Myoglobin Structure & Function

Chime scripting by John Tweedie

This tutorial is designed to be used alongside the material on myoglobin structure in appropriate sections of Biochemistry (4th Edn) by Lubert Stryer. Elliot and Elliot discusses haemoglobin without prior discussion of myoglobin. I think it helps to look at the simpler structure of myoglobin first.

Introduction:

Click on the buttons to load the structures into the frame at left.
The larger buttons mark the beginning of a section. If you wish to return to a section, click on the large button at the start of that section.

The first button displays the oxygen-binding protein myoglobin. In this view the protein is displayed in a form where the helices are displayed as magenta helical strands. The N- and C-terminal ends of the protein are labeled. The structure is displated in approximately the same orientation as in Figure 7-4 on page 149 of Stryer IV.

Structure:

Myoglobin is an unusual protein as it is made up almost exclusively of a-helices joined by short loops. Most proteins have both a-helices and b-sheets.
Use the Chime pull-down menu in the structure frame and select rotation. Watch for a while to get an idea of the 3-D structure. Turn rotation off.

The next button will highlight the helices in turn as magenta helical ribbons, starting at the N-terminus and ending at the C-terminus.

Myoglobin as an example of a water-soluble globular protein. The tertiary (3-D) structure consists of a 8 a-helices which fold to make a compact globular protein. The folding occurs in such a manner that almost all of the hydrophillic (polar) groups are on the outside of the protein, facing the aqueous environment. The hydrophobic groups arte almost all inside the protein and the hydrophobic effect plays a large role in maintaining the stability of the folded protein. The a-helices mostly lie with one side of the helix facing the interior of the protein and the other facing the outside. For this reason the helices are amphipathic with the side facing the interior having amino acids with hydrophobic side-chains and the side facing to outside having polar side-chains.

The next button highlights one of the helices in myoglobin and rotates the structure so that the view is along the length of the helix from the top

Note how the magenta helix lies across the surface of the protein with one face towards the centre of the protein and the other towards the outside. The rest of the protein structure is shown in spacefill mode and the hydrophobic groups are green. The next button will rotate the structure so you are looking directly down on the magenta helix from the top.

Note how the protein surface under the helix is highly hydrophobic (green).

The next button will show the magenta helix in ball & stick representation. The peptide backbone of the helix is shown as magenta sticks and the side-chains as balls & sticks.

The hydrophobic side chains are are yellow and the polar side chains are red.

The next button will rock the structure. This should demonstrate that the yellow, hydrophobic groups are on the uderneath side of the helix while the red polar groups are on the top, facing the outside. Clicking on the button again will repeat the rocking motion.

The next button will hide all of the protein except the helix.

Turn on amino acid labels with the next button.

Use the pull-down menu in the structure frame to turn on rotation. Note the green hydrophobic amino acid side-chains which run as a stripe down one side of the helix.

Turn rotation off before going on to the next section.

Haem Prosthetic Group:

Myoglobin binds oxygen (O₂) in muscle tissue. The oxygen binds to an iron atom (Fe²⁺) which is part of the haem group associated with the myoglobin protein. Haem is an example of a prosthetic group, a non-protein group associated with a protein. The haem group sits in a hydrophobic pocket of myoglobin. The only polar residues in this pocket are two histidines, one on either side of the haem. The histidine side chain atoms are shown as purple-blue spheres. In myoglobin the prosthetic group (haem) is bound to the protein by non-covalent bonds. The structure shown is that of deoxygenated myoglobin (deoxymyoglobin) so there is no O₂ present. We will look at oxymyoglobin later.

The next button will zoom in on the haem group.

The haem group is shown with the atoms colour coded. Nitrogen is light blue, carbon is grey and oxygen is red. The iron atom in the haem group is gold.

We shall now examine the haem group and them look at its interaction with the myoglobin protein.

Haem Structure:

The next button will load just the haem.

Haem is made up of four heterocyclic pyrole rings joined at their corners by methylene bridges. (See the structure on page 148 of Stryer 4.) The four nitrogen atoms (light blue in the structure) bind the iron atom. This can be either Fe²⁺ (ferrous) or Fe³⁺ (ferric). However only the ferrous states can bind O₂. Iron is capable of forming two additional bonds on either side of the plane of the haem and these additional bonds are important in the association of haem with myoglobin and for the binding of oxygen. Note that the pyrole rings of the haem are very hydrophobic, apart from the two carboxyl groups of the propionate side chains (the four red oxygen atoms at the top of the structure).

Use the mouse in the structure frame and select 'Rotation' from the pull-down menu. After a revolution or two use the pull-down menu to select 'Display' and then 'Wireframe'. Notice how the iron atom is slightly out of the plane of the rest of the haem structure. Turn off 'Rotation' using the pull-down menu.

The next button will go back to the myoglobin structure showing the haem.

Haem Pocket in Myoglobin:

In this view the hydrophobic residues surrounding the haem 'pocket' are coloured green. This shows that the hydrophobic haem group is surrounded by hydrophobic amino-acid side chains except for the two histidine groups on either side of the haem. Note also that the haem group is oriented so that the polar part (the carboxyl groups on the propionate side chains- note red oxygens) is to the exterior where it interacts with the water shell of the protein.

The next button will show myoblobin looking down into the haem pocket from the top.

Zoom in on the haem pocket with the next button.

Note once again the hydrophobic nature of the pocket that the haem sits in.

Note also the two histidine side-chains, one on each side of the haem group. The next button will change these to show the atom colours and will hide the hydrophobic groups of the pocket.

Note how the two histidines approach the iron atom in the haem. Histidine 93 on the left (F8 in the numbering system used in Stryer) is close enough to form a bond with the iron atom. Histidine 64 on the right (E7 in Fig 7-8 of Stryer) is too far away. The next button will show the distance between the nitrogens of the histidines and the iron atom. Note also that the Fe2+ atom is displaced out of the plane of the haem towards His 93.

The distance between the bonding nitrogen of His 93 and the iron is 2.09 angstroms which is within bonding distance. The distance between the nitrogen of His 64 and the iron is 4.58 angstroms which is too far for an effective bond. The 'hole' between the iron and histidine 64 is where the oxygen (O₂) binds to form oxymyoglobin.

Oxygen Binding to Myoglobin:

The next button will display oxymyoglobin.

Rotate and zoom the structure with the next button.

In this view the O₂ bound to myoglobin is shown between the haem iron atom and Histidine 64 to the right of the plane of the haem. The software shows a bond between one of the oxygen atoms and the Fe²⁺. Note that now the oxygen is bound the Fe²⁺ atom has moved back almost into the plane of the haem. The next button will show the distances between oxygen and the atoms to which it is bonded. The myoglobin helices have been hidden so that the interactions of the haem with the histidines and oxygen will be clearer. You should always remember that the protein is there and is very inportant to oxygen binding.

If O₂ binds to a free ferrous haem group in solution the Fe²⁺ is immediately oxidised to Fe³⁺ and the O₂ is reduced. Ferric (Fe³⁺) haem cannot bind O₂. The interaction of the haem with the myoglobin protein prevents the oxidation occuring and allows the haem prosthetic group in myoglobin to carry oxygen.

Note that all of the atomic distances shown are well within the distance for bond formation.

The next button displays the oxymyoglobin molecule showing the protein looking down into the haem-binding pocket. The hydrophobic amino acid side-chains lining the hydrophobic pocket are shown in green and their size represents the Van der Waals radius of the atoms.

The next button will zoom in on the haem and hide the helices.

Note the way the hydrophobic groups line the haem pocket with the only polar grouips being the two histidine side-chains. Use the mouse in the structure frame to move the molecule and see how the hydrophobic haem group fits deep into the pocket. The polar propionate side chains of haem are sticking out into the water medium surrounding the protein.