Instructor: Doug Ohlendorf
Paper title: Crystallographic and single-crystal spectral analysis of the peroxidase ferryl intermediate. Meharenna et al (2010) Biochemistry 49: 2984-6.
Type your answers into the worksheet prior to the second class, and then edit a printed copy during the discussion.
No loose sheets. Print double-sided or use a metal staple. Do NOT fold the corners.
1. What is the reaction catalyzed by CCP?
Fe(III)-> Fe(IV)O/R•þ
2. What is (are) the cofactors critical for activity of CCP?
H2O2’s which is used to oxidize Fe(III) and Fe(IV)O/R•þ
3. What are the Fe-O distances for (a) Fe+4-OH, (b) Fe+4=O?
a. About 1.8-1.9 b. About 1.7 A° or less
4. Why was the N184R made (see ref 9)?
In previous studies it was found that the N184R mutant diffracted a lot, giving possibility to attain a low x-ray dose of the compound 1 structure at resolution which are high. This can ultimately help determine structural accuracy between crystal studies and other experimental methods (like solution studies).
5. What is Compound I?
A heme peroxidase containing an oxidized form of iron, more specifically in the form of Fe(IV)O/R•þ.
6. At what wavelength were diffraction data collected (see http://answers.yahoo.com/question/index?qid=20091014232904AAHCfb1 ?
13.0 keV=13000eV
λ=hc/E λ=1240/13000eV=387.5nm 7. Why do x-rays cause reduction of Compound I?
Because x-ray soloution increases the pH of the surroundings making it more likely to donate an H and protonate the iron complex??
8. How were low dose data collected?
About 100 crystals were mounted in an automated fashion. 15 Different runs containing various X-ray dosages were then collected for each crystal. The lowest run (Run 1) consisted of 5 degrees of agiler seperation, with 0.035 MGy of X-ray exposure
9. What structural changes were there between high dose and low dose structures?
The iron group was located at a closer range to the distal pocket, then the higher does structure. The His and Fe bond lengths are also different between the two. Moreover the carbonyl O atom of the heme ligand, His175. is slightly less than 0.1A° closer to Trp191 in the low-dose.
10. What does the difference electron density map shown in Figure 2B mean?
They essentially highlight the structural differences of the molecule seen with different x-ray dosages. The amount of electron density can tell where a certain molecules location may be.
11. Figure 3 shows the Fe-O distance increases with higher x-ray dose. What does this mean?
It also means since the bond length becomes higher with increased x-ray dosages, it is more likely to have a double bond at low x-ray dosages (because double bonds are shorter) then higher x-ray dosages. Since longer bonds (1.8-1.9) are not characteristic of a double bond higher species are most likely to have a single bond.
12. What do the results of this paper suggest regarding structures of other metalloenzymes?
They suggest that the ferryl group associated with compound 1 indicates a double bond with the oxygen (Fe+4=O), rather than a hydroxide group with the oxygen (Fe+4-OH).
Also the results mean low x-ray dosages are more accurate then high x-ray dosages for the crystal structure of compound 1, because their Fe-O bond distances correlate more with other more proven experimental bond length data analysis (like EXAFS). 