BIOCHEMISTRY DISCUSSION 4
Leahh
09HemoglobinanAllostericProtein.pptxBiochemistry A Short Course Fourth Edition CHAPTER 9 Hemoglobin, an Allosteric Protein
Tymoczko • Berg • Gatto • Stryer
© 2019 Macmillan Learning
Created by Brett Barbaro
Biochemistry, chapter nine - hemoglobin, an allosteric protein.
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https://pdb101.rcsb.org/motm/41
HEMOGLOBIN
also see: https://www.youtube.com/watch?v=jVUwn4wWTXI&ab_channel=AndreyK
Created by Brett Barbaro
So we're going to talk about hemoglobin. I thought I would open with a picture of hemoglobin - this is one of David Goodsell's illustrations. And it's cycling between the oxygenated and the deoxygenated form of hemoglobin. So you can see the oxygen there, attaching here to the heme group and detaching. And you can see that that has some large effects on the overall structure of hemoglobin.
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Created by Brett Barbaro
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So hemoglobin is the main component of red blood cells. It's what transports your oxygen. These are red blood cells.
Image courtesy of David Goodsell
Created by Brett Barbaro
And I will show you David Goodsell's, recent drawing - actually he gave this to me personally for this course. And it shows here the membrane, and the spectrin that holds it together, and then all of these little guys inside are hemoglobin. So that's most of what you see inside - and there are some other proteins in here. Some of these are for glycolysis, and some of them have other purposes. And out here in the plasma you can see some antibodies and various fun stuff. But I thought it was important to take a look at the relative sizes of these things.
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red blood cell diameter = ~7.7 um
inset image width = 200 nm (per David Goodsell)
Created by Brett Barbaro
So if you have this picture and made it the proper size relative to an actual red blood cell, this is how big that would be. Ridiculously small. So your red blood cells are full of hemoglobin. They have so much hemoglobin in them. It's kinda ridiculous. It's almost half of the volume of your blood.
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Partial pressure in air
All gasses exert the same pressure, so the partial pressure of a gas is equal to the molar fraction of the gas.
Atmospheric pressure is 760 torr.
The image at right shows the composition of DRY air. Main components are:
Nitrogen 78%
Oxygen 21% (159 torr)
Argon .93%
Carbon dioxide .04%
Other .03%
Created by Brett Barbaro
Now, before we get started in this description of hemoglobin, one of the things we're going to talk about a lot in this is "partial pressure". And partial pressure, which you probably know from chemistry, is equal to the molar fraction of the gas present {times the total pressure}. So {a gas's pressure} doesn't depend on what kind of gas it is. All gases, {as assumed in} the ideal gas law, behave similarly. And therefore, it's just the amount of gas present. Now, atmospheric pressure is 760 torr (millimeters mercury is another way of saying it, or atmospheres - that's one atmosphere is 760). And this image here shows you the composition of dry air. Now, it's important to remember the air you breathe is very rarely actually dry, but it just makes the calculations a lot more easy. So I decided to just go with dry air. So assume you're out in the desert somewhere. And you're mostly nitrogen here, but 21% oxygen. And so you have 21% percent of the gas is oxygen - that's the molar fraction. This is not the weight of the gas, or the volume of the gas. It's the number of particles. And that would make {its partial pressure} about 159 torr. Interestingly, carbon dioxide is very low in here. And the third largest is argon, which I actually wasn't aware of - but apparently there's a lot of argon in our air.
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The partial pressure of a gas dissolved in a liquid is taken to be that partial pressure of gas that would be in equilibrium when that gas is in contact with the liquid.
https://www.compadre.org/nexusph/course/Partial_pressure_-_liquids
Partial Pressure
5.874 mM
.174 mM
oxygen in lungs (100 torr):
Created by Brett Barbaro
Now it talks here about the partial pressure ... of the oxygen in dry air. In the lungs, our partial pressure of oxygen is about 100 torr, which is significantly less - because when you exhale, you don't exhale everything. So when you're inhaling, it's a mixture of what you had in your lungs already and what you just inhaled. So the partial pressure goes down. And I thought this was a really important point to make. The partial pressure of the gas in a liquid is taken to be the amount of gas that would be dissolved in a liquid if it were equal in equilibrium with the gas {at that pressure}. So, yeah, it's kind of weird to talk about the partial pressure of oxygen in blood, because that's not a gas. So it's not really exerting pressure. And oxygen is not very soluble in liquid. So in a gas, this is - I've actually altered this image to make it look more realistic. Has about 33 times as much oxygen per volume as there is in the liquid. So we would call this 5.874 millimolar, versus 0.174 millimolar. So your blood would not normally absorb a lot of that gas.
Hemoglobin enables blood to transport oxygen effectively.
In lungs (100 torr) the concentration of oxygen dissolved in the blood plasma is about .174 millimolar
But due to the hemoglobin, the total concentration of oxygen becomes about 4.5 millimolar
That’s about 25 times as much
Created by Brett Barbaro
However, hemoglobin enables your blood to absorb about 25 times as much. So actually allows it to become almost equal to the {concentration of oxygen} in the air in your lungs. So because of this hemoglobin, you actually end up with a total concentration of oxygen of about 4.5 millimolar. So most of that oxygen is bound to the hemoglobin.
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Chapter 9: Outline
9.1 Hemoglobin Displays Cooperative Behavior
9.2 Myoglobin and Hemoglobin Bind Oxygen in Heme Groups
9.3 Hemoglobin Binds Oxygen Cooperatively
9.4 An Allosteric Regulator Determines the Oxygen Affinity of Hemoglobin
9.5 Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen
9.6 Mutations in Genes Encoding Hemoglobin Subunits Can Result in Disease
Created by Brett Barbaro
Now, on to the meat of the chapter here, we have "hemoglobin displays cooperative behavior", and then "hemoglobin binds cooperatively" here, which is just kind of bizarre, why they did two separate sections that sound almost the same. We're going to compare myoglobin and hemoglobin and talk about heme. And then we're going to talk about the allosteric regulation of hemoglobin. And there're several different things that allosterically regulate hemoglobin. They make it very interesting. Hydrogen ions and carbon dioxide are two of those things. And then finally, we're going to talk about mutations in genes, including hemoglobin subunits. And of course, the most common of those is your sickle cell anemia, which everyone probably has already heard about. So I look forward to going through this chapter with you..
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Section 9.1 Hemoglobin Displays Cooperative Behavior
Learning objective 7: Explain how allosteric properties contribute to hemoglobin function.
Hemoglobin is a red blood cell protein that carries oxygen from the lungs to the tissues.
Hemoglobin is an allosteric protein that displays cooperativity in oxygen binding and release.
Myoglobin binds oxygen in muscle cells. The binding of oxygen by myoglobin is not cooperative.
Oxygen binding is measured as a function of the partial pressure of oxygen (pO2).
~98% of the oxygen in blood is bound to hemoglobin.
Created by Brett Barbaro
So hemoglobin displays cooperative behavior. And this is just a list of facts about hemoglobin. We have that hemoglobin is a red blood cell protein, which we already know. Hemoglobin is an allosteric protein, which you probably know already also. And then myoglobin binds oxygen in muscle cells. So hemoglobin and myoglobin are very similar. And myoglobin is your oxygen reservoir in your muscle cells. Because you have to have lots of oxygen available in your muscle cells in order to do things. Your muscle cells consume a lot of oxygen. So they don't rely entirely on the blood. They have their own supply. But it's slightly different. So we will discuss how that works. And then we'll talk about the binding as a function of the partial pressure of oxygen. Now remember this is the partial pressure of oxygen dissolved in the blood. So it's a little bit different, but we can still talk about it. Just need to make sure we understand what that means. And then 98% of the oxygen in the blood, it's bound to hemoglobin. We talked about there being 25 times as much oxygen bound to hemoglobin as there is dissolved in the blood - that is in the lungs. So all over your body, of course, the amount is going to be different because the oxygen is going to be consumed by your tissues. So it turns out that overall, about 98% of the oxygen in your blood is bound to hemoglobin.
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Curve of Oxygen Binding by Hemoglobin
sigmoidal curve
Created by Brett Barbaro
Figure 9.1 Oxygen binding by hemoglobin. This curve, obtained for hemoglobin in red blood cells, is shaped somewhat like the letter “S,” indicating that distinct, but interacting, oxygen-binding sites are present in each hemoglobin molecule. For comparison, the binding curve for myoglobin is shown in black.
Now, hemoglobin displays cooperative behavior, and that can be seen by this sigmoidal curve. We can see here as compared to myoglobin. Myoglobin is just like your regular Michaelis-Menten kinetics. It's going to bind, and the amount that it binds is going to be pretty well directly related to the amount that is available - until it becomes saturated, and then it can't bind any more. Hemoglobin though, at the very beginning, is not very active. It does not bind oxygen very much. And this is the partial pressure of oxygen here. Here's 100 would be in the lungs, and in your tissues it's about 20%.
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pO2 = 0 torr
0/100 occupied
pO2 = 5 torr
2/100 occupied
pO2 = 10 torr
9/100 occupied
pO2 = 20 torr
38/100 occupied
Cooperativity allows hemoglobin to pass oxygen to myoglobin in the tissues
Created by Brett Barbaro
Figure 9.2 Cooperativity enhances oxygen delivery by hemoglobin. Because of cooperativity between O2 binding sites, hemoglobin delivers more O2 to tissues than would myoglobin.
So we're gonna take a look here at this diagram. And in your lungs, your hemoglobin can absorb quite a bit of oxygen. This is the fractional saturation of the hemoglobin. So almost a 100% of the hemoglobin is bound to oxygen in the lungs. But down in the tissues, because of this cooperative behavior, the hemoglobin is only about 30% saturated. Thirty-three percent, I guess, or thirty-four percent. There's a 66% loss in saturation. Myoglobin does not have this quality - so oxygen will not bind to hemoglobin as much as it does myoglobin, oxygen binds better to myoglobin, at this pressure, than hemoglobin. And that's very important because when you get to the tissues, the myoglobin has to absorb that oxygen. If it was competing with hemoglobin and they were both equally saturated, then the oxygen would not be transferred to the tissues as well. And as far as the mechanism of cooperativity, we have here a diagram from chapter seven, and we can show 25 different hemoglobin molecules here. (This diagram was not initially intended to show hemoglobin.) But there's the tense and relaxed state, the T form and the R form, of hemoglobin - and in the T form (hemoglobin has four subunits, so these are all put together), ... it doesn't bind oxygen that well. But in the relaxed state it does. Now, because of thermal fluctuations, some of these hemoglobin that are not bound to anything will just spontaneously turn into the relaxed state. And when they do that, then they start binding. And if they bind one, then it holds it in that relaxed state - and so it's able to bind three more. And that's what makes this curve so much steeper here. This would be (I did some quick calculations here) probably about 5 torr, 10 torr, and 20 torr. But I actually think that the circular hemoglobins here would be much more occupied than it appears in this diagram.
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Section 9.2 Myoglobin and Hemoglobin Bind Oxygen in Heme Groups (1/3)
Myoglobin is a single polypeptide chain consisting mainly of α helices arranged to form a globular structure.
Myoglobin, like hemoglobin, binds oxygen at a heme, a bound prosthetic group.
http://www.chem.ucla.edu/~rebecca/153A/W11/Lectures/153A_W11_Lec17_Myoglobin&Hemoglobin.pdf
Created by Brett Barbaro
Figure 9.3 The structure of myoglobin. Notice that myoglobin consists of a single polypeptide chain, formed of helices connected by turns, with one oxygen-binding site. [Drawn from 1MBD.pdb.]
So myoglobin and hemoglobin bind oxygen in heme groups. So this heme group is a prosthetic group that is attached to the myoglobin and hemoglobin proteins. Not covalently - it's just sitting there in a very favorable pocket. And here is a comparison between myoglobin and hemoglobin. We can see the structure is generally just a bunch of these alpha helices. They kind of fold up together. So this would be a single subunit of hemoglobin. So the structure is very similar. You can see it curves around here, we've got this group, this group. But of course, hemoglobin polymerizes. It turns into these tetramers, which the myoglobin does not. So the differences between these two are in some ways related to the fact that that happens. But also myoglobin binds oxygen a little bit better than hemoglobin does, even if you don't take into account the cooperativity. And of course, that makes a little bit of sense again - myoglobin has to hold on to the oxygen better than hemoglobin does, because it needs to be able to have a reservoir of oxygen in your muscle cells.
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Section 9.2 Myoglobin and Hemoglobin Bind Oxygen in Heme Groups (2/3)
The heme group consists of an organic component called protoporphyrin and a central iron ion in the ferrous (Fe2+) form.
The iron lies in the middle of the protoporphyrin bound to four nitrogens.
Created by Brett Barbaro
The heme group itself starts off with this interesting thing called the porphyrin... this is a porphin here. They call it a "porphyrin ring" or a "protoporphyrin ring". And that is the heme without the iron. And then the whole thing together. This is a specific type of a protoporphyrin ring. And this is the one that's found in hemoglobin. And this iron sits in the middle of these four nitrogens, and is covalently bound to these four nitrogens. And this is just an interesting structure. I pulled this off of Wikipedia, but you can see here this blue circle is actually a cycle. This is all double bonds. So they're non-localized - they're in resonance with each other. And these pi electrons are actually circulating around this circle. So that's one of the things that stabilizes the molecule in this shape. And I just thought I would share that with you.
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Diagram of Oxygen Binding Changing the Position of the Iron Ion
Iron can form two additional bonds, called the fifth and sixth coordination sites.
The fifth coordination site is occupied by an imidazole ring of a histidine called the proximal histidine. The sixth coordination site binds oxygen.
Upon oxygen binding, the iron moves into the plane of the protoporphyrin ring.
Created by Brett Barbaro
Figure 9.4 Oxygen binding changes the position of the iron ion. The iron ion lies slightly outside the plane of the porphyrin in deoxyhemoglobin heme (left) but moves into the plane of the heme on oxygenation (right).
Now, in addition to the four nitrogens that it bonds to, the iron can bind to two other places. One of the places that it binds to is this proximal histidine, which is right below the heme, and it kind of pulls the iron out of the plane of the heme a little bit. But the other side can bind to oxygen - and when it does, it pulls that iron back up into the plane of the ring. And that change is what makes the entire molecule take a different conformation.
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Created by Brett Barbaro
So this is a little bit more interesting view of that, I think. The heme itself is held in place by these hydrophobic residues which you can see here. And then the oxygen comes in. And this distal histidine is also very important in this. It binds with the oxygen to help stabilize it there.
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Section 9.2 Myoglobin and Hemoglobin Bind Oxygen in Heme Groups (3/3)
CLINICAL INSIGHT
Functional Magnetic Resonance Imaging Reveals Regions of the Brain Processing Sensory Information
The magnetic properties of the heme iron change when it moves into the plane of the protoporphyrin ring.
Functional magnetic resonance imaging (fMRI) can distinguish the relative amounts of oxy- and deoxyhemoglobin.
Functional magnetic resonance can be used to monitor activity in specific regions of the brain by measuring the increase in oxyhemoglobin.
Created by Brett Barbaro
Figure 9.5 Brain response to odorants. A functional magnetic resonance image reveals brain response to odorants. The light spots indicate regions of the brain activated by odorants. [3D model from N. Sobel et al., J. Neurophysiol. 83(2000):537–551; courtesy of Dr. Noam Sobel. 2D slice from R. Osterbauer et al., J. Neurophysiol. 93(2005): 3434-3441.]
Now, an interesting thing about this is that the magnetic properties of that iron change when it's bound to oxygen. When it moves into the plane of the protoporphyrin ring, it has a slightly different magnetism - and that can be detected using functional magnetic resonance imaging. So an MRI can actually tell what state that iron is in, and do a three-dimensional map of which states these irons are in. So it can superimpose that on your brain, and you can take a look and see - the areas that are lit up are the ones that are increased in oxyhemoglobin. There's an increase because when that part of the brain starts to activate, it sends more oxygen and more blood to that part of the brain in order to allow it to function better because it's doing stuff. So if you smell something, they found out that these three areas here, the piriform cortex and the amygdala, the ventral striatum, and the left insular cortex (only on the left side) are lit up - which means that those are the areas that are involved in processing the sense of smell. And at least that's what we take it to be and it appears to be true. So fascinating, amazing stuff we can do with neuroscience, just because of this 0.4 angstrom change in the position of this iron. Yeah, it's unbelievable.
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Created by Brett Barbaro
Now this is stuff that's not in your book, but I thought it was very interesting. Carbon monoxide, and also nitrogen oxide and H2S, can bind to this iron as well. And in fact, carbon monoxide binds 20 thousand times better than regular oxygen. That's free heme - so if you just have heme, not connected to the globin, it really, really prefers binding to carbon monoxide. So why isn't it that all of them are bound to carbon monoxide?
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Created by Brett Barbaro
Well, it's the {globin} itself comes to the rescue here. That distal histidine interacts with the oxygen, but it also prevents the carbon monoxide from binding. Now, if you increase the concentration of carbon monoxide, then of course you can overwhelm the equilibrium and get it to start binding. But it's because you see the geometry here - the oxygen bends off to the side, whereas the carbon monoxide is a linear geometry. And this gets stabilized by this histidine, the oxygen does. And it is resisted by this histidine when the carbon monoxide tries to bind. So those are some of the interesting things that I thought about this.
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Section 9.3 Hemoglobin Binds Oxygen Cooperatively (1/2)
Hemoglobin is a tetramer consisting of two α subunits and two β subunits. Each subunit has a bound heme.
The quaternary structure is best described as a pair of identical αβ dimers (α1β1 and α2β2).
In deoxyhemoglobin, which corresponds to the T state of allosteric enzymes, the αβ dimers are linked by an extensive interface.
Created by Brett Barbaro
Figure 9.6 The quaternary structure of deoxyhemoglobin. Hemoglobin, which is composed of two α chains and two β chains, functions as a pair of αβ dimers. (A) A ribbon diagram. (B) A space-filling model. [Drawn from 1A3N.pdb.]
So hemoglobin binds oxygen cooperatively, and we've stated that that's true - but now let's discuss a little bit more about how that works. So hemoglobin is a tetramer, we've discussed, and there's actually two separate proteins in there. There's alpha subunits and beta subunits. And you could consider that to be one AB dimer bound to another AB dimer, and that's how they like to talk about it. In deoxyhemoglobin we have the tense state here, as we can see. And then here's just a ribbon diagram and the space-filling diagram. And you can see there's quite a bit of space here between these two beta subunits.
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Section 9.3 Hemoglobin Binds Oxygen Cooperatively (2/2)
The transition from deoxyhemoglobin (T state) to oxyhemoglobin (R state) occurs upon oxygen binding.
The iron ion moves into the plane of the heme when oxygen binds. The proximal histidine, which is a component of an α helix, moves with the iron.
The resulting structural change is communicated to the other subunits so that the two αβ dimers rotate with respect to each another, resulting in the formation of the R state.
Created by Brett Barbaro
Figure 9.7 Conformational changes in hemoglobin. The movement of the iron ion on oxygenation brings the iron-associated histidine residue toward the porphyrin ring. The related movement of the histidine-containing α helix alters the interface between the αβ dimers, instigating other structural changes. For comparison, the deoxyhemoglobin structure is shown in gray behind the oxyhemoglobin structure in color.
Now when it goes to the R state, when it becomes oxygenated, it pulls that iron up into the ring, which then pulls this histidine up. And this histidine is attached to this alpha helix. So it ends up pulling up the entire alpha helix. And there's actually a bit of a pivot point over here on the right, so the portion of the helix that is in contact with the alpha subunit over here moves quite a bit. Even though it's only a 0.4 angstrom change, it ends up having a larger effect down the road.
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T state (deoxy) - 2hhb.pdb
R state (oxy) - 1hho.pdb
http://www.chem.ucla.edu/~rebecca/153A/W11/Lectures/153A_W11_Lec17_Myoglobin&Hemoglobin.pdf
The T and R states have shifted contacts between alpha and beta subunits
Created by Brett Barbaro
And you can see here in the T state, this is the helix that gets moved, and this is the alpha helix on the alpha subunit that it's moving against. And this is where it normally sits. But when it gets shifted to the R state, these two residues shift one full residue over, one notch, so to speak, in the alpha helix. And down here, I've showed you in the context of the entire hemoglobin, what that looks like. You can see here, we have sort of an interdigitation of these residues. And over here we have an interdigitation over here, one residue over. And the effect of that is quite dramatic. You can see this whole section here moves up and closes off that gap.
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Diagram of Quaternary Structural Changes on Oxygen Binding by Hemoglobin
T
R
Created by Brett Barbaro
Figure 9.8 Quaternary structural changes on oxygen binding by hemoglobin. Notice that on oxygenation, one αβ dimer shifts with respect to the other by a rotation of 15 degrees. [Drawn from 1A3N.pdb and 1LFQ.pdb.]
And that is analogous to a 15 degree rotation in one of these alpha-beta units with relation to the other. Remember, this is happening on both sides simultaneously. So it whole top of this would basically rotate 15 degrees and will close off this area. And that has a very large effect on the activity of the {complex}.
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Section 9.4 An Allosteric Regulator Determines the Oxygen Affinity of Hemoglobin (1/3)
Learning objective 8: Identify the key regulators of hemoglobin function.
2,3-Bisphosphoglycerate (2,3-BPG) stabilizes the T state of hemoglobin and thus facilitates the release of oxygen.
2,3-BPG binds to a pocket in the hemoglobin tetramer that exists only when hemoglobin is in the T state.
T
R
Created by Brett Barbaro
So the affinity of oxygen to hemoglobin can change, and does change - and it is also regulated by other things in the blood plasma and also the red blood cells. Of course in your red blood cells. Moreso than just the binding of oxygen, oxygen binding does tend to switch it, but it can be allosterically regulated by interactions with molecules in other parts of the protein. So we have here the tense state and the relaxed state. And you can see in the tense state there is this gap. Well, this molecule, 2,3-bisphosphoglycerate, binds in that gap. When it's in the tense state.
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Curves of oxygen binding by pure hemoglobin compared with hemoglobin in red blood cells
SIDE VIEW
1B86.pdb
Created by Brett Barbaro
Figure 9.9 Oxygen binding by pure hemoglobin compared with hemoglobin in red blood cells. Pure hemoglobin binds oxygen more tightly than does hemoglobin in red blood cells. This difference is due to the presence of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells.
Figure 9.10 The mode of binding of 2,3-BPG to human deoxyhemoglobin. 2,3-Bisphosphoglycerate binds to the central cavity of deoxyhemoglobin (left). There, it interacts with three positively charged groups on each β chain (right). [Drawn from 1B86.pdb.]
And the binding of that molecule stabilizes the tense state. So because it's bound, you have to overcome that binding energy in order to transfer to the relaxed state. This is what the area looks like. And this picture, I thought it was - from looking at this, I thought that this molecule was deep inside the hemoglobin, but It's actually not. If you look at the side view here, it's actually way over here on the edge - it's just in between these two beta subunits - which I thought was interesting, so I wanted to share that with you. But looking over here at the effect of the binding - the curve here with no 2,3-BPG shifts quite a bit. So without that stabilization, the hemoglobin much more readily takes up the oxygen. And you would end up only losing about 8% of that oxygen in the tissues. Well, then it would be competing with the myoglobin quite a bit. You want to release as much as possible. So what you need to do, then, is stabilize that tense state. And basically when it gets to the tissues, that molecule wiggles in there and basically squeezes the oxygen out. And that's why you're able to release so much more oxygen in the tissues. It's thanks to this guy in there squeezing the oxygen out of your hemoglobin.
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Section 9.4 An Allosteric Regulator Determines the Oxygen Affinity of Hemoglobin (2/3)
CLINICAL INSIGHT
Hemoglobin’s Oxygen Affinity Is Adjusted to Meet Environmental Needs
Fetal hemoglobin must bind oxygen when the mother’s hemoglobin is releasing oxygen.
In fetal hemoglobin, the β chain is replaced with a γ chain.
The fetal α2γ2 hemoglobin does not bind 2,3-BPG as well as adult hemoglobin. The reduced affinity for 2,3-BPG results in fetal hemoglobin having a higher affinity for oxygen, binding oxygen when the mother’s hemoglobin is releasing oxygen.
Created by Brett Barbaro
Figure 9.11 The oxygen affinity of fetal red blood cells. The oxygen affinity of fetal red blood cells is higher than that of maternal red blood cells because fetal hemoglobin does not bind 2,3-BPG as well as maternal hemoglobin does.
Now, there are other ways to adjust the oxygen affinity of hemoglobin. And a great example of this is fetal hemoglobin. Now, we have normal hemoglobin as your alpha chains and beta chains. In fetal hemoglobin, the {beta} chain is replaced with a different chain. It's a different gene that gets expressed in the fetus than in adults. And it's very similar, but has a greater affinity for oxygen. So it is hemoglobin - it's not myoglobin - but it has greater affinity to oxygen, so the maternal hemoglobin will release oxygen and the fetal red cells will absorb it and transfer it to the baby. And the reason that it has that {increased O2} affinity is because of its reduced affinity for 2,3-BPG. That molecule does not attach as well, so it doesn't squeeze out the oxygen as well. And that's how you're able to transfer oxygen from the maternal blood to the fetal blood.
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Section 9.4 An Allosteric Regulator Determines the Oxygen Affinity of Hemoglobin (3/3)
BIOLOGICAL INSIGHT
Hemoglobin Adaptations Allow Oxygen Transport in Extreme Environments
The bar-headed goose can fly over Mount Everest, where the oxygen concentration is low.
Changes in hemoglobin that facilitate the formation of the R state may account in part for this remarkable ability.
Created by Brett Barbaro
Figure 9.12 Bar-headed goose. [Tierbild Okapia/Photo Researchers.]
And you can have other forms of hemoglobin in other animals, depending on their environment, the needs that they have. This goose, for example, can fly and exist where oxygen concentrations are extremely low. And that's because of changes in its hemoglobin. So there are many, many different variations of hemoglobin in different animals and they're all suited for their environments.
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Section 9.5 Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen (1/3)
Carbon dioxide and H+, produced by actively respiring tissues, enhance oxygen release by hemoglobin.
The stimulation of oxygen release by carbon dioxide and H+ is called the Bohr effect.
Created by Brett Barbaro
Figure 9.13 Carbon dioxide and pH. Carbon dioxide in the tissues diffuses into red blood cells. Inside a red blood cell, carbon dioxide reacts with water to form carbonic acid in a reaction catalyzed by the enzyme carbonic anhydrase. Carbonic acid dissociates to form HCO3− and H+, resulting in a drop in pH inside the red cell.
Now hydrogen ions and carbon dioxide also promote the release of oxygen. So they are also allosteric regulators of hemoglobin. And they are produced - carbon dioxide, of course, is produced in your mitochondria when it is doing {respiration} and it dissolves out into the blood. Once again, probably not that good at dissolving in the blood, but your red blood cells can turn it into this bicarbonate. We'll talk about that in a second. The result of this is the production of hydrogen ions - so that makes the blood more acidic. And this has an effect on your hemoglobin, as we will discuss, which is called the "Bohr effect".
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Diagram of the Chemical Basis of the Bohr Effect
Low pH allows the formation of ionic interactions that stabilize the T state of hemoglobin, enhancing oxygen release.
Created by Brett Barbaro
Figure 9.15 The chemical basis of the Bohr effect. In deoxyhemoglobin, three amino acid residues form two salt bridges that stabilize the T quaternary structure. The formation of one of the salt bridges depends on the presence of an added proton on histidine 146. The proximity of the negative charge on aspartate 94 in deoxyhemoglobin favors the protonation of this histidine. Notice that the salt bridge between histidine 146 and aspartate 94 is stabilized by a hydrogen bond (green dashed line). The green sphere represents the remainder of the 1 chain.
So one aspect of this Bohr effect is the stabilization of the T state by these protons from your carbon dioxide dissolving. It lowers the pH, increases the number of protons, and that increases the binding of these residues here and also this one. But where are these residues? They are right here. Now remember these guys? These are the ones that are right at the interface of your alpha and beta subunits. And this is the one that changes the conformation from T to R. So if you're stabilizing this in the T state, then you are basically reducing the affinity for oxygen. You're preventing the hemoglobin from getting into that R state. And down here between your B subunits, beta subunits, is the place where that 2,3-BPG binds. So I just wanted to show you on the entire molecule where the relative positions of these things are. And also note these little blue guys.
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Curve Displaying the Effect of pH on the Oxygen Affinity of Hemoglobin
Created by Brett Barbaro
Figure 9.14 The effect of pH on the oxygen affinity of hemoglobin. Lowering the pH from 7.4 (red curve) to 7.2 (blue curve) results in the release of O2 from oxyhemoglobin.
So the effect of this reduction in pH, going to pH 7.2 here, is a shift of the curve. And it results in a greater release of oxygen in the tissues. As the blood gets more acidic, it causes the hemoglobin to release more oxygen - which makes sense, because if the blood's acidic, that means that that tissue is doing stuff. It needs more oxygen. So this works out. It's evolved to allow a greater release of oxygen in tissues that are actively metabolizing.
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Section 9.5 Hydrogen Ions and Carbon Dioxide Promote the Release of Oxygen (2/3)
Carbon dioxide reacts with terminal amino groups to form negatively charged carbamate groups. The carbamate forms salt bridges that stabilize the T state.
Carbon dioxide and H+ are heterotropic regulators of oxygen binding by hemoglobin.
Created by Brett Barbaro
Another thing that happens is the attachment of carbon dioxide to the end termini of the beta subunits. And you can see here - oh no, not just the beta subunits, I'm sorry. All of the subunits. These two subunits do get this carbon dioxide attached to them, forms this carbamate. Also increases the amount of protons around. But it also changes this from a regular old amine group to a carboxyl group, which is great deal more negative, and allows the formation of these salt bridges here between the ends of the subunits. So this is a large arginine, and it forms a salt bridge with the N terminus of this subunit. And that further stabilizes the T state and squeezes out even more oxygen.
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Curves Displaying the Effect of Carbon Dioxide
Created by Brett Barbaro
Figure 9.16 Carbon dioxide effects. The presence of carbon dioxide decreases the affinity of hemoglobin for oxygen even beyond the effect due to a decrease in pH, resulting in even more efficient oxygen transport from the tissues to the lungs.
So we talked about the regular (without any kind of interaction like this) curve would be down to here. And you would end up losing about 66% of your oxygen in the tissues. But because of the lowering of pH, as we talked about, it goes down to 77%. And then again, with the component of the addition of those carbon dioxides to the N termini, it squeezes out some more oxygen. And so you end up losing 88% of your oxygen due to these allosteric regulations.
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Diagram of the Transport of Carbon Dioxide from Tissues to Lungs
Carbon dioxide is transported to the lungs as bicarbonate.
Carbonic anhydrase facilitates the formation of bicarbonate ions.
For more on carbonic anhydrase, visit: https://pdb101.rcsb.org/motm/49
Created by Brett Barbaro
Figure 9.17 The transport of CO2 from tissues to lungs. Most carbon dioxide is transported to the lungs in the form of HCO3− produced in red blood cells and then released into the blood plasma. A lesser amount is transported by hemoglobin in the form of an attached carbamate.
And then finally, the carbon dioxide has to get out of the system somehow. And that works by, inside these red blood cells, an enzyme called carbonic anhydrase, which facilitates the formation of bicarbonate ions and {decreases} the pH. So those bicarbonate ions get transported in the blood, and then turned back into carbon dioxide in the lungs and exhaled.
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Section 9.6 Mutations in Genes Encoding Hemoglobin Subunits Can Result in Disease (1/2)
CLINICAL INSIGHT
Sickle-Cell Anemia Is a Disease Caused by a Mutation in Hemoglobin
Sickle-cell anemia is a genetic disease caused by a mutation resulting in the substitution of valine for glutamate at position 6 of the β chains.
Sickle-cell anemia can be fatal when both alleles of the β chain are mutated.
In sickle-cell trait, one allele is mutated and one is normal. Such individuals are asymptomatic.
Sickle-cell hemoglobin is called hemoglobin S (HbS). The substituted valine is exposed in deoxyhemoglobin and can interact with other deoxy HbS to form aggregates that deform the red blood cells.
The sickled cells clog blood flow through the capillaries, leading to tissue damage.
Created by Brett Barbaro
So mutations in genes encoding hemoglobin subunits can result in disease. And this is an example that's the most common that you'll hear about, but it's very interesting from a mechanistic and a structural viewpoint. Sickle cell anemia is a disease caused by mutation in hemoglobin. So you have your normal hemoglobin, usually has a glutamate at position six of the beta chains, and a mutation that causes it to have a valine instead ends up causing sickle cell anemia. Or "sickle cell condition". We'll talk about that. If you have your sickle cell in both of your genes, because remember we've got two genes for everything - if both of them have this substitution, then it can be fatal - it can be really bad. But if you just have one, actually, so basically half of your hemoglobin will be this special form and the other half won't, then you don't have any of those symptoms, so it's not so bad. And in fact... it will still form these fibers, is what it does. It polymerizes and forms fibers. And those fibers will still form if you have one copy of the mutated gene. But they won't be so bad that it will cause trouble. But it will also be forming these fibers which prevent a particular parasite from getting into your red blood cells. And that parasite is the parasite that causes malaria. So having one of these be your glutamate substituted with valine will actually be protective. It's called hemoglobin S. And that will reduce your chances of getting malaria.
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Image of Sickled Red Blood Cell
Created by Brett Barbaro
Figure 9.18 Sickled red blood cell. A micrograph showing a sickled red blood cell adjacent to normally shaped red blood cells. [Eye of Science/Photo Researchers.]
The reason that these hemoglobins cause so much trouble in sickle cell anemia is that it forms these large fibers which distort the shapes of your red blood cells and they clog your blood vessels. And also they're very weak and tend to break open and not live as long. And so you just end up not able to get the blood that you need. And also you start getting these weird clots.
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Electron Micrograph of Sickle-cell Hemoglobin Fibers
Created by Brett Barbaro
Figure 9.19 Sickle-cell hemoglobin fibers. An electron micrograph depicting a ruptured sickled red blood cell with fibers of sickle-cell hemoglobin emerging. [Courtesy of Dr. Robert Josephs and Dr. Thomas E. Wellems, University of Chicago.]
These are the fibers that are creating this condition. And you can see these long things - it's just jam packed with them. This is not your normal hemoglobin. This is not very functional, probably. And it's interesting actually - it's the deoxy form of the hemoglobin that ends up forming these fibers. So this is deoxygenated hemoglobin and I believe it holds it in that position so that it's not able to get oxygenated. So once you have your hemoglobin in these fibers, then you're no longer able to transport oxygen with it.
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Created by Brett Barbaro
(a) Electronic microscopy image of structure of a single sickle hemoglobin (HbS) fiber. (b) Reconstruction of the HbS fiber with a sphere model. (c) Mesoscopic modeling of HbS molecules (left) by patchy particles (right). Green and blue represent lateral and axial intra-double-strand contacts. Red signifies the inter-double-strand contacts. (d) Sequential snapshots of HbS polymerization from a nucleus to a fiber.
Now, what are these fibers? And it's funny, I had always thought that it was just a long string of hemoglobin, but it turns out that it's several long strings of hemoglobin kind of wrapped around each other. This is an electron micrograph of what it looks like, and this is an illustration. So each one of these circles would be a hemoglobin. And they did this model and tried to figure out how such a fiber would grow. And in the end, it ends up being 14 subunits of hemoglobin per layer here, and it layers and causes this large fibrillar structure, which is pretty rigid.
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Structure of Deoxygenated Hemoglobin S
Created by Brett Barbaro
Figure 9.20 Deoxygenated hemoglobin S. The interaction between valine 6 (blue) on a β chain of one hemoglobin molecule and a hydrophobic patch formed by phenylalanine 85 and leucine 88 (gray) on a β chain of another deoxygenated hemoglobin molecule leads to hemoglobin aggregation. The exposed valine 6 residues of other β chains participate in other such interactions in HbS fibers. [Drawn from 2HBS.pdb.]
Now here's your picture from your book about the interaction. And it's a valine here. This is the valine 6 that gets substituted for the glutamate. And this then causes - it causes some changes in the structure overall. But one of the main things that happens is {the residue} becomes hydrophobic - glutamate is hydrophilic. And it also becomes a little smaller. And that allows it to interact with a hydrophobic patch on another hemoglobin (the hemoglobin S). And that's what ends up causing these interactions and the formation of these fibrils.
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Finally, here is a movie for you: https://youtu.be/-1_qkwQkB_c - WEHImovies - Haemoglobin and Sickle Cell Anaemia
Cells are not necessarily sickle-shaped!!!
Shapes vary a lot. These cells were imaged using “soft X-ray tomography”.
Created by Brett Barbaro
But kind of interesting - I didn't realize this, but I found this and I thought I would share it with you. Sickle cells are not necessarily sickle-shaped. They did these soft x-ray tomography. Actually, I know the person that did this. And you get a lot of different shapes that form in sickle cell - but they're all characterized by these large fibril formations protruding through the membrane. And of course, you can see how these would be very fragile, easily breakable, and they are in fact. Now finally, this is a movie. This is WEHI movies - it's a great source of movies - and I am going to show it to you. This is not an exact molecular representation. They just use little spheres or whatever. But you can see this is the oxygens coming in and binding the heme. And then they fly away and you have de-oxygenated {hemoglobin}. Now this is the valine, I think - it's a very strange representation of the valine, because, as a matter of fact, valine is smaller than the glutamine. But when that valine gets exposed, it ends up polymerizing into these shapes. And that is a much better idea of what the actual fibers look like. So anyway, I thought that was cool. I hope you guys enjoyed this, and feel free to ask any questions. Thanks.
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EXTRA SLIDES
Created by Brett Barbaro
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"I would love to see an explanation (even a great movie) of the complete cycle between T and R states that includes oxygen binding and release, the quaternary structural changes"
this was covered on the second slide of Chapter 9, but here is another reference that you might find useful:
https://www.youtube.com/watch?v=jVUwn4wWTXI&ab_channel=AndreyK
"the influences of CO2 and Hydrogen, and 2,3-BPG in human hemoglobin as it moves between the T and R states AND as it moves from the lungs to the tissue in the body. Thanks!"
This is really more of a medical question, but here goes:
The overall picture is summarized pretty well in this image from Wikipedia, where red represents oxygenated blood heading to the tissues, and blue represents deoxygenated blood heading back to the lungs (https://en.wikipedia.org/wiki/Oxygen_saturation_(medicine)):
1. Blood becomes oxygenated in the lungs. When deoxygenated blood arrives at the lungs, most hemoglobin is in the (deoxygenated) T state, stabilized by bound CO2, H+ ions, and 2,3-BPG. To bind oxygen, hemoglobin must overcome the energy barriers created by these stabilizers and reach the R state. When blood passes through the pulmonary capillaries, carbon dioxide is released into the alveoli and oxygen dissolves into the blood plasma to give it a concentration of about 0.174 millimolar. The release of carbon dioxide concurrently reduces the number of H+ ions, thus removing both of these barriers to the R-transition (this is the Bohr effect). Thermodynamic fluctuations cause individual hemoglobin tetramers to dissociate from 2,3-BPG and enter the R state. At this point, oxygen can bind and trap hemoglobin in the R state, allowing its other binding sites to rapidly fill up and create a large energy barrier for transition to the T state. In this way, most of the hemoglobin becomes bound to oxygen. Hemoglobin allows the blood to absorb about 25 times as much oxygen as it would normally, and the pool of oxygen in the hemoglobin keeps the blood plasma oxygenated as it travels through the body.
2. Blood travels to tissues, where the oxygen has been used up and replaced with carbon dioxide. In the capillaries, oxygen travels from the blood plasma into the tissues by simple diffusion, from a place of high concentration to a place of low concentration. Similarly, carbon dioxide diffuses from the tissues into the blood, where it can bind hemoglobin directly, or be converted to carbonic acid, increasing the number of H+ ions.
3. As oxygen leaves the blood plasma, it is replaced by oxygen being released from hemoglobin, keeping the blood plasma oxygenated. This continues until oxygen levels deplete to the point that some hemoglobin tetramers are completely empty. At this point, the tetramers can shift to the T state, which is stabilized by the increasing levels of CO2 and H+. Once in the T state, hemoglobin tetramers can bind 2,3-BPG, which prevents them from shifting back to the R state. At this point they can no longer bind oxygen. This continues until most of the hemoglobin is in the T state and the blood is completely deoxygenated.
4. Blood returns to the lungs, and the process starts over again.
There are many other structural and biochemical processes that occur during this cycle, but we are just focusing on these few important ones.
Created by Brett Barbaro
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