PROFESSOR: Let's talk about what
was probably the first
energy producing system
that evolved.
The thought is when the earth
first formed and the first
primitive organisms, perhaps
resembling a present-day
bacterium in some way came
out, there were a lot of
organic compounds that had
been aided by lightning
strikes and cosmic radiations
triggering chemical
reactions and so on.
So there was food around, but
they depleted those resources
in the same way we're depleting
the petroleum
resources right now.
If life was going to continue,
somehow a way had to be found
to make energy.
Glycolysis, it looks kind
of complicated.
It takes a molecule of sugar and
then there are a series of
10 chemical reactions, each
catalyzed by a separate enzyme
that give two molecules of this,
molecules of pyruvate,
plus two ATPs, plus two NADHs.
Which tells you there must
have been some kind of
oxidation step as part of this
sequence of events, because
electrons got taken off and
got stashed on this NADH.
There are a couple of things
that are important about this.
One is its a pathway.
It evolved probably 3.7 billion
years ago or sometime,
nobody really knows.
But a long time ago.
It's pretty much universal.
Not perfectly so, but it's
in bacteria, it's in
yeast, it's in humans.
And another really important
thing is that it evolved early
in the evolution of earth, so
it evolved when there was no
oxygen around.
So it's a way of making
energy from glucose in
the absence of oxygen.
Which is a really important
thing as you'll
see as we go along.
You're not going to have to
memorize this pathway.
We'll give it to you
if you need it.
But you're going to need to
understand its implications.
And just let me point out
a couple of things.
You're going to see a sequence
of 10 chemical transformations
that in the end are going to
end up with a couple of
pyruvates being produced.
And I'll try to explain
to you why you
should care about this.
There's a concept that you're
familiar with, that if you
want to make something and you
get a little start up company,
what's the very first thing
you have to do?
You actually have to
make an investment
before you can get going.
And you're out looking for
venture capital things.
Well one of the odd things about
this, here's probably
the first sequence of reactions
that arose on earth
within some organism and enabled
that organism to make
energy out of glucose.
And look, the first thing
that happens.
Trying to make ATP, the very
first thing it does is it
spends an ATP.
And it takes glucose, and it
makes glucose 6-phosphate.
Go down a couple of steps.
There's an enzyme that takes
another molecule of ATP.
And now you've got this point.
You're at fructose with
two phosphates on it.
If this was your venture
capital, we'd say, guys, how
about some product?
Stop spending, stop
spending money.
But at this point then, this
is a 6-carbon sugar.
And it gets split into two
3-carbon compounds that are
going back and forth.
Oh I can see it.
It's over there, OK.
In equilibrium over here.
And this particular 3-carbon
compound then
goes on to be oxidized.
You get the production
of NADH.
And at that point, this molecule
has a lot of energy
stored in it, and in the next
transformation this cell is
able to make two ATPs.
And it gets back the
initial investment.
It goes all the way through
the rest of the pathway.
And the very last step, you
get two more ATPs back.
There's your net yield.
So what you get out of this
are 4ATP+2NADH, and your
investment was two ATPs.
So your net 2ATP+NADH.
Why is this cell going and
doing these initial
investments?
Well if we look at the changes
in free energy associated with
what's going on, there's glucose
up in the upper left
starting up there, and there's
pyruvate down there.
So you're going energetically
downhill in the end.
So this is a sequence of events
that, in principle, you
should be able to get some
energy out of it.
But for reasons that may seem
obscure to you at this point,
before it gets to the point of
making energy, it undergoes a
set of transformations that's
pushing the reaction.
It requires the reactants to go
energetically uphill, i.e.
in an unfavorable direction.
So what the cell does is, by
coupling ATP hydrolysis to
this step, it makes
that reaction go.
Here's another unfavorable one
that makes that one go by
coupling ATP hydrolysis to it.
This is an uphill reaction,
but look over here.
This is an immensely favorable
reaction that goes essentially
to completion.
It goes all the way.
So that means this product is
just being continually taken
out of the system, so the
equilibrium is basically being
pulled over the edge by the
removal of that product.
This is where the oxidation
takes place.
You get the NADH made
right there.
And it's finally down here
where you've got to lose.
This transformation gives you
two ATPs and later there's
another one.
Let me just give you
a sense of why you
get ATP at that step.
The compound that you have
at that point is 1, 3
diphosphoglycerate.
Or sometimes this is
called bis, is also
used to describe this.
But what is this compound?
It's a 3-carbon compound.
So glycerate is basically an
oxidized version of glycerol
that has been oxidized up
to a carboxyl acid.
So this is a mixed anhydride
between carboxyl acid and a
phosphate ion.
So that's a very reactive
and unstable compound.
And the other thing that the
cell has succeeded in doing by
all of these transformations
is it's got these two
phosphates with all their
negative charges in.
So this is a compound that would
very much like to move
to a lower energy.
So you can get rid of this
phosphate and move to an
energy level, use that
energy to make ATP.
And there's a similar kind of
logic that explains why you
get energy out of the final
step when you look at it.
So there's several points, I
guess, to make out of this.
One is its pathway.
None of these reactions make
any particular sense by
themselves.
You could have a cell that knew
how to do one of them and
it would gain nothing.
Unless you wanted to use the
product to make something.
This thing only makes sense,
these reactions only make
sense in the context of
this 10 step pathway.
And each step in that pathway
we were looking at is
catalyzed by a different
enzyme.
So for an organism to pull this
off, the first one that
did it had to collect in one
cell all 10 of those enzymes.
And probably there is the reason
that this is such a
complicated system.
If you were sitting as a
designer you might be able to
come up now with a more
efficient way to get ATP out.
But what happened evolutionarily
was some bug
somewhere got all of these
things together, and now
suddenly it could make energy.
So it had a huge advantage
over everybody else.
And once it took over, that
system took over, then it
became universal.
Whether it was the best that
ever could be designed, it
doesn't matter, because it
had an evolutionary edge.
And that's so, to some extent,
we're looking at a living
fossil, biochemical.
But it's in bacteria, it's in
yeast, and it's going on
inside of our body.
Another principle that I think
you can see here, which I've
been trying to say, is in this
case, the energy consuming
reactions are driven by
coupling them to the
hydrolysis of ATP.
The cell spends a bit of its
energy money to get these
intermediates, knowing
that it's invest--
well not knowing, but at least
conceptually anyway, knowing
that it's going to get
its investment back.
And then the reactions that
release energy are used to
drive the synthesis of ATP.
And you'll begin to see, we're
going to just talk about some
other aspects of this
in just a minute.
So, what do you think?
You're the first bug and you've
got this and nobody
else can do it, so you can
start charging away.
What do we need to do?
We just let this thing
cycle away?
The stuff that I had up there,
is it going to work?
There's a problem.
Anybody see what
the problem is?
We're making two molecules of
ATP and two molecules of NADH.
Talk to the person beside you.
Figure out why something
else has to happen.
Go ahead.
See if you've got any ideas.
We're going to keep doing
this, over and
over and over again.
AUDIENCE: [INAUDIBLE].
Where's the first ATP?
Where's the first ATP?
PROFESSOR: OK, let's
imagine for the--
I don't think this process could
have invented ATP, it
had to have been around,
because many of the
enzymes used it.
What else is being used
in this thing though?
Did I hear NAD?
To make this thing work, I have
to keep taking NADs out
of my pocket and putting it in
the reaction, or it isn't
going to go anywhere.
So this isn't such a great
invention at the moment.
We have to do something to get
the NADH back to NAD+ so we
can do another molecule
of glucose.
You guys see?
Do you see this?
This is really, really an
important consideration.
So in order for cells to make
energy using glycolysis in the
absence of oxygen, which is when
it evolved, they have to
do something with that NADH or
it's only going to use up the
few molecules of NAD+ in the
cell, and then it stops.
And so there are two ways
that nature's figured.
Major ways nature had figured
out how to do that.
So here's a molecule
of pyruvate.
I got an extra.
Something was nagging at me
when I did this here.
Sorry about that.
It's always hard to see things
when you're up at the board.
OK.
Molecule of pyruvate.
There's a couple of solutions
that have been arrived at.
One is to take NADH,
2NADH, this is 2H+.
Convert, make these back into
2NAD+, and to take those
electrons and put them on the
pyruvate to give this
molecule, which is
lactic acid.
So by parking the electrons
there, the cell is able to
recycle the NADH.
And lactic acid, we've
run into that.
That's why I showed you this
picture of yogurt.
The lactobacilli that make
yogurt take the sugars that
are present in milk and make
them into lactic acid.
And what's interesting in
their case is they, even
though there's oxygen around,
they don't do respiration,
which you'll see you can
get more energy.
They want it to get very acidic
because that prevents
their competitors
from growing.
And that's why you can leave
yogurt sitting out on the
tabletop and it'll be OK
for quite a while.
Whereas if you left some milk
or something it'll go bad
almost right away.
Here's another example of
when we run into it.
When we do hard aerobic
exercise, when you're running
or skating really hard, things
you see in the Olympics all
the time, you deplete the oxygen
supply in your blood
when you do hard anaerobic
exercise.
And so the cells have the same
problem of regenerating NADH.
The way they solve it is they
make the lactic acid.
And that contributes to the sore
muscles you feel after
you've done hard anaerobic
training.
The other way of handling this
is to take the 2NADH plus two
hydrogen ions to make it
into acetaldehyde, two
acetaldehydes.
Plus two CO2s.
Oops, excuse me.
Let's do this first.
And then take the 2NADH
plus the 2H+.
Convert this to 2NAD+, and what
we get out of this are
two molecules of ethanol plus
two molecules of CO2.
Again, a process that's very
similar to you, familiar to
you, when I was showing
you yeast growing.
What yeast is doing is it's
carrying out glycolysis and
then it's taking those extra
electrons, putting them on the
pyruvate and making ethanol
and carbon dioxide.
I think there's a fermentation
with what we call a
fermentation with yeast.
I think in that case they're
making bourbon whiskey.
Wine making, beer making,
it's all the same thing.
You have yeast, you're
converting the sugars first to
pyruvate, and then making
ethanol and carboxylic acid.
So anyway.
There's no energy gain out
of this, but these
are important processes.
They're called fermentation.
And they can happen when there's
no oxygen around.
If you recall, there's a version
of photosynthesis,
what I called the second release
of photosynthesis that
began to evolve oxygen
as a waste product.
And then over the next ensuing
millennia, the levels of
oxygen slowly, slowly began
to rise on earth.
And as oxygen levels got to
higher levels, and recall the
Cambrian period, which
is down on the fourth
blackboard down there.
We were only still even there
half a billion years ago.
We were only about 5% the
present oxygen levels.
But as oxygen levels arose, new
metabolic opportunities
became available.
And in particular, cells were
able to get at that energy
which is stored in NADH.
In the absence of oxygen,
NADH is just a nuisance.
You've got to get rid of it.
But as you'll see in a minute,
you can do something
interesting if you have
oxygen around.
So just to look at this from a
broad perspective, if we have
glucose and we have all these
little steps going along to
give the two pyruvate, if
there's, in the absence of
oxygen, they get 2 lactate or
we can get 2 ethanol, 2CO2.
And in both cases, 2ATP.
2ATP.
These processes happening in the
absence of oxygen to get
rid of the, or at least not
requiring oxygen in any case
called fermentations.
However, when oxygen is
available, it became possible
to evolve a new system for
handling these pyruvates.
We go into a biochemical
cycle known as
the citric acid cycle.
And I'll say a word about
this in a minute.
Plus something else that's
known as oxidative
phosphorylation.
This is also referred to as
the respiratory chain.
And what these two sets of
processes together, enable the
cell to take these two 3-carbon
compounds and take
them all the way down to six
molecules of carbon dioxide,
six molecules of water.
And to make a net yield of
36 molecules of ATP.
So if you go by fermentation
a molecule of sugar
gives you two ATPs.
If you go by glycolysis and then
follow it by respiration,
you get 36.
So respiration using oxygen, 18
times more efficient than
by glycolysis.
So in order to understand how
this works though, we have to
talk more about how you
change from one form
of energy to another.
And it's interesting, although
this process had to have
evolved billions of years ago,
it was only relatively
recently that we understood
the principal that was
necessary for this kind
of thing to happen.
It's known as the Chemiosmotic
Hypothesis.
It was proposed by Peter
Mitchell in 1961.
He eventually got a Nobel
Prize for it.
It took quite a long time, it
took more than 10 years for it
to be accepted.
In fact when I was in grad
school in the mid '70s, people
were still arguing whether
this made sense or not.
So here's the way it works.
And we have to consider first
three different forms of
chemical energy that can
be all interconverted.
One of them is familiar to you,
we've been talking about
it all along.
It's a chemical bond.
Energy can be stored in
a high energy bond.
And if we break it to get ADP,
an inorganic phosphate, we can
release energy.
However there's another way
of storing energy as a
concentration gradient.
The principal here would be to
have a barrier, which in this
case is the cell membrane, and
to have a high concentration
of whatever it is on one side,
and a low concentration on the
other side.
And there's energy
stored in that.
If you give it a chance it'll
get to be the same
concentration on both sides.
And the trick is to have
whatever the substance is, is
to have a protein in the
membrane that can permit this
thing to go across in a
controlled fashion.
The third form is electrical
potential.
Again, the membrane actually
acts as an insulator, and all
cells, if this is the inside,
and this is the outside,
there's a gradient of hydrogen
ions, so there are more
hydrogen ions outside the cell
than there are inside.
So it creates an electrical
potential.
And these can't cross the
membrane unless, guess what?
There's a protein in the
membrane that's able to permit
their passage under controlled
circumstances.
So there's basically three
different forms of energy that
can be interconverted.
And Peter Mitchell's great
insight, which I will say was
not intuitive for many people,
was the combination, so the
combo of this proton
concentration gradient plus
the electrical potential, could
be used to drive the
synthesis of ATP.
And let me just say
a couple of words.
Because this may feel,
how could this be?
Could you really have energy?
Well the potential across a cell
is about 70 millivolts.
May not seem all that much.
But remember the membrane is
about three nanometers thick.
So that's about 200,000
volts per centimeter.
High tension wires are 200,000
volts per mile or something.
There's a lot of
power in there.
And furthermore, let's see
if I can bring this up.
I've been showing
you this little
movie a couple of times.
The bacteria with these little
nanomotors are spinning those
flagella, and we saw how there's
this machinery that's
a little nanomotor.
You know how it's powered?
It's powered by the
proton gradient.
A proton trickles its way
through this apparatus from
the outside to the inside.
It's coming down the gradient.
That's the source
of the power.
And as I showed you, it's
a pretty powerful motor.
You can basically glue the
propeller to a slide and it
can twirl the bacteria
all around.
In fact, one of my favorite
demos is, years ago people
took a bacterium, and they
managed to pop it open.
So all the cytoplasm, all of the
stuff on the inside came
out of the cell, and you just
got buffer on the inside.
But it had these flagella.
So you had just shells of
bacteria with nothing really
inside them.
But, if you add a drop of acid
to this media, now you've
created a proton gradient with
more protons on the outside
than are on the inside, and
guess what happens?
The flagella motor starts
working, and the bacteria
start swimming, even though all
the air, talk about dead
man walking or something
like that.
It gives you an idea of the
power that's in this
combination of the proton
gradient and
the electric potential.
The combination of this is
often referred to as the
proton motive force.
So here's the principle
of how the cell is
able to exploit that.
And this is what underlies
respiration.
There are two stages.
Stage one, there's a membrane
with some kind of membrane
protein in it, which is actually
a protein, functions
as a proton pump.
So it's a protein that's
designed to be embedded into a
membrane and to work there.
This part here is the
membrane itself.
The proton gets transported from
the inside to the outside
when energy is put into
this proton pump.
So in response to some energy
producing event, the cell
pumps protons from its inside to
its outside, and this then
establishes the proton
gradient.
The second phase, then, is to
take advantage of that proton
gradient, and there's a
different protein embedded in
the membrane.
It's known as an ATP synthase.
And it permits a proton to come
down the gradient, which
you would want to do.
But if that's all that happened,
all you'd do is
you'd just dissipate
your gradient.
So the key here is that this
proton is only allowed to come
down the gradient to the
energetically more favorable
side if ADP and inorganic
phosphate are bound to this
ATP synthase.
And the dropping of the proton
down the gradient's passage
through this ATP synthase, which
is an energy favorable
reaction, drives the
synthesis of ATP.
So much energy is basically
given off with this, you can
make an ATP and the thing
will still go.
Now interestingly, this ATP
synthase, which really lies at
the heart of our energetics for
how we function as human
beings, is derived from it's
crystal structure.
But in fact, evolutionarily,
it's related to
that flagella motor.
And as that proton comes down
the gradient, or actually this
is presented upside down, so
there's the outside as it goes
through in this direction, the
ATP synthase, which is known
as the F1F0 ATP synthase
rotates.
And probably this came first.
It's a little hard in this one
because you don't have the
flagella, so what scientists
have done is they've been able
to attach something like an
actin filament onto this F1
ATP synthase, and show
that as a proton
passages the thing rotates.
So in all likelihood what
happened in evolution was this
came first, and then later the
machinery got duplicated and
evolved to become a nanomotor.
And as I told you the other day,
that apparatus for the
flagella motor got evolved again
into becoming a little
syringe that bacteria like
ursinia are able to use to
pump or to squeeze proteins or
squirt proteins from inside
them into inside of
a mammalian cell.
OK, well.
Thanks to this work by Peter
Mitchell then, we can now
understand how cells were able
to take advantage of that
energy that was in the NADH.
So this process is known
as respiration.
And basically it's
taking the 2NADH.
I'm supposed to see the physical
therapist today, so I
hope we're going to begin to
make progress to lecturing on
two feet sooner or later.
Plus 2NAD+ plus two water.
So as I said earlier,
NADH and protons,
it's basically hydrogen.
It's the equivalent of having
hydrogen gas and adding
oxygen, and we're burning the
hydrogen gas down to water.
So there's a lot
to yield water.
So there's a lot of energy
potentially can be given off.
That's the 50 kcals per mole.
Now if you recall when we talked
about thermodynamics,
so the NADH is up here, by the
time we get down to the 2NAD+
plus the water, the two waters,
energetically we're down here.
And this is about a free energy
changed of about 50
kcals per mole.
In physiological terms, that's
a huge amount of energy.
And I think some of the
textbooks compare it to
letting a stick of dynamite
off inside of a cell.
So it's really more than biology
figured out how to
handle this in a single step.
But do you remember that
important principle about a
thermodynamic property,
when I had the little
picture of the skier?
It doesn't matter which
pathway you take.
You get the same amount of
energy released whether you go
down the black diamond
slope or you go
down the bunny slope.
So in fact, the way biology has
learned, life has learned
to control this amount of energy
is basically taking the
bunny slope.
And so the energy drop occurs
in a series of stages, where
you have the transfer of two
electrons to a lower state
intermediate, transfer of two
electrons to another one,
transfer of two electrons
to another one.
And where this connects with
the stuff that I just told
you, is as these two electrons
are coming down, what's
happening is a proton is
being pumped from the
inside to the outside.
As it moves to the next lower
energy state, another proton
gets pumped from the inside,
the outside.
And the same thing
happens here.
So at the end, you get the two
hydrogens plus the half of an
oxygen and we get
a water molecule
from these two electrons.
But what's happened is these
three protons have changed
from inside to outside.
That enables the cell
to make three ATPs.
So now instead of throwing away
all that energy, losing
the NADH as in the
fermentations, the cell is
extracting energy out of it by
taking advantage of this
principle of the proton
gradient.
So the game changes
if you're this
evolutionary designer or something.
If you were trying to design
life from first principles
now, you could take
advantage of this.
Well of course it doesn't
happen that way.
Experiments happen all the time
in nature and something
happens and sometimes
it's very efficient,
sometimes it isn't.
But if it's there first
it gets going.
In this case, the need now, or
the opportunity was that if an
organism could get more NADH out
of that original molecule
of glucose, it could make more
energy than somebody else.
And so the ultimate way to take
a molecule of glucose is
if you burn it with, oxygen
you end up with six carbon
dioxides and water.
You burn it all away.
So there's a system that,
in essence, does that.
It's known as the citric
acid cycle.
So you have the pyruvate that
comes from glycolysis.
And the way it's processed is
first, one of the carboxyl
group on the pyruvate
is released, and
this produces acetyl.
You can look to see
what CoA is.
At the moment, it
doesn't matter.
What does matter is this
is a 3-carbon compound.
Acetyl, as you probably know,
is a two-carbon compound.
And when you look in your
textbooks at the citric acid
cycle, you'll see this very
confusing circle with lots of
compounds and enzymes
and stuff.
But I want you just keep your
eye on the ball here.
If you'll notice, the compound
over here is in the cycle, is
four carbons.
And what happens is this
2-carbon compound that was
derived from pyruvate gets
added to this to give a
6-carbon compound.
And then that gets converted to
a 5-carbon compound with a
molecule of CO2 being
given off.
That in turn gets converted to
a 4-carbon compound with
another molecule of
CO2 given off.
And then there's some molecular
gymnastics here that
change the nature of the four
carbon compound a bit so you
can get back into the cycle.
But look what's happened to
those three carbons that were
in the pyruvate.
There's one of them, there's
the other one,
there's the other one.
So this citric acid cycle
produces, it actually makes
some ATP, but it makes
quite a bit of NADH.
And it also makes another, one
more reduced electron carrier.
It's not NADH, it's another one
that's used in the cell.
But anyway, the cell is then
able to take all of this NADH
and this electron carrier plus
these to give you, what I'd
said, the net yield you
get from respiration.
36 ATPs from a single
molecule of glucose.
So sort of quite remarkable
to some extent.
We're looking at evolution,
through, if you will, almost
like looking at biochemical
fossils and then when
something works, it's a living
fossil, we still
find it in our cells.
