[SQUEAKING][RUSTLING][CLICKING]
MATTHEW VANDER HEIDEN: Last time, we
discussed oxidative phosphorylation, which
is how to couple the NADH generated from sugar,
fatty acids, et cetera; the oxidation of carbon as a way
to carry out favorable electron transfer to oxygen
and use that energy release to charge a battery--
that is, create this delta psi/delta
pH that can subsequently be used by the battery to do work,
including the synthesis of ADP.
And of course, this can occur at a physiological ATP/ADP ratio
and, therefore, allow the cell to couple ATP
to ADP conversion to other unfavorable processes
in the cell.
Now, all of these reactions occur at the mitochondria.
And I drew, here, a schematic mitochondria for us
just to start.
Remember, the mitochondria has its own genome.
This is a vestige of the symbiotic relationship that
developed long, long ago, where two prokaryotes came together,
leading to this symbiotic relationship-- but of course,
the mitochondrial DNA encoding many
of the key components of this electron transport chain
and ATP synthesis machinery that remains in the mitochondria
today.
Now, ultimately, this process involves the reoxidation
of NADH back to NAD that occurs, that complex I of the electron
transport chain.
Those electrons are transferred into the Q pool, which
then go into complex III to cytochrome c to complex IV,
ultimately being transferred to oxygen as the final electron
acceptor.
And it's this process of favorable electron transport--
that is, electrons moving from a lower
to a higher standard reduction potential-- that
is coupled to the pumping of protons
and the generation of this delta psi/delta pH.
Now, we also discussed complex II of the electron transport
chain, which is identical to succinate dehydrogenase
in the TCA cycle.
This is involving that FAD/FADH2, which can, itself,
also transfer electrons to the Q pool,
ultimately passing those to complex III
to cytochrome c to complex IV, again,
with oxygen as electron acceptor,
ultimately forming what is essentially
two different variations on the electron transport
chain as a way to generate delta psi/delta pH.
Now I show here, on the slide, that there's really
many versions of this electron transport chain,
and another one we discussed is shown down here.
So up here is the typical one to regenerate NAD+ using complex I
to the Q pool to III to c to IV.
Here's the one involving complex II, so dehydrogenase reaction
via complex II to the Q pool, complex III, c, IV.
And then there's yet a third one here from fatty acid oxidation.
So this is Acyl-CoA dehydrogenase.
Again, like complex II, involves FAD/FADH2.
This occurs at the membrane, transfers
electrons to the Q pool to III to c to IV,
ultimately reducing oxygen to water.
And so the net effect of these various electron
transport chains were shown up here
is generating this delta psi/delta pH, which
can then be used to do work.
Now, the cool thing is is that this
is occurring, in this case, at the inner mitochondrial
membrane.
And so you can do work in lots of different ways.
It doesn't have to be next to the complex.
You have a charge across the membrane that
can be used different places in space,
such as so-called complex V or the F1F0-ATPase,
which can use dissipation of that proton gradient
to drive that rotational catalysis machine we described
last time and, ultimately, take ADP plus phosphate
and synthesize ATP.
But that's not the only work it can do.
You can uncouple the whole thing.
We discussed these uncoupling proteins,
which, effectively, just let protons leak
back across into the membranes, that short-circuiting
this potential.
And that's a way to generate heat.
Other type of work we can do is we
can use this proton gradient to power
the transport of other ions.
We discussed this happening with ADP/ATP exchange
because, of course, ADP-to-ATP conversion
needed in the cytosol to favor unfavorable processes there.
Need to exchange those in and out of the mitochondria,
so you can regenerate ATP with the ATP synthase machinery.
And yet another thing we talked about is
you can use this as a way to concentrate ions,
such as calcium.
And the mitochondria ends up being a major calcium
store for cells.
Now, we discussed last time that this is great having all
of these oxidation processes-- the TCA cycle,
fatty acid oxidation--
all occurring in the matrix because it
gives a location for NAD/NADH to happen where
it has easy access to this electron transport machinery
and the ability to regenerate the NAD necessary to keep these
processes going.
But of course, the very first process we talked about happens
in the cytosol, and that's glycolysis.
And we discussed how you can bring pyruvate
into the mitochondria through this mitochondrial pyruvate
carrier.
Then it has access to the pyruvate dehydrogenase
reaction, which, of course, generates acetyl-CoA
and NADH in the right place.
However, if you go way back to when we discuss fermentation,
remember, the reason we had to send pyruvate to fermentation
was to regenerate that NAD in the cytosol that's
necessary to keep glycolysis going.
Now, of course, if we're going to fully oxidize
pyruvate carbon in the mitochondria,
we still have to solve this electron balance
problem for glycolysis.
And as we discussed, we can solve that
by giving those electrons to oxygen.
But now you see that there's this additional complication--
is that these electrons in the form of NADH
need to get inside the mitochondrial matrix
or get to the electron transport chain
as a way to use oxygen to ultimately dispose
of those electrons.
And so, effectively, that NADH needs
to be gotten to complex I, which would
be the matrix in the way we've drawn it here.
Now, really, the way that this happens is not direct.
So you don't transport NADH from the cytosol
into the mitochondria.
And that's partially because one of the benefits, remember,
of having compartmentalized metabolism
is your ability to have different conditions;
different ATP/ADP ratios, NAD/NADH ratios
in different compartments because that
can favor different chemistry.
And so the way that you get electrons into the matrix
or get electrons to the electron transport chains
doesn't occur directly.
You do not transport NAD or NADH directly.
Instead, you use so-called shuttles,
which are analogous to some of the other shuttles we've
talked about, in order to get materials across membranes.
In this case, this is a way to get
electrons generated from oxidation
reactions in the cytosol--
production of NADH in the cytosol, things
like glycolysis--
to the electron transport chain so that they can ultimately
be transferred to oxygen.
And so these shuttles, or redox shuttles,
actually come in several different ways
that cells can deal with cytosolic electrons.
And so these shuttles also, in essence,
help cells also establish these different conditions,
these different NAD/NADH ratios in different compartments.
And so you can imagine that the shuttles themselves could be
regulated as a way to do this.
Now, there's two major redox shuttles
that allow you to move electrons from the cytosol
into the mitochondria, and we'll discuss those two here next.
These are the two that probably are
being looked for if you get this question on an MCAT exam
or something like that.
They're the ones that are described
in the text as the way to get electrons to the electron
transport chain.
But however, as we go through them,
you will see that there's a couple different ways
one can do this.
They basically involve oxidation and reduction
of a pair of molecules on either side of the membrane.
And there's lots of ways you can do that in addition
to these other shuttles.
It'll become more clear what I mean by this,
but I guess I just want you to know that these are not
the only ways one could shuttle electrons
into the mitochondria.
These are just the major ways that have been discussed
and are usually discussed in textbooks.
So the first shuttle system is something called
the glycerol phosphate shuttle.
And so the glycerol phosphate shuttle
involves a reaction that we've already discussed.
So here's our old friend, dihyroxyacetone phosphate
from glycolysis.
Remember when we talked about the generation of
and metabolism of glycerol, in order
to make triacylglycerides, phospholipids--
remember we described that you can
take dihyroxyacetone phosphate.
If you use NADH to reduce that ketone to the alcohol,
that generates this molecule, glycerol phosphate.
And of course, because you're reducing the ketone
to the alcohol, something else has to be oxidized.
You reoxidized NADH back to NAD+,
effectively disposing of those electrons from, say,
glycolysis that generated NADH to regenerate NAD+.
Now, rather than use this glycerol phosphate and lipid
synthesis, turns out there's a complex that
can directly transfer those electrons to FAD
to generate FADH2.
And this complex sits within the inner mitochondrial membrane,
facing the cytosolic side of the membrane.
Here's the inner mitochondrial membrane.
This would be the matrix.
And so transferring those electrons
to FAD to reduce to FADH2 reoxidizes
the glycerol phosphate back to dihydroxyacetone phosphate.
And of course, in this membrane, you can, in this complex,
transfer those electrons by oxidizing the FAD
and sending them to the coenzyme Q pool.
That can take the ubiquinone, makes ubinquinol.
Those electrons can go to complex III.
That can go to cytochrome C. They can go to complex IV,
ultimately ending up reducing oxygen to water using oxygen
as a final electron acceptor.
Again, this is effectively another alternative
to the electron transport chain.
And so I show that here on the slide.
And so we've discussed--
the complex II-- this is the FAD/FADH2 containing enzyme
is complex II.
Succinate dehydrogenase sits in the inner mitochondrial
membrane, converts succinate to fumarate.
Those electrons end up on up FADH2,
can get transferred to the Q pool,
reoxidizing FAD to complex III to cytochrome C to complex IV
to oxygen to water.
We already discussed acyl-CoA dehydrogenase-- same thing.
FADH2 containing reaction in there is also in the membrane--
ultimately allows another version electron transport
chain.
And here's yet a third one involving an FDA using enzyme.
The difference is is that these have the oxidation reduction
reaction occurring on the matrix side--
succinate to fumarate on the matrix side--
the introduction of a double bond
for fatty acid oxidation and the matrix side.
This one here instead faces the cytosolic side.
So glycerol 3 phosphate can be reoxidized
to dihydroxyacetone phosphate--
looks like I have those arrows drawn backwards, sorry--
ultimately sending electrons into this glycerol phosphate
shuttle, reducing FADH2, which can be reoxidized
by giving those electrons to the Q pool to complex III,
to cytochrome C, to complex IV, ultimately
with water serving as the final electron acceptor,
as I've drawn here.
Now, if you notice, if we use this system or any
of these systems, these alternative FAD/FADH2 using
electron transport chains, you don't use complex I.
Remember, complex I will pump a proton, complex II will not.
And that means that if you use any of these FADH2 using
alternative electron transport chains,
you pump fewer protons into the cytosol.
And so if you donate the electrons to complex I
from the matrix, you pump a proton
as you go through complex I to the Q pool
if you go through the glycerol phosphate shuttle,
you avoid complex I, because you give those electrons directly
to the glycerol phosphate shuttle.
And FAD/FADH2 enzyme-- those end up in the Q pool.
Less protons are pumped.
And this is why sometimes in textbooks, you'll
see that you get less ATP per NADH produced in the cytosol
than you get for NADH produced in the mitochondria.
And this is one of the ways that you end up
with these accounting yields for glucose oxidation-- something
we'll talk about in a minute.
But the deeper reason for this, which I want you to appreciate,
is because FADH2 has a higher standard reduction potential.
So the standard reduction potential of FAD/FADH2
is greater than it is for NAD/NADH.
In essence, that means that there's
less change in standard reduction
potential for transferring FADH2 electrons to oxygen as there is
for transferring electrons to oxygen.
And so less energy released, less protons pumped,
and so that's why you get less ATP, if you will,
produced going through the glycerol phosphate shuttle.
All right.
Next, I want to discuss another alternative shuttle
to get electrons into the mitochondria.
And this involves much more what I was alluding to earlier.
And that's oxidation and reduction
of a pair of metabolites that are transported
across the membrane on either side of the membrane,
effectively moving electrons from one compartment
in the cell to another.
Now, to understand how this shuttle works, I have to--
because it's a little more complicated,
I have to first describe that there's
a relationship between amino acids and alpha-keto acids.
And so, what do I mean by that?
Well, here's a alpha-keto acid that we've talked about a lot.
So this is oxaloacetate.
Remember, it's an alpha keto-acid because this ketone
is alpha to this carboxylic acid--
so an alpha-keto acid.
Turns out that alpha-keto acids are
related to amino acids in the following way.
And so if I take that alpha-keto group
and instead make it an amino group,
this here is the amino acid aspartame.
Now, we'll discuss later in the course
the chemistry that allows you to interconvert
this alpha-keto group with this amino group.
But that's effectively where your amino acids come from--
how your amino acids are related to other aspects of carbon
metabolism--
Alpha-keto acids that are generated in metabolism.
And so there's one example--
oxaloacetate and aspartate, an alpha-keto acid, an amino acid.
Here's another one.
So this is alpha-ketoglutarate--
an alpha-keto acid.
And if we change that alpha group to an amino group,
now we get the amino acid glutamate.
All right.
To remind you, the three letter abbreviation for aspartate
is a-s-p.
The one letter is D. The three letter abbreviation
for glutamate is g-l-u.
And the one letter abbreviation is E.
And I just remind you of that in case I use those abbreviations.
But effectively, you can note that if you couple these swaps
to each other--
that is, if I take oxaloacetate and turn it
into aspartate while at the same time taking glutamate
and turning it into alpha-ketoglutarate,
you can see that that is a reaction where
all atoms are conserved.
That is, if I turn this amino acid into this alpha-keto acid
while at the same time turning that alpha-keto acid
into this amino acid, I have not gained or lost any atoms
or molecules in the process.
And we will see later in the course
that these types of interconversions
is exactly how you run these reactions to do
these interconversions between alpha-keto acids
and amino acids.
OK.
Why am I telling you this now?
Because it turns out this is central to understand
the other major shuttle, which is referred
to as the malate-aspartate shuttle as another way
to get electrons and NADH from the cytosol
into the mitochondria.
All right.
So how does this happen?
So here if you have--
here's oxaloacetate.
This is occurring up here in the cytosol.
OK.
And if we take oxaloacetate in the cytosol and we
utilize the malate dehydrogenase reaction
that we heard about from the TCA cycle--
so basically this is the reverse of the malate dehydrogenase
reaction we discussed in the TCA cycle.
We're going to reduce this ketone to the alcohol.
NADH is reoxidized to NAD+.
That will generate malate.
And now we've regenerated NAD+ in the cytosol by putting those
electrons onto oxaloacetate to make malate.
All right.
Now, we can take that malate, send it
across the mitochondrial membranes via transporter
to generate, on the matrix side of the membrane,
malate, whereas from the TCA cycle,
we also have malate dehydrogenase that can now
reoxidize that carbon from the alcohol to the ketone
and regenerate oxaloacetate effectively
by carrying out the same reaction through a redox pair
on two sides of the membrane.
Effectively what I've done is I've
moved the electrons from NADH in the cytosol
to now being NADH in the mitochondrial matrix, where
this can now donate the electrons to complex I,
to the Q pool, to complex III, to cytochrome C, to complex IV,
and ultimately to oxygen, just like it
would for the TCA cycle.
All right.
Now, the simplest thing, of course,
would just be to send the oxaloacetate back out
to the cytosol.
But that's not what happens.
Instead, what happens is you turn that oxaloacetate
into aspartate.
This is an alpha-keto acid.
Turn it into the amino acid.
If you do that, you basically at the same time
turn glutamate into alpha-ketoglutarate all right.
Turns out it's that alpha-ketoglutarate
that is exchanged when you bring malate into the cell.
So malate is exchanged for alpha-ketoglutarate.
And at the same time, you exchange the aspartate
for a glutamate.
All right.
So now you can take that aspartame,
turn it back into oxaloacetate.
To do so you need to balance this.
So you return your alpha-ketoglutarate back
into glutamate.
And effectively, this is the malate aspartate shuttle.
OK.
So electrons from oxaloacetate to malate, and then malate back
to oxaloacetate moves the electrons on NADH
from the cytosol into the mitochondria.
To get that malate across the membrane,
you exchange it for alpha-ketoglutarate
and to maintain carbon balance across the--
and nitrogen balance-- across the whole thing.
At the same time, you also exchange an aspartate
for a glutamate, which allows you to basically interconvert
the oxaloacetate and the aspartate
on either side of the membrane via interconverting
glutamate and alpha-ketoglutarate.
So somewhat confusing systems, but
recognize that the net effect is moving NADH
from one side of the membrane to the other.
And that occurs because you're interconverting to redox pairs
in opposite directions-- in this case oxaloacetate and malate--
on either side of the membrane.
And that's why what I alluded to earlier,
you can imagine that you could easily come up
with other shuttle systems where you have a redox
reaction on one side of the membrane
and the reverse of that redox reaction on the other side
of the membrane.
And as long as you can maintain carbon balance,
the net effect is moving electrons across the membranes
from one compartment to another.
And that is a way to allow you to generate NADH that's
now in the matrix, which has access
to complex I of the electron transport chain
as we discussed now several time.
All right.
Now we're ready to go through and discuss the accounting
that you see--
the accounting that comes up all the time that is in textbooks
and perhaps you memorized from previous classes
about how the ATP yield works for different pathways.
So hopefully it's clear to you by now
that these numbers that you get are estimates.
And that's because there is no direct relationship
between NADH or FADH to NATP.
These interconversions occur via oxidative phosphorylation,
these electron transport chains, delta psi/delta pH.
And so all of these estimates take
into account various assumptions about how efficient
the electron transport chain is, oxidative phosphorylation
works, bounded, of course, by the change
in standard reduction potential, what's the free energy released
from that?
What's the free energy to synthesize ADP to ATP.
Of course, those require assumptions.
And it's, in the end, why if you read different textbooks
and different sources you will find numbers
that are actually a range.
There is no fixed number for what the equivalent is
of an ATP for an NADH.
And that's because it really depends on several variables.
So let's go through what those variables are,
because if we can understand that,
you really understand how this system works.
So the first variable--
at least if we're talking about something like glucose,
where we generate NADH in the cytosol,
we have to consider the redox shuttles.
And hopefully you appreciate now that you have this need
to regenerate NAD in the cytosol to keep glycolysis going.
You got to get those electrons somehow to the electron
transport chain.
And I just described for you two different ways you can do that.
Now, if I use the glycerol phosphate shuttle,
I'm skipping complex I altogether.
And so that's not pumping any protons.
If I'm using the malate aspartate shuttle,
well, I have all the energetics of doing this gymnastics.
But in the end, now I can use complex I and pump protons.
And so the yield of ATP, if you will-- how many
protons I can pump to generate delta psi/delta pH will
be different if I use the glycerol phosphate
shuttle or a shuttle like the malate aspartate shuttle.
The second variable in consideration
is, what is the efficiency of proton
pumping by the various electron transport chain ETC complexes?
That's not so straightforward and, of course,
is going to depend on a couple of things.
Remember, we spent a long time talking about ATP/ADP,
and how it's the ratio of ATP/ADP that was really
the energy there, because, remember,
thermodynamics is dependent on that log
of the reactants over the product's term in the delta G
equation.
Well, that's true also for the NAD/NADH ratio.
And it's also true for how high the membrane potential is.
And so how efficient this pumping
is going to be is going to be depending on what
is the delta psi to begin with, and what is the NAD/NADH ratio
or the FAD/FADH2 ratio, because all of those things
will affect how much free energy there
is to be able to move protons from one side of the membrane
to the other.
Once I get those protons across the membrane,
then there's the question of how--
and for lack of a better word, I'm
going to say tight the membrane is to proton leak.
That is, you can imagine that to make ATP,
I have to send those protons back
through the ATP synthase, complex V, F1F0-ATPase,
whatever you want to call it.
And if some of those protons just
leak back across the membrane, well, that's
how we generate heat.
And so how tight that membrane is,
that is how coupled delta psi is,
delta pH is to ATP synthesis is another variable.
And of course, the last one is, is the efficiency of H+ turning
the F0F1-ATPase, which is the same considerations that we
already talked about for number two.
That is, the higher delta psi/delta pH, the more free
energy that's stored there and the higher or lower
the ATP/ADP ratio is, the easier or harder it is to synthesize
ADP.
And so basically, these different things,
two through four, in general takes into account
a term called coupled respiration.
By coupled respiration is really,
how tightly is oxidation--
moving electrons on the electron transport chain to oxygen--
to phosphorylation-- synthesizing ATP from ADP?
And so, how tight or how well-coupled
is respiration to phosphorylation really
is a variable that is going to very much depend on conditions.
And so ultimately, to give numbers
for what is the ATP produced from various oxidation
processes, one has to make assumptions
about all of these things, how coupled respiration is
and which redox shuttles are used?
If it's something like glycolysis,
it involves the cytosol.
And this is why these numbers are made up.
And it makes no sense in my view to memorize these numbers.
But I think it is important to understand
where those numbers come from.
Because if you understand that, that means you understand
all the assumptions and why the numbers are variable,
you really understand the bioenergetics
of what's going on in metabolism.
All right.
So now let's go through one of these calculations
just to fully illustrate what I'm talking about.
So let's go through the calculation for glycolysis.
So if I run glycolysis and turn glucose into two pyruvate.
So what do we get from that?
Of course, we get 2 ATPs produced
and we get two NADHs produced in the cytosol.
OK.
So this is the cytosol.
This is the mitochondria.
All right?
We're going to completely oxidize that pyruvate.
We send that pyruvate into the mitochondria.
We run the PDH reaction.
That gives us two acetyl-CoA, which
is a yield of two more NADHs.
All right?
If we now take those acetyl-CoAs and we burn them completely,
two cycles around the TCA cycle.
OK, now we get two more FADH2s and six NADHs--
one FADH2, three NADHs from each turn, as well as a GDP.
And so that's a total of two FADH2, six NADHs, and two GTPs.
And so now if I come together, what is our yield?
Well, from the mitochondria I've generated
a total of two FADH2s and six, seven, eight NADHs.
And then up here, there's an additional two NADHs
that are made in the cytosol, and so
have to access the electron transport chain via a shuttle.
A typical number that's given is you get 1.58 ATP
for something that has to access via shuttle,
or on FADH2, that would make sense,
because FADH2 is really the same as a glycerol phosphate
shuttle.
And so if I do that, that would be three ATP from the two
glycolysis NADHs and three ATP from the two TCA cycle FADH2s,
because the NADHs produced in the matrix can generate
POM protons via complex I. Oftentimes, that's said to be,
oh, that's about 2 and 1/2 ATP.
So 8 times 2.5 is 20 ATP.
And if we had all these numbers together,
20 plus 3 plus 3 plus 2 plus 2, that is a total of 30
ATP per glucose that's completely oxidized to CO2.
OK?
So 26 ATP over here plus another 2 from GTP plus another 2
from glycolysis is a total of 20 ATP per glucose.
That is a number.
It made certain assumptions.
Obviously, if I assume that this is not the glycerol phosphate
shuttle but instead the malate aspartate shuttle,
maybe I bump this number up by multiplying that by 2.5.
Maybe I use different conversion factors.
And if you read in textbooks, you
get numbers that range from 30 to 36.
I think the highest I've ever seen is 38 ATP per glucose.
And the fact that you actually see this range really
reflects the different authors of textbooks,
or whoever writes these things, makes different assumptions
about all of these things over here
to understand how to make these conversions.
But the important thing is realizing that these are not
real numbers.
All right.
Let's quickly go through and just do
the similar exercise one last time just to come back to.
I'm not going to go through as much detail.
But we talked about that 6:0 fatty acid
that we did accounting for earlier.
If you look back in your notes, what we wrote down
is that from completely oxidizing that fully saturated
six carbon fatty acid, we got one ATP, 11 and ADHDs, and five
FADH2s.
Remember, all these are being generated
in the-- all these NADHs and FADH2s are already
in the mitochondria.
And so if we just use the same conversion factors
that we used for over there in our calculation for glycolysis,
what we get is 27.5 ATP for the NADHs and 7.5 ATP
for the FADH2s, which is a total of 35 ATP for--
35 plus the one, so a total of 36
ATP from complete oxidation of our six carbon fatty acid,
which is certainly greater than the 30
ATP we got from complete oxidation of glucose.
That makes sense.
Fatty acids are more reduced than glucose--
more calories, should be able to get more from it.
And this calculation illustrates that we get that.
The typical calculation in textbook
usually describes complete oxidation of a 16:0--
a fully saturated 16 carbon fatty acid.
That's palmitate.
That's one of the most abundant fatty acids in the cell.
This, if you completely oxidize it,
the number and a lot of textbooks
is 106 ATP per palmitate completely oxidized to CO2.
If you're so inclined, you can go through
and do the calculations yourself and see if you agree with that.
But the key point of all of this accounting is that none of this
are real numbers.
And I hope you now better appreciate
what energy really means--
that is, how oxidation of carbon by being favorable
can be coupled either to direct charging of an ATP/ADT ratio
as occurs in the GAPDH step of glycolysis,
as well as the succinic thiokinase
step of the TCA cycle.
But most energy transduction involves
this charging up NAD/NADH ratios,
or reactions that are coupled to FAD/FADH2
via these membrane complexes that ultimately lead
to this favorable electron transport
from lower to higher standard reduction potentials.
That favorable electron transport
can be used to create a battery, a delta psi/delta pH, which
can then be coupled to do other work,
including synthesizing ATP at the high ATP/ADP ratio that
can then be used by other reactions
in the cell that would otherwise be unfavorable and allow cells
to basically extract energy from its environment
in order to fuel the processes that are unfavorable and are
necessary to sustain life.
All right.
Great.
So thus far, we have focused almost entirely
on how you oxidized carbon as a way to release energy.
And that's great.
But of course, that reduced carbon
has to come from somewhere to begin with.
And every one of us learned in grade school
that the energy for all life ultimately comes from the sun.
And that's, of course, photosynthesis.
And now I want to turn to beginning
to discuss photosynthesis and how it works.
In other words, how does life harvest solar energy
in a usable form that ultimately involves using atmospheric CO2,
which gets reduced to generate reduced
carbon that, of course, originally
occurred because plants--
or, any photosynthetic organism has the same issues as us.
That is, to fight thermodynamics, maintain,
and survive.
It constantly has to be charging an ATP/ADP ratio in order
to fuel the otherwise unfavorable processes that
are required to maintain order in the cell.
This has to occur during the day when the sun is out
and there's light energy photosynthesis.
But also has to occur at night, or at times
when there is no sun.
And so thus, plants basically stored energy as reduced carbon
so that they had something to eat at night-- that is,
to keep their ATP/ADP ratios high--
at night so they can do all these catabolic processes
at night.
Now, fortunately for us, the fact
that photosynthetic organisms stored all this reduced carbon
allowed other life, like us, to evolve
that is entirely dependent on photosynthetic organisms
either directly or indirectly for food.
And so photosynthesis also is the process that ultimately
oxygenated our atmosphere.
Aerobic life, also something that was necessary for us
to survive because oxygen is such a key electron acceptor,
really key to our energetics--
well, the fact that photosynthetic organisms
put oxygen in our atmosphere also
was really essential for aerobic life, us, to ultimately evolve.
And so photosynthesis is often, in biochemistry courses, almost
a sidelight and forgotten process.
But of course, it is really, really central
to how life works, because no life
could exist without photosynthesis
and the conditions that allowed aerobic--
most multicellular life that we know
of that's not photosynthetic, including ourselves,
could not have evolved without photosynthesis
oxygenating the atmosphere and generating
all this reduced carbon.
That, really, is where we get our energy from as food.
Now, you also learned in grade school,
when you learned that life depends on energy of the sun,
that the process that life uses is
this mysterious photosynthesis.
Well, as we go through this over the rest of today
and in the next lecture, I want you to appreciate
what photosynthesis is.
And that is how life really takes solar energy
and harvests that energy in a biologically useful form,
bearing in mind all of the concepts that we've already
talked about, because that will ultimately help you understand
how that energy from the sun is used to make all
this reduced carbon, this carbohydrate and fat
that is energy that we can use for later, and also, of course,
allows how photosynthetic organisms can use light
from the sun to just power things directly
from that solar battery, and ultimately, of course,
you should appreciate that these things all came first.
Photosynthesis obviously existed first.
And so most of what we already talked about--
well, we'll draw parallels when we talk about photosynthesis
to the stuff we already know.
Of course, photosynthesis came first.
And the stuff you've already learned about
evolved from photosynthesis.
All right.
Now, like all pathways, photosynthesis also
has to follow the same rules and laws of thermodynamics that
were true for all pathways.
So plants and animals have to follow the same rules.
And so what is photosynthesis?
Well, it's ultimately CO2 in the atmosphere that
gets reduced to carbohydrate.
That's classically photosynthesis.
But, of course, plants also make fatty acids.
OK?
This is carbon reduction.
Remember, carbon oxidation generally is favorable.
Carbon reduction is not.
Delta G naught prime for this is greater than 0.
This direction is favored.
That direction is not, which means
you need energy input to reduce that CO2
to carbohydrate or fatty acid.
And of course, it's the sun's energy, the solar energy,
that ultimately must be done to make this favorable.
Now we're reducing carbon.
That means we need to add electrons.
Those electrons have to come from somewhere.
And so we're reducing the carbon.
That means something else has to be oxidized.
Just like when we oxidized carbon,
those electrons had to go somewhere,
something else had to be reduced.
That was pyruvate to lactate and fermentation,
or oxygen to water via all of the oxidative phosphorylation
things we just talked about.
And so in this case, those electrons
have to come from somewhere.
And those electrons ultimately, for photosynthesis--
this reduction, the electrons come
from oxidation, ultimately, of water,
which will generate oxygen. And this is effectively then
the reverse of what you learned about
for the mitochondrial electron transport.
Why this?
Well, water is very abundant, has been for all of life
on Earth.
Water is-- obviously, life depends on water.
And so you can oxidize the water to generate
oxygen. This will obviously generate electrons that
can be used to reduce carbon.
And it's effectively the photosynthetic oxidation
of water to make oxygen that was the source of oxygen
in the atmosphere that, really, today
makes aerobic life possible.
Now, of course, the fact that oxygen is a good electron
acceptor-- we've been talking about this forever.
This means that, of course, you might
guess that delta G naught prime for this process
is not going to be favorable.
To put it another way, oxygen is a great electron
acceptor and water is a poor electron donor.
And so, really, the way photosynthesis--
the magic of photosynthesis, if you will,
is making water a good electron donor, because, ultimately,
that is what is going to make this whole thing
work energetically.
And so you probably could guess that if these processes are
really about these electron transfers,
we need electron carriers.
This is true for photosynthesis, just
as it was true for oxidative phosphorylation.
But remember, we'll describe a lot of the same electron
carriers that are used.
But as we described them, remember
that the photosynthesis came first.
And so the other processes, like OXPHOS, that use these electron
carriers, use them because they were first
used in photosynthesis.
And then they were repurposed for oxidative phosphorylation.
Now, to discuss this, I want to first start
by introducing an electron carrier that's
important for this that's really a variation on NADH.
And it's an electron carrier that's found in--
excuse me-- all plants, all animals, all life,
and is very important in anabolic pathways
like photosynthesis.
Remember that anabolism, building stuff,
has to be distinct from catabolism, breaking stuff
down, for a number of reasons.
We saw this in glycolysis and gluconeogenesis, glycogen
synthesis, glycogen breakdown.
Remember, delta G naught prime can only favor one direction.
And so in the direction that's favorable, it works.
In the other direction to actually make it happen,
we have to make delta G less than zero.
That has to happen by energy addition.
And so now we had to have these separate pathways.
You also need separate pathways because that
allows separate regulation, avoids futile cycle,
and enables cells to match needs with processes.
And as we already discussed, one way
to keep these anabolic and catabolic processes distinct
is by building separate compartments, right?
It's useful to have separate compartments,
different conditions in different compartments
that can favor different things happening.
However, not all cells have separate compartments.
Prokaryotes don't.
And you might think of situations
where you clearly want to have two
different types of reactions happening
in the same compartment.
And so what if you want to both do oxidation and reduction
in different reactions in the same compartment?
Well, clearly you need some way that
will favor some reactions being oxidation
and other reactions being reductions.
And if you simply rely on NADH/NAD+ as your oxidant
or your reductant, this now becomes a problem.
All right?
So a solution is, well, let's make a distinct electron
carrier for the oxidation reactions in the cells.
So that's what this is.
Turns out, you keep your NAD/NADH
to largely favor the oxidation reactions you want to do.
And let's create a different currency for reduction.
And this currency ends up being a different molecule,
NADPH/NADP+.
And you use this as a cofactor pair for reduction.
Well, what is NADPH?
Well, it's effectively the same thing as NADH.
But now you mark it as a separate pool
with a two prime phosphate on the ADP portion
of the molecule.
Let me draw it out for you.
OK.
So this here is NA--
so if I took this phosphate off, this would be NADH,
put a two prime phosphate on, now I
mark this as a separate molecule pool, NADPH.
A is an adenine.
Remember, it's a nicotinamide adenine dinucleoside.
OK?
So here's a adenine, phosphate, phosphate, other nucleoside
with the nicotinamide group.
This here is in the reduced form.
Seen this now a million times, but we
can oxidize it as possible.
That generates this two electron hydride ion.
So remove two electrons and now this nicotinamide form is here
in the NADP+ or oxidized form of the molecule.
And so effectively by marking this as a separate pool with
that two prime phosphate, you can have a different NADH/NAD+
ratio in cells.
Then you have NADH/NADP+ plus ratio and cells.
Use one co-factor fair pair to favor
oxidation and the other co-factor pair
to favor reduction.
And so I want to give a quick aside on NADPH
because it's really also NADPH that
can be very important in keeping the cytosol of cells reduced.
So what do I mean by that?
Well, you remember you learned from Professor Yaffe.
We talked about proteins that you can have
these inside the side cytosol.
You'd have your cystine residues on proteins
be in this reduced form.
And outside the cell, you'd form these disulfide bonds, which
is basically oxidized cystine.
And so in the cytosol, you want your cystine residues reduced.
And outside of the cell, you end up
with these disulfide bonds on proteins.
Why does cystine tend to get oxidized outside the cell?
Well, there's lots of oxygen in our atmosphere.
Oxygen's a good electron acceptor.
It's very good at oxidation.
In fact, that's what gives it this property that makes
oxidative phosphorylation work.
And so it turns out that these cystines,
these sulfhydryl groups, like to give their electrons to oxygen.
And that leads to these more oxidized things
outside the cell.
In fact, oxygen is very good at accepting electrons
from lots of donors.
So if you have iron 2+ sitting around outside the cell,
oxygen is very good at oxidizing that iron 2+ to iron 3+.
That's rust.
And this process, in the end, ends up transferring electrons
to oxygen and can generate lots of oxygen species,
including oxygen radicals.
And so you can get things like this O2- superoxide,
ultimately hydrogen peroxide, also
comes from oxygen picking up electrons.
And as you know, very reactive molecules like this
can damage membranes, damage proteins, kill cells.
What do we use hydrogen peroxide for in daily life?
Use it to clean wounds, kills a bunch of bacteria.
And ultimately, production of these so-called ROS,
or reactive oxygen species, in excess
can be very bad for biological systems.
And this is one of the arguments for why
antioxidants are good for us.
They protect us and ourselves from these reactive oxygen
species.
OK.
Now, cells, of course, have endogenous ways
to deal with this, to keep cystines in our cytosol
reduced, to keep our cytosol in this reducing environment,
prevent this ROS damage.
And really, if you're going to have
things to deal with these reactive molecules, what do
you want?
Well, you want something that's going
to buffer the damage that these things can cause.
That is, if these things are going
to react with lipids and proteins and cause damage
and you want to keep the cytosol reduced,
you really want to have a series of molecules
that will actually, instead, undergo this process before it
can damage critical structures in the cell.
And a key system cells use to do this is
something called glutathione.
So what is glutathione?
Well, glutathione is a small molecule tripeptide.
All right?
And it's among the most abundant small molecules found in cells.
And effectively, what glutathione does
is it serves this purpose of reacting with nasty stuff
before it can damage other biomolecules and hurt the cell.
And so by tripeptide, what it is is it's
a tripeptide of glutamate with a gamma peptide bond
to glycine and then cystine--
so glutamate glycine cystine tripeptide
that looks like this.
OK.
So this here is the amino acid glutamate.
Here's the alpha carbon with the carboxylic acid
and amine groups.
So you would typically think of the peptide bond of glutamate
was in a chain as being from this nitrogen
to the next amino acid and that carbon to the next amino acid.
But in this case, it uses the nitrogen on the side chain,
the so-called gamma linkage here,
to form a peptide bond with--
oops, sorry.
This would be glutamic acid.
It uses this gamma carboxylic acid
on the side chain of glutamate to form a peptide bond
with glycine.
OK.
So this is glutamate.
This is glycine.
And then this has a peptide bond to cystine,
which contributes that sulfhydryl group that
can either exist in this reduced form.
Or, if there's two of them together,
you can go into this oxidized form.
And so, often, this is drawn as glutathione GSH in the reduced
form.
And if you have two glutathione molecules that then basically
form a disulfide bond to give you this GSSG
oxidized form of the molecule.
And NADPH plays a key role in the cell,
because that's an oxidation reduction
reaction in keeping glutathione in the reduced form.
So if you have two GSH molecules that
can cycle to a oxidized GSSG--
so this is reduced, this is oxidized.
On the other side you can cycle those electrons.
If something gets oxidized, something else
has to be reduced.
So you can have NADP+ and NADPH.
This is reduced.
This is oxidized.
And so by maintaining a high ratio of NADH to NADP+,
you can keep a high ratio of reduced to oxidize glutathione
in the cell and keep the cell in a protected reducing
environment where you have these glutathione molecules to react
with other things before it causes damage to the cell.
All right.
A little bit of a diversion, but a very important system
that cells use related to oxidation reduction and NADPH,
and needed a place to talk about it somewhere in the course.
All right.
Now back to photosynthesis.
Now, for photosynthesis, this reduced
NADPH molecule's of course useful,
because it can serve as a source of electrons
to donate carbons to reduce CO2.
So if you're going to reduce CO2 to make carbohydrate or fat,
you need this source of electrons.
Those can come from NADPH, which can be oxidized to NADP+.
And so perhaps at this point, you've
already guessed or maybe already know
that photosynthesis uses the favorable electron transfer
from water to generate NADPH.
And to do that, you use the energy of the sun.
Now, of course, if you're going to do a favorable electron
transfer, that can be coupled, just like we talked about
for oxidative phosphorylation, to make delta psi/delta pH.
And if we can use that favorable electron transfer
to make delta psi/delta pH, we can also
use it to do other work, including making ATP exactly
as we described for oxidative phosphorylation.
And at the highest level, this is exactly how photosynthesis
traps solar energy and uses that energy
to both remake reduced carbon and generates
a battery for the photosynthetic organism that
can be used to make ATP or do other work,
provided that sun is available.
And so classically, photosynthesis
can really be separated into, I guess, two distinct processes.
All right?
So you have this process of taking solar energy,
using that solar energy to charge up
a battery, delta psi/delta pH, which of course can be used
to generate ATP or do other work, and at the same time
gives you an electron donor, NADPH,
that is useful to generate reduced carbon.
Once you generate, high ATP/ADP ratio, high NADPH/NADP ratio.
Now we can use that to be couple to do something
else unfavorable.
And that would be something like reduced CO2
to generate a carbohydrate.
And typically, when we talk about photosynthesis,
the way it's often described is how you
reduced CO2 to carbohydrate.
And we'll do that too.
But of course, we'll then see once you have carbohydrates,
obviously, it's just further reduction of carbon
to make fatty acids and lipids.
We'll discuss later how we can use
those to make amino acids, which would
allow you to make proteins.
And, of course, we can also store those carbohydrates
as disaccharides or polysaccharides, storage
sugars, which is what plants do.
They make starch, they make sucrose, et cetera,
as a way to store that reduce carbon
so that they can have a source of energy,
do the oxidation reactions, to deal with their energetic needs
when the sun is not shining.
All right.
So let's break down these two processes.
And so to just be very explicit, the first part
is basically trapping energy to make a battery.
All right?
And so this means that we need to generate delta psi/delta
pH via favorable electron transport, exactly what we
describe for OXPHOS.
But, of course, photosynthesis was first.
This would then allow us to use ATP to fill our energy needs.
And we get NADPH if we set it up in the right way, which is
important for carbon reduction.
Then we need to use NADPH and ATP via pathways
to describe how we can use that to reduce CO2, and ultimately
produce glucose, which allows us to store energy for later.
All right.
So that is, at a very high level, what photosynthesis is.
And, of course, this occurs in plants.
All right?
But perhaps more importantly, it also
occurs in algae and other unicellular eukaryotes
as well as in prokaryotes-- photosynthetic prokaryotes,
bacteria.
And, of course, photosynthetic bacteria,
unicellular eukaryotes, algae--
these really are the heroes of life.
These are the things that life started from, and generated
the oxygenated atmosphere, and ultimately
led to the evolution of higher plants,
but also enabled the formation of animal life,
and really anything that lives off of eating other organisms.
Just a few facts about photosynthesis
because it's really quite an amazing process--
and so about 10 to the 17th kilojoules, at least
by some estimate, of energy is harvested on our planet
from photosynthesis every year.
Just to give you a perspective of what that number means,
that is 10 times the worldwide energy use
by all people on Earth.
So if you add up all the energy that we as humans use
in the world, it is tenfold less than the amount of energy
that is harvested by photosynthesis,
by photosynthetic organisms around the world.
Photosynthetic organisms, when they do this, consume CO2.
All right?
So that is removing CO2, the greenhouse
gas from our atmosphere.
And this occurs on a massive scale.
So this is better than any man-made engineering solution
that we have come up with by far.
And I think this really illustrates
the awesome power of biology to solve
some of the biggest problems that face our society today.
So world energy problems--
photosynthesis harnesses a ton of energy.
Global warming, greenhouse gases--
photosynthesis, better than almost anything
at dealing with these CO2 that is produced.
However, this also illustrates what some of the problems are
and what some of the issues are with fossil fuels,
because what are fossil fuels.
Well, they're effectively reduced
carbon that is trapped in the ground somewhere, that is
effectively old photosynthesis.
So these are organisms in many, many, many, many years ago
that did photosynthesis, made reduced carbon that
was effectively trapped in the ground.
And so the fact that photosynthesis transducers 10
to 17th kilojoules of energy per year and we're using 1/10
of that amount of energy, if that's all coming from fossil
fuels, that means we're using 1/10 of a year's worth
of photosynthesis-- and, of course,
a year is not entirely stored in the ground--
per year.
So that's a lot of energy.
And it really comes down to why people say fossil fuels can't
last forever, and also why the net release of these things
can change our atmosphere, because, of course,
once upon a time, there was much more CO2 in the atmosphere.
And photosynthesis, over millennia,
trapping these things as fossil fuels really
has lowered CO2 in our atmosphere to the levels
that they exist today, and how, basically,
burning fossil fuels on the scale that we are
is able to change our atmosphere.
So you can view this both as glass half full and glass
half empty.
It really says understanding photosynthesis
could be a key to solving some of these problems.
But it also points out why some of these things
might be problems to begin with.
All right.
Now we want to discuss first how photosynthesis traps energy.
And you'll see that it is similar to oxidative
phosphorylation in many, many ways.
So it's going to occur at a membrane.
All right?
It's going to involve a series of protein complexes
where we're going to favor, we're
going to couple favorable electron
transport along a electron transport chain
to favor proton pumping.
That is going to generate a membrane potential delta
psi/delta pH.
In order to do that, of course, we
need favorable electron transport
from something that is lower standard reduction
potential to something that is higher standard reduction
potential.
That is what is going to make it favorable,
allow proton pumping.
That, when it generates delta psi/delta pH,
we can now harness that as a way to synthesize
ATP or do other work, as we've now talked about a lot.
Looks a lot like oxidative phosphorylation
and it makes sense.
Oxidative phosphorylation came from photosynthesis.
However, there are some really big differences
between the two.
And that is in photosynthesis, as I've already alluded
to, the electron donor is going to be water,
which will generate oxygen. And the electron acceptor is NADP+,
which will generate NADPH.
And you might now be asking, well, how on Earth
can this be possible?
Because, remember, the standard reduction potential of NADH
is less than the standard reduction potential
of our oxygen water pair.
And it's really this that makes the change
in standard reduction potential positive, which
means that delta G is going to be negative,
which is why electron transport and oxidative
phosphorylation is favorable and can be coupled
to make delta psi/delta pH.
Now, adding a phosphate to NAD/NADH
does not change its standard reduction potential
in a way that is at all meaningful to impact
what goes on.
And so this is really where energy input from the sun
becomes critical.
And that is-- it is this light energy from a photon that
in the end makes water a good electron donor,
such that all of these other things are satisfied that
electron transport is favorable going from lower to higher
standard reduction potential with NADP+ ultimately being
the final electron acceptor, such that the change
in standard production potential is positive.
And that really is the magic of photosynthesis.
And, of course, once you do that,
now you have delta psi/delta pH.
You can use that to make ATP, do other work
for the photosynthetic organism.
And you also get NADPH, which is an electron
donor for carbon reduction.
And the net result is that you get this familiar formula
that you learned in grade school for photosynthesis--
CO2 plus water with light goes to oxygen plus carbohydrate,
which is, of course, the exact opposite of combustion.
We talked a lot about how burning wood combustion is
the opposite reaction that's favorable-- lots of energy
released.
And so the energy input here is obviously
the opposite of that to do this opposite of combustion
and stores lots of energy.
All right, so again, I want to say why
use water as an electron donor?
Well, because it's abundant.
There's nothing particularly special about water other
than its abundance, just like we talked about in oxygen is not
really being special other than that it's
a good electron acceptor and abundant.
You could obviously build systems that do something other
than oxygen and water.
And so there are extremophiles out there
that instead do hydrogen sulfide to make
reduced sulfur as another--
sorry, oxidize sulfur as a way of doing photosynthesis.
Exact same concepts would apply is what
we're going to talk about here.
All right.
Now, the parts of photosynthesis,
as you might guess, are spatially distinct.
That is, we're going to generate delta psi/delta pH and NADPH.
Those are basically this electron
transport chain reactions, charge the battery, et cetera.
And then imagine if we're going to fix CO2 as carbohydrate.
Well, effectively that's more like what we talked about
with gluconeogenesis.
That's the chemical reactions that we just
have to build a pathway for, like gluconeogenesis.
Now these two things are often referred
to as the light and dark reactions of photosynthesis.
And that's because the first set of reactions
generating the membrane potential and making NADPH
requires solar energy inputs or requires light from the sun.
This fixing of CO2, which will just use electrons from NADPH
to reduce CO2, can occur--
of course, it can occur in the light.
But it doesn't need light.
So it can occur in the light or the dark,
hence called the dark reactions.
And so these light reactions can really be broken up into water
plus NADP+ goes to NADPH plus oxygen.
And the dark reactions are NADPH plus CO2 goes to carbohydrate
plus NADP+, which of course gives us our net--
water plus CO2 goes to carbohydrate plus oxygen.
And in the next lecture, what we'll talk about is
we'll go through the details of how
these light and dark reactions work.
But when we do so, bear in mind that we're really
going to follow all of the concepts and rules
that we talked about for past thermodynamic considerations
of pathways as well as what we've
been discussing about how oxidative phosphorylation
works.
Thanks.