And you can think of this electron transport chain
as being entirely analogous to what we already
talked about when we talked about how
pyruvate dehydrogenase or alpha-ketoglutarate
dehydrogenase works when we talked about the TCA cycle.
Remember, these had these different protein complexes,
this E1, E2, E3, where electrons were
transferred across this chain.
It's exactly the same idea here, a number
of multi-polypeptide encoded protein complexes
that come together to hold a bunch of electron carriers
that break up the energy release, if you will,
as we oxidize NADH and reduce oxygen and transfer
those electrons along the chain with the complexes themselves
coupling the favorable electron transfer to the moving
of protons against a gradient across the membrane to work.
And so for this electron transport chain to work,
we obviously need a bunch of electron carriers.
And why you need more than one electron carrier
should now be obvious.
Because having different carriers that
sit at different standard reduction
potentials is going to be useful if we
want to build a chain such that we can have favorable oxidation
and reduction to move electrons in a favorable direction
and couple that to processes that allow
us to charge this battery.
And so it turns out that for this electron transport
chain to work--
well, we know about some of our electron carriers.
So we've discussed NAD, NADH.
There's FAD, FADH2.
There was lipoic acid.
Lipoic acid is not part of the electron transport chain.
NAD, NADH is a donor.
You'll see FAD can be part of the electron transport chain,
but there's a different--
or there's additional electron carriers.
I want to discuss those now.
So another important one is something called
FMN or Flavin Mononucleotide.
What FMN, or Flavin Mononucleotide is, is it's
effectively FAD.
But it's a FAD not in a dinucleotide state,
but a mononucleotide state.
So if you take FAD and you remove an AMP group from it,
what you're left with is FMN.
So just to remind you to be explicit about this--
so.
So you have this flavin group attached to this ribitol group
with a phosphate on it.
If I put another phosphate and an adenine nucleotide here,
an AMP group attached here, that would be FAD.
This would be FMN, nucleotide instead of a dinucleotide.
This is FMN in the oxidized form.
And just like FAD, it can accept an electron pair.
So here's our hydride ion, two electrons
if we just draw here the middle part of the molecule.
And so this here would be FMNH2 or the reduced form
of this electron carrier, OK?
So flavin mononucleotide-- part of the electron transport
chain.
Now, note FMN, FAD, NAD, all of these things
are two electron carriers, right?
We disguise the mechanism of all of them,
just reminded you of it, these hydride ion
transfers, two electron carriers.
It turns out the electron transport chain also
uses metal ions as electron carriers.
And so-- lots of minerals in our diet, things like iron.
It turns out elemental iron is a great electron carrier.
And it's very important for electron transport chain.
Now, elemental metals are one electron carrier.
And so there's a few ways you incorporate iron.
One is something called iron sulfur clusters.
And so iron sulfur clusters is, within a protein,
you have some cysteine residues.
And those cysteine residues can coordinate an iron atom.
And so that iron atom can sit in the 3+ versus 2+ state,
oxidized, reduced.
So reduction of the iron--
oxidation of the iron, OK?
So one electron carrier--
move one electron that way.
Also, ion sits within molecules called cytochromes.
So what are cytochromes?
This is an iron atom that's basically
chelated into a porphyrin very similar to what
you saw for hemoglobin.
And so you have an iron 2+ or 3+ that's chelated here into this
porphyrin ring structure.
OK.
Lots of conjugated double bond systems in it,
this should look very similar.
Porphyrin is the thing on the outside, coordinates iron
atom in the middle.
That can move between the 2+ and the 3+ state just like we do
for iron sulfur clusters, so carry electrons be oxidized
or reduced, 3+ or 2+.
And ultimately, this porphyrin can
have some R groups added here, can be associated with it
or bound to cysteine residues within proteins.
Effectively, these molecules varying these R groups
and how it's attached to proteins
gives you different families of cytochrome is referred
to as cytochrome A, the B type cytochromes, and cytochrome C.
These are associated with enzyme complexes,
cytochrome C. It's actually covalently bound
to the polypeptide itself.
Basically, these are named because you
have these different conjugated ring structures here.
That and how the R groups are will slightly change.
So this will absorb visible light.
That will slightly change the absorption spectra.
And so classically, based on absorption spectra,
these were classified into A, B, and C type cytochromes.
And you can imagine, by these properties
having slightly different--
affecting the iron in ways that it slightly
affects its electron transport properties.
Of course, hemoglobin almost certainly
evolved from these molecules as oxygen carrying ways
in multicellular life.
But of course, the original use was as electron carriers
for the processes here that we're
going to talk about, to hold that iron as a way
to control electron transport across these electron transport
chains.
All right, so that's iron.
It turns out iron is not the only metal that
can be used to do this.
And so cells also use copper.
And so histidine can coordinate a copper that can move between
an oxidized and reduced state, so copper 2+ reduced to copper
+, copper + to copper 2+ or copper + oxidized to copper 2+,
another one electron carrier.
And so iron and copper can act as one electron carriers.
And so you have these two electron carriers.
Here's a bunch of metals as one electron carriers.
Obviously, you need ways to carry both one and two
electrons if you're going to move
electrons between these different carriers.
And an important molecule to do that is something
called coenzyme Q. Coenzyme Q--
sometimes referred to as ubiquinone ubiquinol,
because it's ubiquitous, often abbreviated co-Q. Many of you
have probably heard of the supplement
CoQ10, very popular supplement out there.
CoQ10 is basically a version of coenzyme Q,
ubiquinone, ubiquinol.
What this looks like is as follows.
So this here is ubiquinone, the oxidized form.
So this middle part is the quinone.
And then you have these decorations on the outside,
including this R group.
This R group is typically a long acyl tail.
If it's 10 carbons long, that's CoQ10 supplement.
Having this long acyl chain makes this
a very hydrophobic molecule.
And it actually lives within the membrane itself
as an electron carrier.
And so this ubiquinone can pick up an electron as well as
a proton.
Whoops.
And so by picking up one electron,
it can then basically go here to this semiquinone stabilized
free radical state and then pick up
a second electron to go to the fully reduced form.
ubiquinol.
OK.
So one electronic carrier and two electron carrier--
so basically, it can carry two electrons
from the oxidized, the reduced form,
but can transfer them as single electrons
by going through this stabilized free radical ubiquinone state.
And so, basically, these various electron carriers
are associated with these protein complexes
within this electron transport chain that ultimately works
to couple favorable electron transport down
the chain to pumping protons to generate this potential
across the membrane.
All right, so this process occurs, again,
at the bacterial membrane or the inner mitochondrial membrane
and, in animals and most organisms,
operates with four large multi-subunit protein
complexes, all right?
Each of these protein complexes has multiple polypeptides,
as I said.
It's coming together to form these complexes just like we
described for pH, for instance.
Most of the components of this are
encoded in the nuclear genome, but a subset of these
are encoded in the mitochondrial genome.
Now, what exactly is encoded in different mitochondrial genomes
varies by organisms.
But across organisms, it's basically
mostly complex components of these protein complexes.
And so this is why mitochondrial DNA is retained.
It's kind of this vestige of this symbiotic relationship.
The mitochondria have retained some of the ability
to control their own energy transduction, sort
of core components of these complexes.
There is one soluble protein.
So these complexes are embedded within the membrane.
There's also one soluble protein that's
associated with the membrane.
And then there's coenzyme Q, which also
floats within the membrane.
So that's in the membrane.
These are embedded in the membrane.
And then there's a soluble protein that's
associated with the membrane.
OK, now I want to discuss the details of what these protein
complexes are and also few comments about how they work.
So if this is the inner membrane space
and this is the matrix side, that'd
be out or in if we're discussing a prokaryote.
The first is we have something called complex I, also
referred to as NADH oxidase because it oxidizes NADH back
to NAD.
NADH oxidase is a giant complex greater than 900 kilodaltons
in total.
It has more than 25 individual protein subunits,
contains some flavin mononucleotides as well as
some iron sulfur clusters as electron carriers.
And it is the least well-understood
of all of the protein complexes.
In fact, it is still a major field of study
to try to understand how NAD oxidase really works.
But it effectively is one of the complexes that can pump protons
across the membrane as those two electrons are transferred
through the complex from NADH ultimately to coenzyme Q that
exists within the membrane.
OK, the next complex discuss is complex II.
Complex II is succinate dehydrogenase, the exact enzyme
that we heard about from the TCA cycle.
So what does succinate dehydrogenase do?
Remember, it converts fumarate to succinate.
It gave those electrons to FAD to make FADH2.
Well, that occurs within this complex,
which can also reoxidize the FADH2 with those two electrons
and give them, also, to coenzyme Q
that sits within the membrane.
So I want to point out that this, ultimately, you will see,
is why it is that electron transfer from NADH and FADH2
has different energy yields.
And so the electron transfer from NADH to ubiquinone
is more of a change in standard reduction potential
than from FADH2 to ubiquinone.
Why use FADH2?
Well, because, remember when we did electron transfers,
we used, when we oxidized a carbon, say,
in a carbohydrate to a ketone, we tended to use NAD+/NADH
as the electron transport pair.
So this change in standard reduction
potential is such that we can make that reaction work
and ultimately drive that.
It turns out that putting a double bond in--
this going from a not oxidizing an alcohol to a ketone,
but oxidizing the carbon to a double bond--
that basically you need a different change
in standard reduction potential to carry out that reaction
and make it work.
FADH/FADH2 sits at a higher standard reduction
potential than NAD/NADH.
And so that means to further transfer them
to something further downstream, like coenzyme Q,
there's a bigger change in standard reduction potential
here.
That can be used to drive protons.
There's not enough of a change here.
You can't use that to drive protons.
And it's because of these differences
in what's actually being oxidized that ultimately leads
to this difference in energetics when we think about FADH2
versus NADH and how much ATP equivalence
that they might have for cells.
All right, once electrons are in the ubiquinone pool
or coenzyme Q pool, they can now be transferred to complex III.
So complex III is also referred to as cytochrome C reductase.
You'll see why in a second.
It's a 250 kilodalton complex.
There are 10 subunits.
It has a bunch of B type and C type cytochromes,
as well as some iron sulfur clusters.
Shown here on the slide from a textbook
is a picture of complex III and what it actually looks like.
Fairly well-understood how it works,
this coupling of basically electron transfer through the Q
pool can pump protons.
And this ultimately allows transfer
of electrons to this small soluble protein that
lives in the intermembrane space or associated with the outside
of the bacteria called cytochrome C--
or outside in the intermembrane space called cytochrome C.
Cytochrome C is a 13 kilodalton protein.
It's a very small highly conserved protein
with a C type cytochrome commonly used
because it's found across many, many different types
of organisms.
And so it's been sequenced from many, many organisms
as a way to identify evolutionary relationships.
And then cytochrome C, this is also
why it's called cytochrome C reductase because it's
reducing cytochrome C to transfer electrons
along the chain, ultimately to the final complex, complex IV.
Complex IV-- also referred to as cytochrome C oxidase.
And this is ultimately the place where electrons are transferred
to reduce oxygen to water.
So this is 160 kilodalton protein complex
with 13 subunits, uses A type cytochromes and copper
as electron carriers and, ultimately,
allows you to get electrons from cytochrome C to oxygen.
And so here, again, from the textbook
is a picture of complex IV and what it looks like.
Also, fairly well-understood-- also couples electron transport
to proton pumping.
And so this is effectively the electron transport chain
as it exists in mitochondria.
And you can draw it a different way as I will do here.
So here's complex I, allows oxidation of NADH back to NAD+.
That oxidation can be coupled to reduction of FMN.
Ultimately, those are passed through some iron sulfur
clusters leading to the reduction of ubiquinone.
And in the process, this electron transport
can pump on the order of four protons,
or at least that's what's estimated.
Complex II, which is succinate dehydrogenase from the TCA
cycle, really converts succinate to fumarate.
That oxidation reaction within the complex
leads to the reduction of FAD to FADH2.
That can be reoxidized through iron,
ultimately driving the reduction of coenzyme Q
from ubiquinone to ubiquinol.
That ubiquinone to you ubiquinol in something
called the Q pool within the membrane
can then be reoxidized in complex III.
That reoxidized in complex III puts electrons
through complex III that can be coupled to pumping
on the order of two protons.
Those electrons are transferred to cytochrome C. Ultimately,
those electrons are transferred to copper in complex IV
to cytochrome A and, ultimately, oxygen to water,
reducing oxygen to water and pumping
on the order of four protons.
And so you can see here that, basically, this
is a series of oxidation and reduction reactions
that happens across these complexes, such that complex I,
the transfer electrons from the Q pool through complex III.
And complex IV can pump protons across the membrane
and generate this delta psi delta
pH that can be used to do work.
And it's really the favorable transfer of electrons
either from NAD/NADH or the oxidation of the succinate
to fumarate with those electrons being given to oxygen to drive
this process.
And so really just to schematize this, in a way, is that,
if this is our membrane, NAD+ to NADH gives electrons to complex
I, which goes to coenzyme Q, which goes to complex III,
which goes to cytochrome C, which goes to complex IV
and reduces oxygen to water.
That is one way to carry out this set of reactions.
The other way is we can convert fumarate--
sorry, succinate to fumarate.
That's the FAD.
That's complex II, also gives it to the Q pool.
And so you can see that the electron transport
chain would be complex I, Q, III C, IV or II Q, III, C IV.
It is not I, II, III, IV.
It is actually I to Q or II to Q, ultimately going to III.
Now, the way protons are pumped is a process
that I don't have time this year to discuss in class.
I will post something on Stellar that is just
a short section from the textbook that describes
at least what is known about how that process works,
such as the Q cycle, basically how oxidation
and reduction of coenzyme Q, which will take protons
on and off the molecule on either side of the membrane,
can be used to show how this can be coupled to proton
pumping across the membrane.
And you can read that if you're interested.
OK.
Now, a couple other points about this, this
works because you're moving from a lower
to a higher standard reduction potential across the chain.
And it turns out oxygen is a particularly good electron
acceptor.
And this is really why oxygen and respiration is really
so vital to a lot of the way energy transduction
works in metabolism.
Oxygen is now abundant.
And being a great electron acceptor is why it's used.
But of course, oxygen was not abundant
2 and 1/2 billion years ago.
And it doesn't have to be oxygen for this to work.
If you have another electron acceptor that
basically makes this process favorable,
that can work just as well.
And in fact, that happens still in some very extreme
environments.
This so-called chemosynthesis basically
can use other electronic acceptors
to drive the energetics of cells.
And you really just need the right ways
to build an electron transport chain to make this whole system
work, such you can build a battery and use it to do work,
OK?
So oxygen-- commonly used because it's abundant and good
at it, but it's not the only way that this can work.
And there are examples in biology
where oxygen is not used, that there is other acceptors that
are used instead.
Now, I also want to note, as I've alluded to in discussing
complex II, this FAD/FADH2.
So when we talk about that in the TCA cycle
or in fatty acid oxidation, we discuss it
as if it's kind of like NAD/NADH, but it's not.
NAD/NADH are basically cofactors that
move around between enzymes.
Fad/fadh2 are not free.
They're electron carriers that are part of protein complexes,
like what we described up here.
And they're part of enzymes like succinate dehydrogenase.
So complex II just happens to be an FAD/FADH2 containing enzyme
that's part of the TCA cycle and part of the electron transport
chain as a way to transfer electrons in the TCA cycle
into the Q pool via this succinate
to fumarate conversion.
Acyl-CoA dehydrogenase, the FAD/FADH2 enzyme
we discussed in fatty acid oxidation,
could easily be drawn instead in electron transport chain that
goes acyl-CoA dehydrogenase, coenzyme Q, III, C, IV,
just like we can say succinate to fumarate
is the same as complex II, Q, III, C, IV, OK?
And so there's really lots of alternative electron transport
chains that involve these FAD/FADH2 containing enzymes
that really sit in the membrane and directly transfer electrons
to the Q pool.
Ultimately, the goal of all of this
is, of course, to create this delta psi delta
pH, which can be used to charge this battery that then can then
be done to do work.
And so this work includes the phosphorylation of ATP.
And so if we charge this battery,
we can now use this as a way to couple current in this battery
to phosphorylate ADP to make ATP, the phosphorylation part
of oxidative phosphorylation.
Now, this occurs also by a large protein complex sometimes
referred to as complex V. But remember,
complex V, or this ATP synthase machinery,
is not really part of the electron transport chain.
It is separate in space and is basically something, a way,
to utilize this delta psi delta pH across the membrane
to drive ATP synthesis.
All right, how does this complex V or ATP synthesis work?
So this is also a large protein complex,
sits within the same membrane, of course,
because it has to utilize the membrane potential to function.
And the so-called complex V is also
referred to as the ATP synthase or the F0 F1 ATPase.
And basically, if this is the intermembrane space,
this is the inner mitochondrial membrane.
This is the matrix.
You have a protein complex that sits
within the membrane called F0.
It's actually FO.
The O stands for a Oligomycin because there's
a drug called oligomycin that inhibits the FO,
or F0, part of the molecule.
And then there's this F1 component
that is associated with the F0 component that
sits on the matrix side of the membrane.
And it is effectively the machine
that phosphorylates or interconverts
ATP and ADP on the matrix side of the membrane.
Now, it's pretty well-understood how this system works.
And basically, F1 works by a process
called rotational catalysis.
How this works was awarded the Nobel Prize in 1997,
figured out not all that long ago.
And basically, this is F1 is a large protein complex where
you have a gamma subunit that is attached
to F0 the couples, basically, proton pumping to this gamma
subunit to literally turn the F1 component
and change the confirmation of attached regions, parts,
of this F1 machine.
Because it turns out you can have
three different conformations of the rest of F1,
the so-called L, or Loose, conformation, the T,
or Tight, conformation, or the O, or Open, conformation.
And so in the open conformation, ADP and phosphate or ATP
can exchange in and out of this complex, all right?
In the loose confirmation, you basically
have ADP and phosphate bound.
And in the tight conformation, this
is favored to synthesize ATP.
And so you can envision that what actually happens here
is that you first have ADP and ATP
bind in this open conformation.
Protons are moved.
You get rotation around the gamma subunit.
That changes the conformation of the O subunit
to the loose subunit.
So now, you have ADP plus PI bound.
Another proton moves, changes the conformation
of the loose subunit into the tight confirmation.
Now, ATP synthase is favored.
Another proton moves, comes back to the open conformation.
ATP is released from the molecule,
and you can run another rotational catalysis cycle
again to ultimately drive ATP to ATP synthase using
this transfer of protons across the F0 subunit
to ultimately rotate this machine to synthesize ATP.
Now, the way F0 works is within the membrane--
and this is always hard to draw--
you basically have a central complex
with another set of complexes around it
that, within that, have two half-channels that
are open to either side of the membrane.
And so if you look at this from the top view,
so looking at it straight down, you'd
have this central piece with this external piece
with the two half-channels that basically can rotate around
the central stalk.
This is hooked up to the gamma subunit of F1
in which drives rotational catalysis.
And how this works is that attached
to this inner piece that is open to the half-channels
is an aspartate residue.
So this is an aspartic acid residue that's there.
And recall that aspartic acid, it's an acid.
So it can pick up a proton or release a proton
from its acid group depending on the pH
on either side of the membrane.
And so, remember, there's a different pH
on the two sides of the membrane.
And it's really that difference in pH
that will allow favorable turning around this center
stalk.
And so the pH in the intermembrane space is lower.
So that means you're more likely to favor
protonation of the aspartate.
It can then rotate, such as the channel, that aspartate, is now
exposed to the other channel, to the matrix side.
There, the pH is higher, so favors deprotonation.
And basically, this will drive motion of this piece
around the central stalk.
That being coupled to the gamma subunit of F1
will cause the rotation of the F1 subunit changing
the attached conformation of the rest of the complex to favor
ATP synthesis.
And it's really this is how moving of protons back
from the intermembrane space to the matrix
can drive the synthesis of ATP, the phosphorylation
part of this process.
And so now you have a full picture
of how carbon oxidation really releases energy in a way that's
coupled to do work in biology.
So we can, of course, couple this directly,
as we did at GAPDH or succinic thiokinase.
But mostly, it's captured to charge up these NAD/NADH ratios
where favorable electron transport can
be used to make this delta psi delta pH in a membrane that
can be used also to synthesize ATP despite a high ATP/ADP
ratio because that battery can be used to drive this machine.
Now, I want to point out that the ATP synthase is just
like any other enzyme in any pathway.
And that is, if conditions are right,
there's no reason that this machine
has to drive ATP synthesis.
It could also consume ATP as a way
to create a proton gradient.
That is use ATP hydrolysis to pump
protons in the opposite direction
into the intermembrane space.
Well, why would you ever do that?
Well, you would do that because having this battery
is useful because ATP is not the only way that cells
can use energy to do work.
It turns out that having a membrane potential
is useful for other things.
So if you're in course nine, you know
that action potentials are carried, basically electricity
along cells.
What is that?
That is basically changing membrane potentials
across cells.
More generally, membrane potentials
can be used to do lots of other types of work.
And so ATP is only one way that cells can use biological energy
because it can be coupled to otherwise
unfavorable reactions.
But you can also couple ion gradients
to unfavorable reactions and use that to do work--
and so lots of useful ways that delta psi delta
pH can be used by cells.
And so what are some of these?
Well, the first one is this can be used to generate heat.
So if you're checking your phone right now,
your phone is obviously a very useful device
to do all kinds of work for you.
You can read whatever you want on your phone,
use it to call people.
Well, what happens if you short circuit
the battery in your phone?
Well, it'll get really hot.
And the same thing happens here.
Rather than use this delta psi delta pH to generate ATP,
if I just put a hole in the membrane,
something referred to as an uncoupling protein, UCP,
Uncoupling Protein, this will just allow the leak of protons
back across the membrane.
Well, that's short circuiting the battery.
Energy has to be dissipated somewhere,
and it's dissipated as heat.
And this is effectively ways in which biology can create heat.
And so uncoupling proteins are expressed in a tissue called
brown fat in mammals.
Brown fat is a way to generate heat.
And it's one way that we maintain our body temperatures.
Now, the system is imperfect.
And lots of cells, all metabolism,
generates some heat.
If you've ever done composting, you
know your compost pile gets hot.
That's because the organisms there
that are metabolized in the material in the compost pile
are generating some heat.
And that's because membranes aren't perfect.
They're somewhat leaky.
Now, as humans, we work hard to maintain a constant body
temperature.
And there's actually a huge amount of energy
that goes into just maintaining our body temperature.
But you can imagine how efficiently
we couple these processes will determine how many calories we
need to maintain heat.
And that's actually a theory of weight control,
that those of us out there who have
less well-coupled mitochondria can
eat more because they waste more energy in generating
heat than those of us who are really efficient in doing this.
And in fact, there's drugs that allow
you to uncouple electron transport
generation of delta psi delta pH from utilizing
that membrane potential.
And these are great weight loss drugs.
Dinitrophenol was used middle part of the last century
as a weight loss agent.
It was very effective.
The problem is it's also incredibly dangerous
because people will die.
Because it turns out, if you uncouple this process
and you don't have enough ATP, you die really fast.
And so don't use that drug, but it illustrates
how you can use this battery to generate
heat, another form of energy.
Another big one is membrane transport.
So if I want to concentrate something-- and this
could be moving proteins.
Or it could be moving other ions,
like in an action potential.
Those are unfavorable processes, right?
I'm causing dissociation of ions across membranes
or moving protein from one side of a membrane to another.
One can couple those transfer processes to these delta psi
delta pH as well.
And in fact, protein import in the mitochondria
depends very much on a membrane potential
to get those proteins in.
The mitochondria is an important site
of calcium storage for cells.
Calcium is concentrated in the mitochondria.
And that concentration can be driven
by this membrane potential.
And of course, this potential can also
use to power lots of shuttles, like the carnitine shuttle,
and actually create situations where
you have different conditions across membranes as a way
to favor different chemistries.
And so the last thing I want to mention
is, in fact, these shuttles, which is just ways
that one can couple membrane potential to drive
other things, can also be really important for how
metabolism works.
And I just want to illustrate one aspect of this.
So if I draw here my mitochondria--
let me draw it up here.
If I draw a mitochondria, so here's
all these processes are happening here
where we have electron transport generating
a potential across the intermitochondrial membrane
that can be used to synthesize ATP.
Well, this synthesis is occurring in the mitochondria.
But ultimately, I need to get that ATP out
into the cytosol for it to be useful for the cell
to run otherwise unfavorable processes in the cytosol.
And so one needs a transporter for this.
There's some transporter called the adenine nucleotide
transporter.
And this partially takes advantage of the fact
that there's a charge difference between ATP and ADP
to actually couple membrane potential to exchange
of ATP/ADP between the cytosol and the mitochondrial matrix.
Now, there's other issues we'll talk about next time as well.
And so, remember, some metabolism
is happening in the cytosol.
So glycolysis turning glucose into pyruvate
is occurring in the cytosol.
We've already discussed.
That means you got to get pyruvate
in the mitochondria for the PDH reaction,
but this is also generating NADH in the cytosol.
And if that NADH needs to transfer its electrons
to oxygen, we also have to get that
NADH into the mitochondrial matrix or, more correctly,
transfer those electrons into the mitochondrial matrix.
It turns out cells maintain distinct NAD/NADH pools
in the mitochondria and the cytosol.
That's part of the conditions that
make different reactions favorable in each compartment.
And how cells do that, transfer those electrons
into the mitochondria, ends up being another important thing
that has to be transport that's driven across these membranes
where one can use delta psi delta pH.
And we'll talk about that at the start of the next lecture.
Thanks.