Myoglobin, Hemoglobin and Allosteric Regulation

BIMS/Biochemistry 503
David Auble


This tutorial shows how myoglobin and hemoglobin bind oxygen. Aspects of the changes in hemoglobin structure that accompany oxygen binding are also shown to provide a molecular framework for understanding Hb allosteric regulation. While the molecular details differ, many multisubunit enzymes are allosterically regulated by ligand-induced changes in subunit conformation that are propagated via changes in intersubunit contacts, so while specific mechanisms vary, an understanding of hemoglobin allostery provides a good model for understanding allosteric regulation in many other systems. Much of this material is found in mosty any biochemistry text, but the following minireview discusses how the model for hemoglobin cooperativity has become more complicated as recent results have indicated that neither the MWC or KNF models adequately describe how Hb works. If you'd like to take a look, follow the link to download it from the password-protected site.

Ackers, G. K. and Holt, J.M (2006) "Asymmetric Cooperativity in a Symmetric Tetramer: Human Hemoglobin" J. Biol. Chem 281: 11441-11443. pdf

The globin fold is found in many related proteins, including myoglobin and hemoglobin. Usually it has eight α-helices, designated A-H (see the example below). These eight helices pack into two layers, making an angle of about 50o between them. In each layer, the α-helices are arranged in an antiparallel arrangement forming a characteristic pattern called a Greek key helix bundle. Using Jmol, the globin fold in sperm whale myoglobin (1bz6) can be visualized in a variety of ways. See the options to the left of the window below. In the spacefilling model, the globular protein domain (magenta) provides a pocket for the heme prosthetic group (gray). Note that the heme is well buried with only an edge visible from the exterior of the spacefilling model and little or no obvious space between the heme and protein residues lining the pocket. As discussed in class, this implies that there is at least some conformational flexibility in the molecule. The overall shape of the globin fold can be seen by displaying myoglobin as a cartoon model. The wireframe model shows something similar but with more detail. You can see how the helices are arranged by selecting the residues that define a particular helix and changing their color or other display properties. (See the Index for information on how to customize this display.) Notice that neglecting side chains, all atoms in the tetrapyrrole ring of the heme lie nearly in the same plane. In the display of just the heme group, the image has been enlarged so it's easier to see. Reset the zoom to show it at the same scale as myoglobin.

Click descriptions on right to render image. Click and drag across image to rotate display. Control-click on image to view Jmol menu.

Helix A : SER3 -- GLU18
Helix B: ASP20 -- SER35
Helix C: HIS26 -- LYS42
Helix D : THR51 -- ALA57
Helix E : SER58 -- LYS77
Helix F : LEU86 -- THR95
Helix G : PRO100 -- ARG118
Helix H : GLY124 -- LEU149
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Spacefilling model of Mb

Cartoon model of Mb

Wireframe model of Mb

Heme alone

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The heme pocket is formed by helices E and F. To see this, highlight the heme pocket in myoglobin or view the isolated heme pocket composed of the E and F helices and the bound heme.

To see how oxygen is bound in the heme pocket, take a look at oxymyoglobin (1mbo). The relationships among oxygenated heme, E-7 (distal His), E-11 (Val), and F-8 (proximal His) residures are highlighted to the right in oxymyoglobin, but it's much easier to see how these groups are arranged in the isolated heme pocket .

Notice that the iron atom of the heme binds to the Nε2 atom of the proximal histidine, F8. The distal, E7, histidine is not bonded to the heme, leaving room for the oxygen molecule to bind to the iron. If you look closely, you will see that the bound oxygen molecule lies in between the iron atom and the Nε2 atom of E7.
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Cooperativity in oxygen binding by hemoglobin is driven by ligand-induced conformational changes in an individual subunit that are propagated to other subunits in the tetramer. Relatively small conformational changes in a subunit cause substantial changes in the subunit-subunit interfaces in the tetramer. (Refer to the handout for a diagram of how subtle the change in heme conformation is when oxygen is bound.) The alpha-beta subunit interface can be observed on the right , which reveals the spacefilling structure of an oxygenated form of human α/β hemoglobin dimer (1hho). By viewing the structure as a cartoon, you can see that the overall folds of the alpha and beta subunits are very similar to each other and to that of myoglobin. (This despite the fact that the alpha subunit is missing the D helix.) The cartoon depiction also allows you to see how the subunit secondary structural elements in one subunit are oriented with respect to the secondary structural elements of another subunit.

Also, notice that similarity between subunits and to myoglobin extends to the heme group, which is again found in a very similar environment to that described above.

Since changes in intersubunit contacts underlie cooperativity, the following images show some of the intersubunit contacts in hemoglobin and how they are altered when oxygen binds. First, take a look at the deoxy, T, or Taut form of human hemoglobin (4hhb; each chain is shown in a different color). This shows the two alpha chains in red and two beta chains in yellow.

The T form is stabilized by intersubunit contacts that are broken during the conformational change that accompanies oxygen binding, leading to the Relaxed, or R form. For example, two pairs of salt bridges involving the C-terminal groups (Arg141) of the a-chain at the α12 interface are present in deoxyhemoglobin. The negatively charged carboxyl group of Arg141 of each a-subunit makes a salt bridge with the positively charged amino group of Lys127 of the other α-subunit. Additionally, the positively charged guanidino group of Arg141 interacts electrostatically with the carboxyl group of Asp126's side chain.

Contacts between alpha subunits in the deoxy form

The binding of protons by hemoglobin lowers its affinity for oxygen, a phenomenon known as the Bohr effect. The Bohr effect has important physiological significance. In tissues where oxygen is needed and the pH is low, protons promote oxygen release from hemoglobin. In the lungs, the pH of blood is higher because CO2 is being exhaled, and hemoglobin can be readily oxygenated. The C-terminal His146 residues of the two β-chains have been implicated in Bohr effect. At lower pH, the histidines tend to from salt bridges with Asp94, contributing to the stability of the T-form of hemoglobin in tissues. In the lungs, however, these salt bridres are disrupted upon oxygen binding when Asp94 moves away. Hence, the T ->R transition is promoted. Take a look at these beta chain residues in deoxyhemoglobin (1hba). Notice how close the His146 and Asp94 residues are. The salt bridge can readily occur between them. Consequently, the T-form is stabilized by these ionic interactions.

 

 

In contrast, here you can see a β subunit from the oxy or R-form of hemoglobin (1hho) in which this interaction has been disrupted. Oxygen binding thus causes the disruption of multiple interactions both within and between subunits, leading to the relaxed conformation.

 


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