Protein Function - Hemoglobin II

Yesterday we saw hemoglobin, its structure, and its O₂ binding behavior. We saw that Hb exists in two states, the T state and the R state, and that they have different O₂ affinities; the T state has lower affinity for O₂ than the R state. Binding of O₂ to one of the subunits of Hb promoted a conformational change in the subunit, which cascaded towards a complete conformational change of the whole protein into the R state. Therefore, the protein showed cooperative binding, which allowed it to bind O₂ with high afinity when pO₂ was high (lungs) , and the reverse change to the T state allowed it to lower its affinity and release O₂ when the environment was depleted of oxygen (tissues)

This all made a lot of sense (I hope), but we never discussed how the T z R transition took place. We have to remember that we have a multisubunit protein, and we only talked about it being either all T or all R. How does the T R transition occurs in the different subunits, and what, if any, mixed states (Hb tetrames with a mixture of subunits in the T and in the R state) will we have? A complete detailed picture of what happens while the protein is in the cooperative stage (when nH > 1) is still in debate, but there are two models that solve certain aspects of this problem.

In the first T z R transition model, proposed by Jaques Monod, Jeffries Wyman , and Jean-Pierre Changeux in 1965, is called the MWC model, or concerted model. The model was derived from data from Hb, but is applicable to any allosteric protein. This model makes the following assumptions:

- The subunits of the protein are functionally identical

- Each subunit can exist in (at least) two conformations

- All the subunits of a the protein undergo the conformational transition (that's why it is called concerted).

Therefore, when we the pO₂ is small (yellow), the T z R transition (equilibrium) will be shifted towards the T state (all circles). Thus, the first row in the diagram above is shifted towards the left. When we increase pO₂, more subunits will bind O₂ (orange), and the concentration of protein in which all the sites are in R configuration (squares) will start increasing. In the diagram, this means that we are moving downwards. In each of the rows, the K value for the T z R transition equilibrium grows. At the bottom, when we have high pO₂, the T z R transition will be shifted completely to the right.

In general terms, the T - R transition process can be viewed as following the green path shown in the diagram as we increase the pO₂ (as we go down in the diagram).

The second T z R transition model was proposed a year later by David Koshland, and is called the sequential model. In this model, individual subunits in a allosteric protein can undergo the T z R, which means that we can have proteins with different ratios of subunits in the T and R states (mixed states). In this model, each subunit in the protein can change conformation independently of what the other ones are doing. However, as we said before, a change in cinformation in one subunit will promote the change of conformation in other subunits. Again, a change from the T to the R state will increase the affinity of the site for O₂ (or the ligand in general)

In this model, the most likely path for the protein to follow when we increase the pO₂ (in the case of Hb) it the one in green.

The two models are not mutually exclusive: We see that if we consider the outer most columns in the sequential model, we get the MWC model. However, the probability of having these ones alone is low in the sequential model, while these ones are the only ones allowed in the MWC model. In either model, the K values for the conformational equilibria will have to increase as we move towards the green part of the diagram.

New studies have shown that neither model is completely correct, and we actually have a mixture of models.

Hemoglobin and effectors

Hb is not only used to transport O₂, but it is also used to transport two by-products of cellular respiration: protons (H⁺) and carbon dioxide (CO₂). About 20% of the H⁺ and CO₂ formed in the tissues is transported to the lungs (CO₂) and the kidneys (H⁺) by Hb. The formation CO₂ in the cells is a by product of oxidation of nutrients. However, CO₂ is not very soluble in water, and it has to be transformed to something else unless we want it to bubble in our blood. An enzyme called carbonic anyhdrase catalizes the conversion of CO₂ into bicarbonate, which is readily soluble in water:

As you can see, H⁺ is a by product of this reaction.

Not only is Hb used to transport these two species, but its O₂ binding affinity is affected by their concentrations in a way that favours its prinicpal task. When the CO₂ and H+ concentration is relatively high (tissues), they will bind to Hb. The binding of these two species promotes the transition of Hb to the T state, which as we saw before, has lower affinity for O₂, and therefore has the effect of increasing even more the ammount of O₂ released in the tissues:

When we get to the lungs, where the concentration of CO₂ is lower, CO₂ will be released from Hb and later extreted from the organism. This not only lowers the CO₂ concentration, but also the H⁺ concentration. Now, these two were stabilizing Hb in the T state, and since their concentration decreases, there will be a shit towards the R state. We have the same old story: Now Hb will have higher affinity for O₂, and will bind a lot of it because it is abundant in the lungs.

This dependence of the O₂ binding affinity of Hb on the CO₂ and H⁺ concentration is called the Bohr effect (Niels Dad). CO₂ and H⁺ are called negative effectors, because their presence decreases the O₂ affinity of Hb.

Now, as we asked ourselves with O₂, why is that CO₂ and H+ affect the binding of O₂? We have to look how is that these two species stabilize the T state of Hb. Neither of them bind in the same binding site than O₂ does.

H⁺ binds to a number of amino acid residues in the protein. In particular, one of the major contributions to stabilization of the T state by H⁺ is that it binds (protonates) His HC3 of the b chains (His146, the carboxy terminal histidine of the b subunits). In the protonated form, His HC3 forms a salt-bridge to the Asp FG1 of Hbb. This is one of the salt-bridges that stabilizes the T state, and higher concentration of H⁺ guarantees that this histidine will be protonated, and the salt-bridge will be in place.

Not to be a bore, but back to pKs for a second. The ion pair between His HC3 and Asp FG1 stabilizes the protonated form of His HC3, and increases its pK_R. Therefore, it will become prototonated at lower H⁺ concentrations (higher pHs) if we are in the T state, because it is in this conformation that both residues are close to one another. Upon transition to the R state, the His HC3 and Asp FG1 are displaced from one another, and the pK_R of His HC3 falls back to the normal value of 6.0, so we will need higher concentration of H⁺ (lower pH) to protonate it.

Now, what's the story with CO2. Carbon dioxide binds to the N-terminal amine of all the chains of Hb, forming a carbamino derivative:

Formation of the carbamino derivative means that we have the creation of a negative charge on the N-terminus of the chains. As you are probably thinking, this negative charge forms additional salt-bridges with positively charged amino acid residues that further stabilizes Hb in the T state.

The last effector that we will discuss id 2,3-biphosphoglycerate (BPG):

BPG is an example of an heterotropic allosteric modulator. This molecule is in relativelly high concentration in red cells (which are 30% Hb by weight), and therefore most of the Hb will be bound to BPG. It is so hard to remove from Hb, that all the binding curves we have seen so far are in the presence of BPG.

BPG binds to the whole that is formed between the a₂b₂ subunits in Hb. As we know, this binding pocket is present in T state hemoglobin, but it is substantially reduced in R state Hb. This means that upon binding of O2 and transition to the R state, BPG is spitted out from Hb:

BPG regulates the binding affinity of O₂ in relation to pO₂ in the lungs. An example is the best way to describe it. If you go to Denver (~3000 meters high), the pO2 is 7 kPa, a lot lower than at sea level (13 kPa). The result is that your Hb fill only 85% of its sites, as opposed to filling ~ 95% of them at sea level. The pO₂ in the tissues is the same, so this means that instead of delivering 40% of the O₂ it can, Hb will deliver only 30% - You get dizzy, tired, vomit your guts out, etc., etc.

After a very short time, your body starts making more BPG. This will bind in the tissues to Hb in the Tstate (it only binds to the T state) favouring the release of O₂. The result is that the the T state is stabilized, lowering the affinity of O₂ when pO₂ reaches the levels found in the tissues. This affects more the low affinity state than the high affinity state, and restores the delivery of O₂ to normal levels of (delivery of 40% of the bound oxygen). This is why after a couple of days you feel OK again.

The interaction between Hb and BPG is due to the formation of several salt bridges between the negatively charged groups in BPG and several positively charged amino acids that are present in the binding pocket between the a₂b₂ subunits.

Next time we will go over molecular diseases briefly, and we will start with enzyme function (chapter 8 in LNC).

Prepared by Guillermo Moyna, 1999.