Cytochrome C

An interactive tutorial for 122.102, Biochemistry of Cells

This is an interactive tutorial to allow you to manipulate the way that the electron tranport mobile carrier Cytochrome c (cyto c) is displayed. The overall structure of the protein and its haem group is shown in the rotating figure in the frame at the left.

By following the instructions you will get an idea of the 3-dimensional image of the structure and the way that it inteacts with the haem prosthetic group. Once you have worked out how to use the interactive aspects of the software you should experiment as much as you like!

If you find this text is too small for you to read without strain you should use the browser controls to increase the displayed font size. If you are using Netscape Navigator Use the View menu to increase the size. (You may have to do this several times to see an effect.)

Once the molecule in the frame to the left has stopped rotating press the first button (box with an X in it) below which will reload the file of structural co-ordinates for horse heart cytochrome c and display the output in a pseudo 3-dimensional form in the structure frame to the left of the screen.

Overall Structure of Cytochrome c

This view shows the protein backbone with a-helices as magenta-coloured ribbons and b-sheets as yellow ribbons. Turns are light blue and coil structures are white. As you should know cytochrome c is an electron carrier with an iron-containing haem prosthetic group. The iron atom can be either in the oxidised (ferric, Fe³⁺ or the reduced (ferrous, Fe²⁺ form. The next button will add the haem group to the cytochrome molecule in the structure frame.

Before looking at some details of the cytochrome c structure it will help we look at the structure of the haem group itself.

Structure of Haem Group

The single iron atom of haem is the orange sphere in the center of the molecule. This is linked to the four nitrogen atoms of the four pyrole rings that make up the porphyrin ring of the haem. The next button shows the bonds of the pyrole rings in green and the bonds between the iron atom and the nitrogens in red.

The iron atom is in the ferrous (Fe²⁺) state in the reduced form and in the Ferric (Fe³⁺) state in the oxidised form of the cytochrome.

The haem group in cytochrome c is linked covalently to the protein by links from the vinyl groups of the sidechains on the two pyrole rings on the upper right of the structure. The links are to the -SH groups of the side chains of two cysteine amino acids in the cytochrome c protein. In the free haem molecule there is a double bond between the two carbon atoms of the vinyl side chains (-CH=CH₂. When linked to the protein this bond becomes a single carbon-carbon bond and the electrons form a thioether bond to the sulphur of the cysteine side chains (-C-(CH₃)-S-CH₂-). Note that the bond to the sulphur is from the innermost carbon of the vinyl side-chain.

The next button labels the vinyl side chains.

The two propyl groups are on the lower left (the oxygen atoms of the carboxyl groups are shown in red). The next button labels the propyl side chains.

Note that the carboxyl groups of the propyl (-CH₂-CH₂-COOH) are acidic and will carry a negative charge at neutral pH (-COO^-).

To get a better idea of the overall shape of the haem turn on "Rotation" using the pull-down menu in the structure frame and look at the molecule as it turns. Make sure that "Rotation" is turned off before going to the next section.

Cytochrome c Function

The answer to this question has a number of levels. Free iron itself is a very reactive atom. In fact there is very little free iron in biological systems. If it is not combined with functional proteins such as the cytochromes it is bound in protein storage complexes such as ferritin or is bound to iron transport proteins such as transferrin. Ferrous iron (Fe²⁺) is quite soluble. However free ferrous iron is rapidly oxidised to ferric iron. This rapidly forms ferric hydroxide which has an extremely low solubility coefficient and precipitates as rust. To prevent this the iron is complexed with the tetrapyrole porphyrin ring which is in turn bound to the cytochrome c protein. In this environment the iron atom is able to cycle between the ferrous and ferric states. In particular the oxidised (ferric) form is sequestered in such a way that the insoluble iron hydroxide does not form. The redox potential of the iron atom is also modified by the haem group and the protein environment. While different cytochromes have similar haem groups the redox potentials can be quite different depending on the protein. It is also instructive to remeber at this stage that the oxygen transporting molecules myoglobin and haemoglobin also have iron-containing haem groups. With these proteins the functional requirements are quite different. The ferrous iron in myoglobin and haemoglobin has to bind to oxygen (O₂) without becoming oxidised to the ferric form.

Cytochrome c Structure & Bonds to Haem. Iron Ligands

The next button loads the cytochrome c molecule with the protein shown as ribbons or strands and the haem group in ball-and-stick format. The bonds to the iron atom are shown in red.

The next button will rotate the molecule by 360^o. This will show that the haem group is located in the center of the molecule. Clicking on the button again will repeat the rotation.

"Rotation"

In cytochrome c the haem is covalently linked to the protein by thioether links between the vinyl groups of the haem and the sulphurs atoms of two cysteine side-chains of the protein. The next button will show the cysteine side-chains with the sulphur atom in yellow.

Click the "Rotation" button above to get a better idea of the relationship of the groups.

The iron atom has six possible coordination links. Four of these are shown as the links to the nitrogen atoms of the pyrole rings. In cytochrome c the two addional links are to one of the imidazol ring nitrogens of histidine 18 and to the sulphur of the side chain of methionine 80.

Click on the next button to minimise the protein backbone and zoom in on the haem group and the iron ligands. Use the "Rotation" button again and look at the links between the haem sidechains and the cysteines at position 14 and 17. Look also at the two ligands to the iron from histidine₁₈ and methionine₈₀.

The next button will zoom out again to show all of the protein backbone.

The Haem Pocket in Cytochrome c is Hydrophobic & the Iron Atom is Shielded from the Solvent

Now highlight the hydrophobic amino acids of the protein with the next button.

Note how the pocket that the haem sits in is lined by the side chains of hydrophobic amino acids. The only polar side chain in the pocket is the histidine (His₁₈) that is the iron ligand. This amino acid is highlighted by the next button along methionine 80, the other protein ligand to the iron. (His₁₈ cyan, Met₈₀ green.

The next button rocks the structure so that the relationship of the haem atoms in the pocket is clearer.

This view might suggest that the iron atom is accessable to the cytosol down the haem binding cleft. However if the haem group atoms are shown at their van der Waal's radii (next button) then it is clear that the iron is well shielded. The haem group colour is changed to red.

Use the rotation button below to spin the molecule to see the shielding of the iron.

"Rotation"

Cytochrome c Structure & Function

The differences in the structure of the cytochromes also mean that the redox potential of the ferrous to ferric redox couple can be tuned to the requirements of the particular cytochrome function. Examination of a table of redox potentials of biological electron carriers (eg in Biochemistry by Lubert Stryer Table 21-1, p532 4^th Edition) will reveal that the various cytochromes have quite different redox potentials.

It is also instructive to compare cytochrome c with the oxygen-transporting haem proteins myoglobin and haemoglobin. The function of myoglobin requires that the ferrous iron atom is bound to oxygen but is not oxidised to the ferric form. Once again the function of the iron atom is modulated by the environment provided by the porphyrin ring of the haem and the surrounding protein.

j.tweedie@massey.ac.nz