Protein Function - Hemoglobin

So far we have studied protein structure as an independent property. However, I have mentioned a couple of times the close relationship between structure and activity. Furthermore, we saw that the structure of a protein is not a single structure, but a number of closely related structures that look a lot alike but have slight differences. We saw this when we discussed protein folding and said that proteins were on the energetic 'edge'. This allowed them to have small changes of conformation without great changes in energy, and made them behave as if they were 'breathing': The protein will be wobbling around a certain tertiary structure fold, but never fixed at a particular fold.

This property is what makes proteins so versatile. After an enzyme binds to a ligand or subtstrate, there will be small conformational changes that may affect how the enzyme behaves from then on, affecting its function afterwards. Today we will start discussing these properties, using as a template myoglobin and hemoglobin, two oxygen-carrier proteins that suffer small conformational changes after binding molecular oxygen.

Before we start start our discussion of oxygen binding proteins, we will define some of the concepts laid out above:

- When a molecule interacts with specific amino acids or prosthetic groups in a protein, and the interaction is reversible, the molecule is called a ligand. Most interactions between molecules and proteins are of this kind, and are crucial to the function of the protein.

- The ligand, as said above, binds specifically to certain amino acids, or a combination of amino acids and a prosthetic group. This group of amino acids is called the binding site of the ligand in the protein. A protein can have one binding site for a single ligand, or several binding sites for the same ligands, or different binding sited for different ligands.

- As we said over and over, proteins are flexible and dynamic. Both the backbone atoms and the side chain atoms will move, in what is known as the breathing of the protein. Protein dynamics not only affect the fucntion of a protein, they are usually crititical and necessary for the protein to work well.

- When a ligand binds to its binding site in a protein, there will always be changes in the conformation of the residues around the binding site. These conformational changes can cascade to conformational changes of the whole tertiary structure of the protein, and the phenomenon is know as induced fit.

- As a result of these two last facts, the binding of a certain ligand to a protein can affect its shape, and therefore it can affect the affinity of a distant binding site for another ligand. This is the way in which proteins regulate their function. We will see an example of this with hemoglobin.

As you all know, transport of O₂ is crucial to maintain life in almost all organisms. The presence of oxygen allows to get 15 times more energy from a molecule of glucose than if we do things anaerobically. When we think about this, we usualy forget that the O₂ we breath has to go through many hoops before getting to the cells, were it is actually used in the transformation of nutrients into energy and biological building blocks. One of the problems we have with oxygen is its low solubility in water, and therefore in blood. Also, oxygen diffuses poorly through membranes, so it cannot be delivered properly from blood to the inside of the cell.

How do we go around this? As you probably figured out already, we use proteins which are designed exclusively to transport oxygen in blood and in the tissues. However, proteins alone can't cut it, because there is no amino acid among the twenty natural amino acids that can bind O₂ for the purpose of transporting it. What about using a prosthetic group? As you remember, prosthetic groups are non-amino acid components that have properties amino acids don't have, and therefore become crucial to the activity of certain proteins.

If we could use iron, as Fe²⁺, as a prosthetic group we would be in the clear, because oxygen binds tightly to Fe²⁺. The problem with this is that Fe²⁺ alone is a nasty thing to have floating around, because it is way too reactive and it promotes the generation of free-radicals, which are baaaad things. Furthermore, Fe²⁺ is very prone to oxidize to Fe³⁺ (and on the process make radicals):

What we do is we lower the activity of iron by sequestering it with some molecule, thus lowering its reactivity. A great molecule to do this is heme (or haem). Heme is a porphyrin (proto porphyrin IV), a large aromatic ring structure composed by four pyrrole rings. Heme has four nitrogens that can coordinate with Fe²⁺ by donating their electrons and forming a coordination complex:

From the point of view of the Fe²⁺ ion, we can see that before binding to heme, we have six coordination sites, and after we bind to the four nitrogens in heme, which are all on the same plane, we end up with only two possible coordination sites for other molecules: one above the porphyrin ring system, and one below:

Now, we still have some problems. First, we have two coordination sites free on the Fe²⁺, and this can lead to non-reversible conversion of Fe²⁺ to Fe³⁺, which is not a good O₂ binder as is Fe²⁺ (actually, its a very poor O₂ binder), and can also lead to the generation of radicals. Second, since both faces of the heme are completely exposed, both coordination sites are completely exposed. This means that many ligands apart from O₂ can bind to the heme Fe²⁺. The most prominent one is carbon monoxide (CO), the dreaded silent killer from furnaces and gas stoves, which binds almost 25,000 times tighter to heme than does O₂. That means that we need 25,000 times less CO to get the same binding than we do with oxygen. This is bad if we had heme alone in the blood, because there are several cellular processes that generate CO as a low level by-product.

What Nature did is to put the heme group as a prosthetic group deep inside a certain class of proteins, called globins. The two that we will study are myoglobin (Mb) and hemoglobin (Hb). The globins, as the name implies, are globular proteins.

Myoglobin is perhaprs the best studied protein ever. Its X-ray structure was the first X-ray structure obtained for a protein, and many other biochemical studies have been performed. Mb is used in mammals for oxygen storage in the muscles. Mb releases its bound oxygen to oxygen-depleted tissues upon prolonged physical activity. Therefore, whales have a lot of it, and the Mb used by Kendrew when determining the X-ray structure was from sperm whale.

Mb is a small protein of MW ~ 16,700, and consists of a single polypeptide chain of 153 amino acids. It is very compact, with only one major gap into which the heme prosthetic group fits:

Some better pictures of Mb can be found in in the CHIME page. As you can probably tell, this is a single domain protein, with 75% a-helical content. There are eight a-helices in the protein connected by loops. Each helix is named A, B, C, etc., and the loops that connect them are named by reference to the a-helices they connect: AB, BC, CD, etc.

The residues in Mb can be referenced by their position in the primary structure or by their location in the a-helix or loop to which they belong. For example, histidine 93, which we'll see below is crucial for the function of Mb, can be named His93 (the 93^rd residue in the primary structure, or His F8, the 8^th residue in the a-helix F.

How does Mb makes the Fe²⁺ to be even less reactive, as well as to improve the binding of O₂  to heme with respect to CO? First, one of the two free coordination sites of Fe²⁺ that come out of the heme plane is occupied by coordination to the nitrogen of the side chain of a histidine residue, His F8 (or His93):

Second, there is another histidine residue, His E7 (or His64) lurking on the other side of the heme plane (opposite to His F8). This histidine residue, usually called the distal histidine, is not coordinated to the Fe²⁺, but instead acts as a flap the covers it. It all has to do with the geometry of coordination of O₂ and CO (or NO): Due to the electronic structure of O₂, each oxygen has sp² hydridization. The lone pairs in O₂ will therefore coordinate at an angle with the heme Fe²⁺. In CO (or NO) we have an sp hybridixation, and therefore the coordination has to be linear. Since the Histidine is almost on top of the coordination site, this means that if the bound ligand is linearly coordinated it will butt against His E7, increasing the energy and lowering the affinity. A ligand that coordinates at an angle, like O₂, can avoid this:

By the way, what is the angle? Furthermore, there is a h-bond between the labile hydrogen of His E7 and the O₂ molecule that further stabilizes the interaction. The binding site of O₂ in Mb is completed by two hydrophobic residues, Val E11 and Phe CD1, which make a tunnel through which the molecule has the get in and out of the binding site. With all this taken into consideration, CO ends up binding only 200 times better than O₂. Thus, we improve the selectivity for O₂ a lot, but we still have to make sure that there is a window open at night if we have gas heating...

Now, this shows how is that O₂ ends up bound to Mb instead of CO. Now, how does O₂ gets here, if the molecule is as compact as we said, and furthermore, how does it get out, if it is so tightly bound and stabilized by h-bonds? First, we have to remember that the protein is not static, and it will be "breathing", i.e., it will be changing its conformation and the conformation of its side chains slightly all the time. This allows O₂ molecules to slip through cavities that are created transiently (e.g., for short periods of time), and get to the coordination site in heme. Second, these same fluctuations of the conformations of the side chains are resposible for the release of oxygen. It has been shown that the side chain of His E7 flips (rotates around its CH2-imidazole bond) once every nanosecond (10^-9 seconds), breaking the h-bond to the bound oxygen and allowing it to escape the protein.

Quantitative description of O₂ binding in myoglobin

Myoglobin has been so extensivelly studied that all these qulitative concepts can be described quantitatively, i.e., we can put numbers to all the phenomenological descriptions we have just discussed above. This will not only help us to understand how O₂ binds to Mb and Hb, but it is also the foundation for describing the binding of any ligand to any protein or enzyme.

A protein binds to its ligands following exactly the same rules that describe any chemical equilibrium. For a certain protein P and a ligand L, we can define an association constant K_a as:

As with any other equilibrium constant equation, the larger the value of K_a, the more affinity the ligand L has for the protein P. SInce [P] tell us the concentration of protein whose binding sites are free and [PL] indicates the concentration of protein whose binding sites are saturated or occupied, we can rewrite the above expression to see the fraction of bound to free protein:

We see clearly that the ratio of bound to free protein is directly proportional to the concentration of ligand. After we reach a certain concentration of [L], [P] and [PL] will reamain almost constant, and the changes of concentration of ligand will not affect the binding. This is common with all proteins (and enzymes) that bind a ligand.

Now, it is more useful to evaluate the ratio of bound protein ([PL]) to total protein ([PL] + [P]). This is the same as the ratio occupied binding sites to total binding sites. We define this ratio as q (theta). Working on the previuos relationships, we get to:

This equation has the form y = x / ( x + k ), and it's called an hyperbolic function. Therfore, q varies hyperbolically with [L].

Lets analyze this function and what it means a bit more. The fraction of occupied binding sites (q) approaches full occupancy (q = 1) asymptotically. That it, it never gets there but gets really really close for very large [L]. Second, the concentration of ligand [L] at which half of the binding sites are occupied (q = 0.5) is equal to 1 / Ka.

There is one small detail. We usually talk about the dissociation constant of a ligand and a protein complex (PL), instead of its association:

If you do the math, you'll find out that K_d = 1 / K_a. When you hear discussions about a drug binding a protein or a receptor that has a small K_d, this means that it dissociates very poorly, and thus it binds very tightly to the protein. In the future, you will see that all medicinal and pharmaceutical chemists try to find drugs with smaller and smaller K_d's. Therefore, we can re-write our occupancy equation as:

If we use K_d instead of K_a, we see that q = 0.5 when [L] equals K_d. In other words, K_d is the molar concentration of ligand ([L]) at which half of the protein binding sites are occupied. You have probably heard about the famous IC₅₀'s. In these case, we refer to the concentration of an inhibitor at which 1/2 of the enzyme id dead (inhibited).

Now, how about going back to myoglobin. Mb binds O₂, and therefore our equation is re-written as:

Since K_d is the the concentration at which 1/2 of the binding sites are occupied, we rename it [O₂]_0.5. and our equation ends up as:

Finally, we are working with oxygen, wich at normal pressure and temperatures is a gas. As you know from PhyChem, the concentration of a gas disolved in any liquid is poropotional to is partial pressure over the liquid it is dissolved in. Since the proportionality constant is the same for all the concentrations, we re-write (for the last time...) the equation as:

These equations (and the way we analyze them) are the foundation we will use to describe how a more complicated oxygen binding and transport protein, hemoglobin, works in the blood. We will see in the next classes the how the changes in the 3D structure of hemoglobin affect the way O₂ binds, and how this affects the way O₂ is delivered to the cells and tissues.

Prepared by Guillermo Moyna, 1999.