Dear Tatiana, Having worked also worked on various Fe(II) dependent hydroxylases, may I suggest trying some other less redox sensitive divalent cations? My old lab routinely used to screen hydroxylases with (to my knowledge) Co and Mn, which essentially inhibited enzymatic turnover, so they were also able to add peptide substrates and get structures with inhibitory metal ion, cofactor and substrate bound. I know there were also instances where purification on a Ni column resulted in pretty much irreversible binding of an Ni(II) ion in the active site, which unfortunately prevented functional characterisation as it had a higher binding affinity than the physiological cofactor Fe(II)!
Also, from your description I am guessing there is a fair chance you are working with 2-oxoglutarate dependent hydroxylases? If so there are various cofactor analogues available (N-oxalylglycine among others) which prevent enzymatic turnover and may help trap your iron in the ferrous oxidation state. We often used to add ascorbate, as it prevented a phenomenon called "uncoupled turnover" where the Fe would end up as a Fe(IV)=O species due to a side reaction that didn't involve substrate hydroxylation. Feel free to message off-board if you want more details on any of the above. Best Regards, Tom On 2 October 2014 18:22, ISABET Tatiana < [email protected]> wrote: > 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 > -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
