Hemoglobin

HEMOGLOBIN STRUCTURE

Quaternary structure

In contrast to myoglobin, the quaternary structure of hemoglobin is that of a heterotetramer (a protein consisting of four polypeptide subunits that are not identical). Adult hemoglobin is composed of two similar tyes of subunits, α and β, so that the tetramer can be represented as α2β2. The α subunits associate more strongly with the β subunits than with each other (and vice-versa), and consequently hemoglobin's quaternary structure is sometimes described as a dimer of αβ dimers.

Shown here is the hemoglobin α2β2 tetramer, with one αβ dimer in blue and the other αβ dimer in green. The α subunits are depicted with a darker shade than their β counterparts. Take note: similar color (blue or green), and not similar shade (dark or light), indicates stronger association.

View the following animations to explore the dimer and each subunit:

Subunits
αβ dimer
α subunit
β subunit

Secondary and tertiary structure

The three-dimensional secondary and tertiary structural characteristics of the individual subunits of hemoglobin are virtually identical to those of myoglobin—perhaps surprisingly, given the little similarity between these two globins in terms of primary structure (amino acid sequence). View the following animations to witness this:

Subunit comparison
hemoglobin α chain
hemoglobin β chain
myoglobin
align all

OXYGEN BINDING AND ALLOSTERY

Despite the structural similarities between myoglobin and hemoglobin subunits, hemoglobin has a markedly different function in the body. For hemoglobin to function in its oxygen transport role effectively, it must bind to oxygen in the lungs where the concentration of oxygen is high, yet release it at the tissues where the oxygen is being consumed and the concentration of oxygen is relatively low. In other words, hemoglobin's oxygen affinity must be regulated by the concentration of oxygen surrounding it. Myoglobin could not serve to deliver oxygen, for it has a stronger affinity for oxygen than hemoglobin and would not release it to the tissues, even in the lower oxygen concentration environment near respiring cells. We shall see that hemoglobin's oxygen affinity is indeed regulated by the local oxygen concentration, as well as by additional factors.

Allostery

What feature of hemoglobin sets it apart from myoglobin, and how might this be responsible for its different biological niche? The answer is quaternary structure. In contrast to single-subunit proteins such as myoglobin, proteins with multiple subunits can exhibit allostery. The function of an allosteric protein is regulated by ligands (called effectors) that interact with the protein. In hemoglobin's case, oxygen is both a ligand and an effector. Because hemoglobin serves as a good model for allosteric proteins, its structure is perhaps the most studied to date. Another allosteric protein is discussed in the phosphofructokinase exercise.

The allosteric effects of oxygen on hemoglobin can be described simply. Hemoglobin can bind one oxygen molecule in each of its four subunits. For a hemoglobin devoid of oxygen (deoxyhemoglobin), the binding of oxygen molecules enhances the binding of additional oxygen to the remaining empty (deoxy) subunits of the same hemoglobin molecule. Another way to describe this is to say that hemoglobin binds oxygen cooperatively, meaning that the binding of one oxygen molecule increases the oxygen binding affinity of the remaining subunits.

For hemoglobin, oxygen is a positive homotropic allosteric effector (positive indicates that activity is enhanced, and homotropic means that the same molecule is both substrate/ligand and effector). Other substances, including carbon dioxide, protons, and 2,3-bisphosphoglycerate (BPG) are negative heterotropic allosteric effectors (negative because they decrease activity, and heterotropic because different molecules serve as effector and ligand/substrate).

Allostery and quaternary structural conformations

The allosteric behavior of hemoglobin, and indeed all allosteric proteins, is the result of quaternary structural changes that occur upon ligand (effector) binding. The importance of quaternary structure for allosteric behavior is illustrated by the observation that the individual, separated subunits of hemoglobin behave similarly to myoglobin, yet behave quite differently when they are part of a tetrameric complex. Quaternary structural changes must occur in hemoglobin, because the three-dimensional structures of oxy- and deoxyhemoglobin, which have been solved by X-ray analysis, differ markedly.

Comparisons of oxy- and deoxyhemoglobin show that there is a 15° rotation of the αβ dimers relative to each other. Also, the oxygen-bound structure is more compact, which is readily apparent when you look down the central cavity of hemoglobin. Because deoxyhemoglobin has many more salt bridges that constrain the tetramer, the deoxy form has been termed the T (for tense) state, and the oxy form has been termed the R (for relaxed) state. The T and R designations are now generally used in discussions involving allosteric proteins, with the T state used to describe the form of the protein with the lower affinity for ligand (or substrate in the case of enzymes).

T to R transition of hemoglobin

Use the buttons below to explore the large conformational changes associated with changes in oxygen-binding affinity. Remember, relaxation of the protein (going to R state) leads to increased oxygen-binding affinity of empty subunits, while becoming more tense (going to T state) lowers the affinity for oxygen.

Ribbon Display
deoxyhemoglobin T state
oxyhemoglobin R state
Simulation of T to R transition


Spacefill Display
deoxyhemoglobin T state
oxyhemoglobin R state
Simulation of T to R transition


Alignment Display
deoxyhemoglobin T state

For transport from the tissues to back to the lungs, carbon dioxide binds to the N-terminal amino groups of hemoglobin in the form of negatively charged carbamates.

These carbamates participate in salt bridges, making hemoglobin more tense and stabilizing the T state. Thus, this stabilization of the deoxy form also aids in oxygen delivery, for the T state now has a lower affinity for oxygen and will offload it to the respiring tissues.

HEME CONFORMATIONAL CHANGES AND ALLOSTERY

The large-scale quaternary structural changes of hemoglobin between the tense and relaxed states are derived from small changes in the conformation of the heme group, much like how small changes in the position of the end of a lever nearest the fulcrum lead to large changes in position at the other end of the lever. For a closer inspection of this phenomenon, we must focus in on the heme group and iron's coordination partners.

In deoxyhemoglobin, the iron atom is coordinated by only five nitrogen atoms (one from His F8 and four from the porphyrin rings), which assume tetrahedral pyramidal geometry with the iron atom ~0.6 Å out of the plane of the heme group. When oxygen is bound to the heme iron, forming a sixth coordination partner, the geometry of the coordination partners changes to octahedral, with the iron atom now lying within the porphyrine plane.

Use the buttons below to examine the conformational changes in the heme group and the proximal and distal histidines upon oxygen binding and release.

Heme conformational changes
deoxyhemoglobin
oxyhemoglobin
Simulation of deoxy (T) to oxy (R) state transition
deoxy (T) - oxy (R) state alignment

Structural communication: Oxygen binding leads to changes in quaternary structure

How do the very small changes in conformation at the heme group upon oxygen binding affect the large quaternary conformational changes associated with the T to R state transition? These small changes lead to larger movements within the oxygen-bound subunit, which in turn create major shifts at the subunit interfaces.

As we have seen, upon oxygen binding, the iron atom moves into the porphyrin plane of the heme group, pulling with it the proximal histidine bound to it. This small movement effects a concomitant movement of the entire F helix (note: α helices are fairly rigid), as well as the corner between the F and G helices. This FG corner on the β subunit of one αβ dimer is the area of contact with the α subunit of the other αβ dimer. When it gets pulled, the interface between the two αβ dimers slips like the movement during an earthquake of two plates along a fault. This "α1β2" contact is referred to as the "hemoglobin switch."

Use the buttons below to witness the large changes that occur in the hemoglobin switch. Note how β2 residue 97 alternates its position: between α1 residues 41 and 44 (in the T state) and between residues 38 and 41 (in the R state):

Dimer interface changes
deoxyhemoglobin
oxyhemoglobin
Simulation of deoxy (T) to oxy (R) state transition

Many salt bridges at the C-terminus of the β chains that held hemoglobin in the T state (deoxy form) are broken during this slippage. Consequently, the αβ dimers rotate 15° relative to each other, and a few new intermolecular interactions are formed as the structure settles into the R state (oxy form).

BPG: A negative heterotropic allosteric effector

BPG (2,3-bisphosphoglycerate) is a compound present at roughly equimolar amounts to hemoglobin in red blood cells. It is produced by the cells to lower hemoglobin's oxygen affinity enough to enable hemoglobin to offload oxygen at the tissues. Interestingly, regulation of BPG concentration is one method the body uses to acclimate itself to the lower atmospheric concentration of oxygen at higher altitudes. At higher altitudes, how and why would the body change the concentration of BPG in red blood cells?

BPG easily binds in the central cavity of hemoglobin in its more open conformation (T state or deoxy form). BPG also crosslinks the opposing β subunits by forming salt bridges between its negatively charged phosphate and carboxylate groups and positively charged amino acid residues (making the molecule more tense). Therefore, BPG stabilizes the T state (tense, deoxy form) and lowers the affinity of hemoglobin for oxygen (by a factor of ~26 in red blood cells!).

Dimer interface changes
ribbon
spacefill