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JOANNE STUBBE: The last time, we were finishing the first part

of the reactive oxygen species module, focused

on how we as humans fight bacterial or viral infections

using neutrophils, the white blood cells.

And I introduced you to this cartoon.

So here's our neutrophil, all of this blue stuff.

It has an unusual-looking nucleus.

Neutrophils have weird nuclei.

They somehow sense the bacteria you saw, the bacteria getting

chased by the neutrophil.

And then somebody asked me this question.

Somebody asked me this question last time

about where the NOX2 proteins end up.

And let me reiterate again that they can

be found in multiple membranes.

The predominant membrane in neutrophils

is in these little vesicles within the cell,

OK, but the bacteria out here, so they need

to somehow engulf the bacteria.

So they can also be found in the plasma membrane.

And they need to engulf the bacteria to form phagosomes.

And so here you see them again.

So this is the phagosome with the NOX2 protein,

with this predisposition where the NADPH is in the cytosol.

OK?

So you see that the location here, the NADPH

is also in the cytosol.

And so you need to think about where you're located.

And we'll see in a few minutes that there

are lots of different kinds of NOX proteins,

and they all have different locations

and all have different factors that

control the regulation of all of these things.

OK, so what I want to do today is start out by looking,

talking about the NOX complex, what the chemistry is,

because it's not only involved in killing bacteria,

but we'll see-- and you've already seen in last

week's recitation--

in the signaling process.

It's the same protein.

And so once the bacteria are engulfed,

you now have a phagosome within the neutrophil.

And here's the NOX complex.

Remember, it's composed of two proteins,

the 91-kilodalton glycoprotein and a 22-kilodalton protein,

both membrane-bound.

I drew those on the board last time.

And the NADPH/NADP is in the cytosol.

But all the chemistry is going to happen

in the lumen of the phagosome.

OK, so somehow the reducing equivalents from the NADPH

need to be transferred into the lumen, where oxygen is going

to be reduced to superoxide.

So I want to talk a little bit about how that happens first,

given what we know about the cofactors in NOX2 that

are essential for this process.

And then what we'll do is look a little bit at how

the superoxide that's generated in the phagosome

gets converted to hydrogen peroxide,

and ultimately with another protein

we're briefly going to discuss, a heme-dependent protein,

myeloperoxidase, which uses chloride,

can form hypochlorous acid.

OK?

So that's where we're going.

This is a cartoon I drew on the board

the last time, where you can see that we

have a flavin domain, a flavin domain that's

located in the cytosol.

And then you have two hemes.

And the unusual part about this system is that these hemes--

we've seen heme before with reversible binding of oxygen

in hemoglobin-- they're both hexacoordinate.

So there's no binding site for oxygen.

So the chemistry is not happening by reversible binding

of oxygen to the heme.

In fact, you know, how close this is located to the surface,

we don't have any structure of this,

but somehow this reduction has to occur

by an electron transfer process that

probably occurs through the edge of the perforin system.

So what you have here-- and I think

this is an important teaching point,

because I think there are only two ways inside the cell

that you control all the redox balance.

The redox balance within NADPH and flavins

really play a central role in everything,

so you really need to understand how these cofactors work,

and how they're controlled.

And so if you have a flavin--

and this is also written-- you don't have to write this down.

This is written on the next handout,

so you don't have to draw all this stuff out.

OK, so I'm going to draw the business end of the flavin.

OK, so this can be flavin adenine dinucleotide.

So this is the oxidized state.

And the two important places where redox chem--

redox chemistry, but look at flavin.

If you don't know anything about heterocyclic chemistry,

it's confusing.

But in fact, we know a huge amount about flavin chemistry

from studying model reactions in organic chemistry.

Decades ago, Tom Bruce did that.

And so the two places where the chemistry in general happen

are either the inside--

so this is 1, 2, 3, 4.

This is the N5--

or the C4A position, and this is the C4A position here.

And I'm not going to go through this in detail

but this is the oxidized form, and what

we have is the reaction of this oxidized form with the reduced

form of NADPH.

And I'm not going to draw out the whole structure here

either.

But hopefully you all know now that NADPH and general

works by hydride transfer, so it's almost always a two

electron transfer.

The one electron chemistry is really

outside the realm where it would normally

happen inside the cell.

So basically the chemistry involves

transfer of a hydride, a hydrogen

with a pair of electrons to the N5 position.

And so you go from the oxidized state to the reduced state.

And so again I'm not going to draw out the whole flavin.

The rest of the--

I guess I don't have a picture there but--

well, let me just show you where we're going.

So the key is, which is interesting about this,

we need to get across a membrane.

So how do you get across a membrane?

So the flavin domain is way over here,

and we need to have two hemes.

Remember you can do electron transfer over 10 to 15 axioms

with very fast rate constants.

Somehow these reducing equivalents from NADPH

need to be transferred to the flavin.

And the major function of the flavin inside the cell

is to mediate two electron one electron chemistry.

And here's an example that.

The two-electron chemistry is being provided

by the NADPH ass a hydride.

But what we have to do is the hemes in this system are

in the plus 3 oxidation state, so what

we need to do is be able to convert-- ultimately

we want to reduce oxygen to super oxides,

so we need an electron.

So that electron is coming from NADPH.

So we need to have an electron transfer--

a single electron transfer to the heme because it only can--

iron can only be reduced by one electron.

So we end up then with this system.

So this is the reduced state of the flavin.

And you can draw a resonance structure.

This is deep-- you can draw all kinds of resonance structures

with flavins.

That's why I can they can do one-electron chemistry.

So one-electron chemistry, you can make the one electron

oxidized a reduced state depending

on which state you're starting in

and the electrons are delocalized.

They turn out to be blue, or they turn out

to be red depending on the prognation states.

So what you can then do is--

let me just write that over here.

So I'm just drawing a resonance structure of that

so you can see--

let me not.

And again let me just show you that that's there so you

don't need to write that down.

You don't need to write this down.

It's all there.

So just pay attention to me.

So you're going to do an electron transfer to reduce

an iron 3 to an iron 2.

Then you've got another iron 3 because we got

to get through this membrane.

So that heme-- and again, this is where the redox potentials

become critical--

can transfer an electron because now in the reduced state

to the other heme so that becomes in the iron 2 state.

So the one out here is in the iron 2 the state.

Now it can transfer electron from oxygen

to form super oxide.

But at the same time during this process,

we're transferring the two electrons

that the Flavin received from the NADPH one at a time,

so then we can repeat that process.

And that's why you get the stochiometry

of the overall reaction.

You get two super oxides produced.

So you have a resonance form of this.

Let me see.

I think I need to write over here.

So anyhow you have a resonance form of this where you--

And now we're ready to do-- so this is the same as--

so this is a resonance form.

These are the same structures.

In the flavin, this is attached either to adenine

or to a ribose biphosphate FMN versus FAD.

And now what you're ready to do is you have the heme.

And so the heme again is embedded in the membrane.

And so now what you're doing is electron transfer.

So you do an electron transfer reaction

and you get this structure.

So make a big dot for the radical.

And now we've reduced one of the hemes to iron 2.

And if you look at the redoxx potentials--

I haven't ever read these original papers--

but they're close to being matched

in terms of redox potentials.

I haven't read how they measured these kinds of things,

but the system needs to be set up so that you

can do transfer the electrons across the membrane

and ultimately reduce the super oxide over here.

And so now what happens so you've gotten to this stage.

So now you have a semi quinone form of the flavin.

this guy can then be re-oxidized by the next heme,

generating the iron 2 form and regenerating the iron 3 form.

So this guy then--

so let's-- to distinguish between them--

again I don't think this-- hopefully most of you were

seeing this, but you have another one,

so it donates an electron to covert the iron 3 form so we

have an iron 3 form to the iron to form.

And it itself becomes re-oxidized.

And so now what's happening is you're

set up to do another electron transfer where this is going

to go to the iron 3, transfer it again, and in the end

this guy is now in the lumen--

adjacent to the lumen.

I don't know where it's located.

We don't have a structure but this guy

is probably through the hemage going to convert oxygen

into super oxide.

So is everybody following that?

The main point here is you all hear about the flavin

being the major mediator between one-electron chemistry

and two-electron chemistry.