Myoglobin & Hemoglobin

Because myoglobin and hemoglobin each bind oxygen, we can assume that there should be some similarities between their structures. However, there should also be some dissimilarities because of their different roles as oxygen binding proteins. One obvious difference between the two proteins is that myoglobin is a single polypeptide chain while vertebrate hemoglobins are tetrameric.

Myoglobin

Human myoglobin has 153 amino acid residues.

Myoglobin has a highly folded and compact structure that has 8 separate and distinct alpha helical secondary structures (labeled A through H).

Located at the surface are the polar, hydrophilic residues. The animation will cut slices through myoglobin, allowing you to "slab" through the protein, allowing you to see that the interior of globular proteins such as myoglobin is comprised of mostly hydrophobic residues, while the water-exposed exterior is predominantly hydrophilic.

Notice the distribution of non-polar residues (gray) verse polar residues (red) in this representation as the molecule is sectioned.

Hemoglobin

In human hemoglobin, the overall structure is made up of four individual polypeptide subunits.

Human hemoglobin is comprised of two alpha chains made up of 144 residues and two beta chains made up of 146 residues. The alpha and beta subunits associate more strongly with each other than similar subunits (alpha-alpha or beta-beta). For this reason, hemoglobin is sometimes referred to as a "dimer of dimers". Each of these subunits have structural characteristics similar to that of myoglobin:

This is interesting in light of the fact that these three polypeptide residues have only a 27% identity in their amino acid sequence.

alpha subunit    ---    beta subunit

The subunits of hemoglobin are arranged in a tetrahedral array with a tight spherical overall appearance and, like myoglobin, each individual polypeptide is folded in such a manner to maximize polar residues being on the exposed surface and non-polar interactions being internal, making this large protein water soluble. The arrangement of polypeptides is held together by hydrogen bonding, hydrophobic interactions and multiple ionic interactions, that take place at the contact points between subunits. These subunit interactions play a critical role in the binding of oxygen to hemoglobin.

Oxygen Binding in Myoglobin And Hemoglobin

The heme is a small but important non-protein molecule, or prosthetic group, that is associated with these oxygen binding proteins. The heme is comprised of protoporphyrin IX (an organic ring structure) which has a chelated (bound) iron atom.

The heme resides in a small hydrophobic cleft within each polypeptide. The hydrophobic residues will be shown in green, while hydrophilic are colored red.

There is one heme associated with myoglobin.

There are four hemes found in hemoglobin, one in each of the subunits.

Therefore, myoglobin binds reversibly to only one oxygen molecule, while and hemoglobin binds four. These differences help to explain the different biological functions of hemoglobin and myoglobin. Hemoglobin is used to transport oxygen over large distances (from the lungs to all tissues), whereas myoglobin is present in muscle and behaves as a local "storage" reservoir of oxygen.

Also, due to the intrinsic nature of hemoglobin, it can more precisely control the binding and release of oxygen in the appropriate areas of the body. The internal arrangement of heme in this cleft is very similar in myoglobin and hemoglobin. The heme prosthetic group is made up of a protoporphyrin IX ring structure that is chelated to an iron atom in the ferrous (+2) oxidation state. Incidentally, when a metal ion binds to a porphyrin, which has a great number of conjugated double bonds, it will lead to a compound that is highly colored. This is why blood is red. In plants, the porphyrin is chelated with a magnesium ion, giving chlorophyll a greenish-blue color.

This prosthetic group is physically held in the cleft by hydrophobic interactions as well as a coordinate covalent bond between the iron atom and a nearby nitrogen atom, belonging to the side chain of a histidine residue.

This histidine residue is often referred to as the proximal histidine.

On the side of the heme group opposite the proximal histidine, where oxygen reversibly binds, is another histidine residue called the distal histidine. This residue serves two very important functions in the polypeptide. First, it prevents oxidation of the iron ion to a higher oxidation state by any number of possible nearby oxidizing agents. At a higher oxidation state, the iron (Fe+++) is unable to bond to oxygen. Secondly, this histidine residue acts to reduce carbon monoxide (CO) binding to the heme. If the distal histidine was absent, even low levels of CO would greatly interfere with oxygen binding. The heme group alone (in the absence of the surrounding protein of myoglobin or hemoglobin) has a much greater bonding affinity for carbon monoxide than for oxygen.

Allostery and Hemoglobin

The physical process of binding and releasing oxygen by hemoglobin has a marked effect on its three-dimensional structure. Hemoglobin in its deoxygenated state has a low affinity for oxygen compared to myoglobin. When oxygen is bound to the first subunit of hemoglobin it leads to subtle changes to the quaternary structure of the protein. This in turn makes it easier for a subsequent molecule of oxygen to bind to the next subunit. Thus, with the initial oxygen binding to a subunit, the remaining unbound subunits become more receptive to oxygen. This phenomenon is called an allosteric (through space) interaction and is clearly illustrated in the sigmoidal curve shown above for oxygen binding to hemoglobin.

The mechanism for this effect is initiated when upon binding to oxygen, the iron (Fe++) atom is pulled into the plane of the ring. (In the unbound state, the iron (Fe++) atom lies just to one side of the porphyrin ring.) Since this iron atom is attached to the proximal histidine, this causes the local helix to move also. This movement within the polypeptide subunit causes subtle but substantive changes localized in the regions between subunits. These changes include breaking and reforming hydrogen bonding and ionic linkages. The alpha1-beta2 and alpha2-beta1 subunit junctions are the segments most affected.. In the following depictions shown with Chime, this subunit motion is illustrated by the differences between oxyhemoglobin and deoxyhemoglobin.

Use the links below to toggle between the two structures. BE PATIENT, IT MAY TAKE A LITTLE WHILE TO SWITCH BETWEEN EACH STRUCTURE.

Deoxy Hb   ---   Oxy Hb

Notice the position of the orange residue (histidine97 on the beta subunits) relative to the three other yellow residues (on the alpha subunits). Additionally, notice the change in the central space formed between the four subunits, it is smaller in the oxygenated form of hemoglobin - the importance of this will soon be made apparent.

Other Factors Affecting Oxygen Binding

The binding of oxygen to hemoglobin can be dramatically altered by a small group of substances called allosteric effectors. Hydrogen ions (protons), carbon dioxide, and 2,3-bisphosphoglycerate are effectors that can promote the release of oxygen by favoring the deoxygenated form of hemoglobin. Since these allosteric effectors bind to sites that are specific to each kind of compound, their effects are cumulative.

Hydrogen ions and carbon dioxide are found in high concentrations around actively metabolizing tissues. In the capillaries, the environment favors the release of oxygen from hemoglobin and the binding of these allosteric effectors. The overall result is to facilitate oxygen release into blood plasma and subsequent uptake of oxygen by the high affinity myoglobin in the tissues. The specific reactions of the hydrogen ions and carbon dioxide with hemoglobin causing the release of additional oxygen is called the Bohr effect .

The reactions of the Bohr effect are reversible. When deoxygenated hemoglobin returns to the lungs, the concentration of the hydrogen ions and the partial pressure of carbon dioxide is low. This causes these compounds to be released from hemoglobin. The carbon dioxide is expelled out of the body through expired air. In effect hemoglobin not only carries oxygen to the cells but it also carries waste products from the cells to the lungs, eventually to be eliminated out of the body.

In addition to hydrogen ions and carbon dioxide, a very important allosteric effector is 2,3-bisphosphoglycerate (2,3-BPG). It is a small organic molecule with a large overall negative charge due to the presence of a number of phosphate groups. This species is present along with hemoglobin in red blood cells.

BPG affects oxygen binding affinity by binding in a small cavity at the center axis of deoxygenated hemoglobin. It is held in place by a number of electrostatic interactions with positively charged residues in hemoglobin. In oxygenated hemoglobin, this cavity is too small to effectively accommodate 2,3-BPG. It is only in the deoxygenated state that this molecule has the ability to bind into this cavity. When bound, 2,3-BPG greatly diminishes the binding of oxygen to hemoglobin and facilitates oxygen unloading to actively respiring tissues.


QUESTIONS

The following three interactive questions require you to interact with the tutorial to arrive at the correct answer. You may use any of the visualization controls or the dropdown menus to help you to answer the questions - direct manipulation of the structure may be required. Click the LOAD STRUCTURE link to get you started, the structure that is loaded will contain everything you need to answer the questions.

QUESTION ONE - Which helical segment is the proximal histidine connected to in HEME?
LOAD STRUCTURE
ANSWER ONE

QUESTION TWO - In deoxygenated hemoglobin, the residues most likely to interact with 2,3-Bisphosphoglycerate come from what subunits?
LOAD STRUCTURE
ANSWER TWO

QUESTION THREE -Which helical segments are the most likely to interact between the alpha-2 and beta-1 subunits?
LOAD STRUCTURE
ANSWER ONE


For more specific instructions on how to use Chime, visit Chime Help.