1. Cytochrome c oxidase Mitochondrial inner-membrane protein
Oxidizes reduced cytochrome C, reduces O2. Pumps H+
Creates a pH gradient across the membrane, which in turn drives ATP synthesis through ATP Synthase.

2. CcOx is ancient history
Cytochome C Oxidase (CcOx) may have evolved before oxogenic phototrophs. It occurs in both archea and bacteria, while the photosynthetic reaction center occurs only in bacteria (and chloropasts, which are descended from bacteria).

3. Vertebrate CcOx is “decorated” by short chains from the host chromosome. Bacterial CcOx contains mostly just the core.
Core of vertebrate enzyme
bacterial enzyme

5. O2 is the electron sink
High energy electrons from the TCA cycle travel back and forth across the membrane setting up a pH gradient, finally being donated to O2 to make water (absorbing two more H+). H+ diffuse back into the matrix driving the synthesis of ATP.

6. Transmembrane helices
In gram-negative bacteria and mitochondria, all inner-membrane proteins have transmembrane helices, all outer membrane proteins are beta-barrels.

7. Non-ionic detergent used to crystallize CcOx
Membrane proteins such as CcOx, porin, acetylcholinesterase, colicin, and the photosynthetic reaction center have been crystallized in an artificial membrane of pure non-ionic detergent. b-octyl or b-nonyl glucopyranosides.
nonyl-ß-D-glucoside

8. 1OCO.pdb Download 1oco.pdb from www.rcsb.org
Look at the PDB header. Find:
resolution: ___________
data/parameters ratio: __________
NCS? (yes/no) Where?_______________
Names of heteroatom groups:_____________

9. 2.8Å is “medium” resolution
Backbone is easy to trace, but sidechains may be flipped. Density may be disconnected.

10. Guided tour of Cytochrome C oxidase
Download and display 1OCO.pdb
backbone color-->chain select cys spacefill color cpk Find te non-crystallographic 2-fold
What chain has a disulfide close to the non-crystallographic 2-fold axis?

11. Guided tour of Cytochrome C oxidase
Write two scripts called “main.ras” and “script.ras” in the same directory where you started RasMol:
main.ras
script.ras
restrict (:A|:B|:C|:D|:E|:F|:G|:H|:I|:J|:K|:L|:M)
backbone
color chain
echo "Chain A"
select :A
script script.ras
echo "Chain B"
select :B
...etc....
echo “chain M”
select :M
spacefill
pause
spacefill off
backbone
Which chains do not have transmembrane helices?
Run the script using: script main.ras Hit <return> to advance the script after each “pause”.

12. Guided tour of Cytochrome C oxidase
In the scripts on the previous page:
Change “color chain” to “color structure”
and “backbone” to “cartoon:
Which chains have beta sheets?
Restrict :A and helix Options-->slab mode Adjust slab to show just the transmembrame helices.
How many transmembrane helices are there in chain A?

13. Guided tour of Cytochrome C oxidase
In the scripts on the previous page:
Change “color chain” to “color structure”
and “backbone” to “cartoon:
Which chains have beta sheets?

14. Transmembrane electron channel
select all wireframe select polar color red select !polar color blue select charged color yellow select hetero color cpk spacefill
Center on heme group, use slab mode.
What sidechains that hold the metals? Can you see a pathway for the electrons?

15. You should see this

16. Measuring distances Align the molecule as in the picture.
set picking distance
select polar residues on either side of the membrane.
How thick is the membrane?

17. Possible pathways for electrons
heme irons
Copper ions
Electrons can tunnel or flow through conjugated systems (i.e. aromatic rings, guanadyl groups, carboxylates) and sulfurs.