Six individuals were found dead at the scene of a crime.  It’s your job to find out how they died. 

In the first part of this project, you will need to investigate the structure of the normal protein and find the active site so that you can recognize it later when you example samples from the victims.  In the second part, you will look at the active site in the structures from the crime scene samples. If there’s something bound to the active site, you’ll need to identify it and determine if it could be a poison.

Before you begin, you will need to download and install Cn3D from the NCBI. 

Part I.  Investigating the active site of cytochrome c oxidase

1. 1.Download the structure (1OCC.cn3 or the gzipped version 1OCC.cn3_zip, gzipped versions download more quickly).
   

2. 2.Open the structure in Cn3D. 
   

3. 3.Cytochrome C oxidase is found in the inner membrane of the mitochondria.  Turn the structure around and identify the part of the protein that’s normally inside the membrane by looking for the green alpha helices.
   

4. 4.Find the parts of the molecule that are located outside the membrane by looking for the tan beta sheets. 
   

5. 5.Open the Style menu, choose Coloring Shortcuts, and Molecule. 
   

6. Each of the different protein chains and all the other molecules in the structure will shown in a different color.  The amino acid sequences of the protein chains are shown in the Sequence/Alignment Viewer window.  
   

1. A.How many protein chains are in this structure? 
   

2. B.The protein in this structure also happens to be a dimer.  How many protein chains are in each monomer?
   

6. 6.Notice the letters that appear in the structure.  These letters represent different elements.
   

7. 7.Zoom in and find a copper ion located close to an iron.  The iron is part of a structure called heme.  Hold the shift key down and select both the heme group and the copper ion by clicking them with your mouse. 
   

8. 8.Open the Show/Hide menu and choose Select by Distance > Other molecules.  Set the distance at 3 angstroms and click OK.  You should see some of the residues become highlighted in one of the protein chains in the Sequence/Alignment Viewer window.
   

9. 9.Scroll through the Sequence/Alignment viewer to see which residues became highlighted. 
   

1. A.Which chain contains the highlighted residues?
   

2. B. Which residues were highlighted?
   

10. 10.Open the Show/Hide menu and choose Show Selected Residues.  You will see the heme, the copper, and the amino acid residues from the protein chain that binds to these substances. 
    

11. Heme is a prosthetic group and copper is a cofactor.  Both of these are very important for the enzymatic activity of cytochrome C oxidase.
    

11. 11.Open the Style menu and choose Rendering Shortcuts, then Ball and Stick. 
    

12. 12.Click your mouse inside the Sequence/Alignment Viewer to deselect the highlighted residues. 
    

13. You will see ionic bonds (white) between the copper, and the iron, and some of the amino acids.
    

13. 13.Select the bound residues with your mouse and look for the highlighted amino acids in the Sequence/Alignment Viewer to identify the names and position numbers for those amino acids.
    

1. A.Write down the names and PDB position numbers for any amino acids that are bound to copper.
   

2. B.Write down the names and PDB position numbers for any amino acids that are bound to iron.
   

14. 14.Open the Style menu and change the Coloring Shortcut to Element. 
    

1. A.Which element forms an ionic bond to copper?
   

2. B.Is this the same element for all the bound amino acids?
   

3. 
   

4. 
   

Part II.  What killed the victims?

The copper ion and the iron that you looked at in part I are both important for cytochrome c oxidase activity.  During oxidative phosphorylation, molecular oxygen (O2) binds to the iron, and is reduced, leading to the production of two molecules of water.  If other substances are bound to the iron, they can block binding by the oxygen and inhibit the activity of the enzyme.  Your job is to determine if there might be other substances bound to the active site in the structures from the crime scene.  

1. 1.Download one of the structures from a victim.
   

2. 2.Open the structure in Cn3D. 
   

3. Each file shows the protein chain from part I aligned to structures from the crime scene. 
   

3. 3.Find the heme and copper that you identified in part I as the active site. 
   

4. 4.Is there another substance present one of the structures?  Use your arrow keys to toggle back and forth between the normal version of the protein chain and the version from the crime scene. 
   

5. Hint - you can tell which structure is normal (part I) by highlighting the sequences from 1OCCI in the Sequence Viewer.  When you’re looking at the 1OCC structure, you’ll see the yellow highlights in the structure window.
   

6. 
   

5. 5.If there is another substance besides the heme and copper in the victims’ structure, use the color of the substance to identify the elements.  Then, go to Wikipedia (www.wikipedia.com) and search Wikipedia with the name of the substance to find out more about it and whether or not it could be used as a poison.
   

6. 6.If the substance is poisonous, read about the chemical properties of that substance and propose a theory to explain how it could have been administered to the victim.
   

7. Some of the files are large (17Mb).  If you’re having trouble downloading files, try using the zipped versions.
   

1. Harder versions
   

2. Victim A    material A             material A zipped
   

3. Victim B    material B            material B zipped
   

4. Victim C    material C            material C zipped
   

5. Victim D    material D            material D zipped
   

6. Victim E    material E             material E zipped
   

7. Victim F    material F             material F zipped
   

1. Easier versions
   

2. Victim A    material A EZ           material A EZ zipped
   

3. Victim B    material B EZ           material B EZ zipped
   

4. Victim C    material C EZ           material C EZ zipped
   

5. Victim D    material D EZ           material D EZ zipped
   

6. Victim E    material E EZ            material E EZ zipped
   

7. Victim F    material F EZ            material F EZ zipped
   

Funding for this project was provided by the National Science Foundation, grant DRL-0833779, as part of a collaboration between Digital World Biology, the Northwest Association for Biomedical Research, and the EdLab Group.