I know I promised a couple of posts on enzyme mechanics, but before I get to that, I’ve realized that I need to go back and review some information about hemoglobin and myoglobin. We got this lecture right before our first test, so I wasn’t paying a terrible amount of attention during it, and most of my notes consist of, “Ooh, Synnecrosis? Kizh? Cal?” which means I was mostly paying attention to details that would affect my SciFi book. Oh well, at least now the one paragraph that I wrote about oxygen transport will be accurate. Or something.
So yeah! Why don’t we spend a lil time talking about our favorite lil oxygen carrier and its cousin?
I knew you’d be down. 😉
(I swear, I get worse and worse at writing these intro bits…)
Oxygen is, as I’m sure you’ve learned by now, as an aerobic organism, quite important to our existence. We use it as a final electron receptor in respiration, which means we need it to, you know, make more than two ATP per glucose molecule. However, oxygen doesn’t exactly like to just sit around in our blood—my notes say that, if oxygen had its way, there would only be 0.3 mL of it in each deciliter of blood.
That’s where hemoglobin comes in. With its four heme groups, it binds molecular oxygen and transports it throughout our blood rather efficiently. Thanks to hemoglobin, we can count on there being about 20mL of oxygen per deciliter of blood under saturating conditions. That’s a bit more than 0.3 mL. Just a bit.
So, how does this oxygen transporter work? Well, it turns out, that’s a bit of a complicated question right off the bat. Thankfully, hemoglobin has a much less complicated cousin called myoglobin.
Myoglobin is a monomeric (one-unit) oxygen-carrying protein that’s found in muscles. Its structure is significantly easier to study than hemoglobin, but it looks ridiculously similar to one hemoglobin’s subunits. (In fact, my biochemistry professor said he was tempted to award an immediate A to anyone who could distinguish the two based on visual inspection alone.) As you would expect, this means that myoglobin works very similarly to hemoglobin, which makes sense, since it earns its keep by providing muscles with oxygen when hemoglobin is falling down on the job (you know, whenever the cell is respiring).
The strictly protein part of myoglobin, without the heme, is referred to as “apo-myoglobin,” and is composed of eight alpha helices in an “all alpha” structure. These eight helices make a nice little pocket in which a heme, which contains an iron dication coordinated with four pyrrole nitrogens, can situate itself. The Fe2+ bonds to the imidazole of histidine 8 on the sixth (F) helix, which is called the proximal histidine. Opposite this is where oxygen will bind—this site is near His 7 of the fifth (E) helix, the distal histidine. The presence of the heme actually stabilizes the protein because it is hydrophobic, and the interior of the protein is hydrophobic. The additional hydrophobic groups help keep the hydrophobic interior of the protein happy.
So, how is this stuff relevant? Well, it turns out that this very precise arrangement affords the protein the ability to bind oxygen in a way that I can only describe as “neat-o.” You see, the Fe2+ in the deoxy-Mb (myoglobin without oxygen) is being pulled out of the plane of the heme by about an angstrom (about the length of an O-H bond) by the histidine bound to it. Since Fe2+ likes to have six ligands (instead of the five it has in deoxy-Mb), it will bind oxygen in that binding site opposite the proximal histidine. However, the distal histidine, the one that’s just kind of hanging out there above the binding site, is just close enough that the oxygen can’t bind to it straight-on, but instead has to bind at an angle. This prevents the very tight binding of poisonous carbon monoxide, which prefers to bind linearly rather than in a bent conformation.
Here’s an image of the whole setup, courtesy of Smokefoot. The green bit at the bottom is the proximal His, and the distal His isn’t shown.
When the oxygen binds the Fe2+ in the heme, it pulls the iron back into the plane. The iron pulls on the proximal histidine, which pulls on the helix that it’s attached to and ultimately changes the structure of the protein.
Like I said, neat-o!
So, how would we describe this binding of oxygen to Mb? Well, you can thank a few chemists who liked math, because they figure that out with an equation. [uproarious whooping in the background, I’m sure]
It’s not too difficult to imagine that deoxy-Mb is in equilibrium with oxy-Mb. If you define Y as the fraction of Mb bound with oxygen, Y is equal to the partial pressure of oxygen (essentially, the concentration of oxygen) divided by the sum of the partial pressure of oxygen and the partial pressure of oxygen required to saturate 50% of the myoglobin, which in this case, is 2 Torr. Yeck, not a fun definition, right? I know. Here’s an easier way to look at it, though:
Y = pO2 / [pO2 + P50]
Quick, before I say anything else, sis! What kind of curve is that?
(If I don’t see you comment, I’m going to be annoyed… =3=)
You were right, you smart lil’ cookie! It’s a hyperbolic curve!
What that means, practically, is that, when pO2 is a lot smaller than P50, increasing the partial pressure of oxygen will increase the fraction of Mb bound linearly. Double the partial pressure of oxygen? Double the fraction of Mb bound. Easy peasy. Then, when the partial pressure of oxygen equals P50, half of the Mb is bound. Since that’s the very definition of P50, that’s not too tricky. Finally, when your partial pressures of oxygen get a lot larger than P50, Y approaches pO2 / pO2, and the fraction of Mb bound is essentially 1. Since the partial pressure of oxygen in our tissues is way above 2 Torr, myoglobin is essentially 100% saturated in our tissues. Cool, huh?
(Yeah, I hate the math, too. It’s important, unfortunately, because hemoglobin is, as Sapphire’s fiance says, “a problem child.”)
Okay, okay, that’s all well and good, but what about hemoglobin? That’s the main player, right?
Right you are. Let’s take a look at it, shall we?
Unlike myoglobin, hemoglobin is made up of four pieces, called subunits, two alpha and two beta. This makes it a tetramer, although, because it’s made up of two alpha-beta pieces, it can also be described as a “dimer of dimers.” Since the alpha and beta subunits of hemoglobin are so incredibly similar to myoglobin, Hb can be said to be made up of four Mb-like structures. Since, like myoglobin, each subunit contains a heme, a single hemoglobin protein contains four hemes, and can bind up to four molecules of oxygen. The subunits are held together by noncovalent forces—oppositely charged groups on different subunits interact to form salt bridges, hydrogen bonds form between H-bond acceptors and H-bond donors on different units, and hydrophobic pieces interact with other hydrophobic pieces.
We talked about the binding of oxygen to myoglobin above. Would you expect the same kind of thing to work for hemoglobin? If you’re like me, you would—after all, each little piece is basically a myoglobin protein, right? Turns out, though, that God got even more creative than you’d think. Rather than oxygen binding of hemoglobin being dependent only on the amount of oxygen the protein has available, like with myoglobin, it’s dependent on the amount of oxygen and whether other subunits have bound oxygen already.
This is called “cooperativity,” and it’s characteristic of hemoglobin. To explore this idea a little better, let’s look at an equation for the binding of oxygen to Hb based on partial pressures of oxygen. That equation looks like this:
Y = pO2n/ [pO2n+ P50n]
where P50 for hemoglobin is 26 Torr.
You’ll notice that this equation looks almost like the one for Mb, except now we have those suspicious-looking exponents. Those coefficients mean that the equation for the binding of Hb is sigmoidal, not hyperbolic, when n > 1. They also mean that, at low pO2, increasing partial pressures of oxygen doesn’t linearly increase the binding of hemoglobin.
That’s all well and good, but why the heck are those freaking coefficients there?
The coefficients are called “Hill coefficients,” and they describe the degree of cooperativity between the subunits of the hemoglobin. When n = 1, the four subunits behave like four independent Mb units, and the curve is hyperbolic. When n > 1, though, the binding of one oxygen increases the ability of hemoglobin to bind another oxygen. The maximum value of the Hill coefficient is the number of binding sites in the protein. The experimentally derived value of n for hemoglobin is 3.3, which means that hemoglobin displays nearly-perfect cooperativity. What that means practically is that, each time an oxygen molecule binds to hemoglobin, it betters the ability of the protein to bind the next molecule of oxygen.
Why is that? Well, you remember that whole bit with the iron getting pulled into the plane by the oxygen binding it in myoglobin? That happens in hemoglobin, too, except, in hemoglobin, the other subunits are helping keep the iron pulled out of the plane in the first place. The binding of the first heme in the first subunit, therefore, is relatively unfavorable (said to be in the “T-state”, or “tense-state”). When the first oxygen binds to the first subunit of Hb, it tugs on that Fe2+, which in turn tugs on the proximal His, which then tugs on the helices. This has the effect of weakening the interactions between the subunits, making it easier for the next oxygen to bind to the next heme. By the time three hemes are bound with oxygen, the fourth is pumped up and just begging for an oxygen molecule (“R-state” or “relaxed-state”). Wikipedia user Habj made an awesome gif of the T-state versus the R-state:
(No, June, it’s not, you’re just a nerd, Junhi is right, you and Sapphire are the lamest people on the planet, yadda yadda…)
So, hemoglobin does a really cool thing. But why does it do the thing? What’s useful about having oxygen binding being dependent on something other than oxygen concentration?
Well, let’s think about it this way. In the arteries close to the lungs, oxygen tension approaches 100 Torr (way more than the P50 of 26 Torr for hemoglobin). That means that, in the arteries, the amount of oxygen bound to hemoglobin is at a maximum. Once you move to peripheral tissues, however, the oxygen tension quickly drops to 40 Torr (resting). Between 100 Torr and 40 Torr, only about 20% of hemoglobin’s oxygen gets dropped off (that’s that sigmoidal curve afoot). However, when you start exercising, the oxygen tension in these tissues drops to somewhere around 20 Torr. Since the slope of the curve of oxygen binding of hemoglobin is the Hill constant around P50 (20-30 Torr), oxygen binding linearly decreases in this range. That means that between 40 Torr and 20 Torr, 70% of the oxygen in hemoglobin gets released!
Here we can see the usefulness of this mechanism: when hemoglobin moves from the lungs to resting tissues, which don’t need a huge amount of oxygen, it doesn’t drop off a lot of oxygen, even though the difference in oxygen concentration between the lungs and peripheral tissues is astounding. However, the small drop from resting to exercising pressures within tissues, which means that rigorous respiration is taking place, is enough to make hemoglobin drop off plenty of oxygen for the exercising tissues, while still retaining enough (30%) for emergencies. This is a feat that myoglobin, with its single, independent unit, can’t achieve.
It doesn’t stop there, either. The Bohr effect relates the concentration of CO2 or protons in tissues to oxygen binding of hemoglobin. Turns out, when oxygen binds to Hb, it releases protons. That means that, when there are more protons in a solution, the equilibrium is shifted toward unbound Hb, meaning that the T-state becomes favored. The same happens with CO2 for two reasons; firstly, carbon dioxide reacts with water to make carbonic acid, which releases protons and acidifies the environment, and secondly, it covalently bonds to the amino end of Hb and produces both protons and a carbamate anion that stabilizes the T-state. In either case, acidic environments with excess CO2 indicate respiration is taking place, and more oxygen than normal is released for respiring tissues. In the case of CO2, the Hb also helps transport CO2 and protons to the lungs, where they’ll be eliminated.
(It’s soooo coooooooool!)
There is also a nifty little molecule that affects the P50 of Hb, and it’s called 2,3-bisphosphoglycerate. RBCs make it, and it binds to positive surfaces on the beta subunits of deoxy-Hb. This has the effect of stabilizing the T-state, which in turn raises the P50 and ensures that more oxygen is made available to tissues at lower oxygen partial pressures. Our bodies use this to help us adjust to high altitudes; when a human goes to a high altitude, over a period of days or even weeks, her RBCs will produce more 2,3-BPG, tweaking her hemoglobin so that it makes oxygen more available to her tissues. That’s freaking nifty, isn’t it?
(That was the part where I started scribbling notes about SciFi…)
Okay, so we’ve come this far, and I know what you might be thinking. What’s a really important thing that affects hemoglobin? Sickle cell anemia, perhaps? Wouldn’t you like to know what causes it?
Oh, come on, you want to know. The answer’s really simple. It’s caused by a single mutation in the beta subunit of Hb that changes a glutamine to a valine. The glutamine, a hydrophilic residue, is situated near water, and in normal hemoglobin, everything’s a-o-good. However, when you substitute it for valine, which is hydrophobic, the valine seeks out a hydrophobic environment. It ends up finding a pocket on a beta subunit of another tetramer. Ultimately, you end up with long chains of hemoglobin aggregating, which force red blood cells to conform into their characteristic spindle shape.
Phew! Well, I’m thoroughly exhausted! I think I’m going to take a break now. Eat something, maybe. Take some deep breaths, so that my oxygen partial pressures will go up a little bit…?
[is shot, dies]
Now that we’ve looked at hemoglobin and myoglobin, we can apply what we know about oxygen binding to another oddly similar mechanism—the binding of substrate to an allosteric enzyme.
You know what to do, yo ^^