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.

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).

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

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.

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

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

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:_____________

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

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?

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”.

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?

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?

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?

You should see this

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?

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.