Dear all, Sorry for a non purely crystallographic question.
I am working on an enzyme which binds Fe2+ cations to catalyzes an FeII-dependent hydroxylation reaction. Because of fast oxidation in presence of the enzyme, it is very difficult to soak Fe2+ ions into the crystals. We succeed only under anaerobic conditions (glove box). I use a combination of dithionite as a reducing agent and Fe2+SO4 or (NH4)2Fe(SO4)2 as Fe2+ source. Despite these precautions, the Fe2+ is most often disordered in the active site. When I add Fe2+ under aerobic conditions, Fe2+ oxidizes immediately upon contact with the protein solution (despite 1mM Dithionite for 5mM Fe2+ and protein concentration = 230uM). Furthermore, the hydroxyl donor molecule, which should bind Fe2+ (before the substrate) and one residue of the protein, is not seen in the electron-density maps in the active site. I have tried several soaking conditions. When I try a co-crystallization approach, adding Fe2+ and this hydroxyl donor molecule directly to the protein solution under anaerobic conditions, the protein precipitates. Does anybody have an idea or experience with this type of results? or how to fix the molecule to such a site? What type of phenomena could occur at the active site preventing the binding of the product? Thanks for your help Best regards Tatiana
