Reconstitution, Preparation and Investigation of Metal Derivatives

In stating the metal content of the enzyme and of its metal derivatives only one subunit will be considered. The Cu site is presented to the left and the Zn site to the right (Cu,Zn) e.g. for the native enzyme. 

Cu and Zn can entirely be removed from BESOD by chromatography on a Sephadex G-25 column by elution with 10 mM EDTA at pH 3.8 At this pH the apoprotein is stable and a total reconstitution was achieved by the stoichiometric addition of Cu and subsequently of Zn, followed by a dialysis against phosphate buffer pH 7.8. Zn could also be replaced by Co, Cd, and Hg yielding active products. The (Cu,—)-derivative with an empty Zn site was only partially active . It is important in such experiments to remove EDTA entirely from the apoprotein, like by a dialysis against 0.1 M NaClO  

A very interesting (Cu ,Cu )-derivative of BESOD was prepared with 2 Cu per subunit. The EPR spectrum between 5 and 10 K indicated an antiferromagnetic interaction of the two Cu with a coupling constant of 52 cm , which seems very good evidence for an imidazolate-anion bridge as borne out with model complexes Similarly the (Cu , Co )-derivative of BESOD showed only a weak EPR spectrum reduction of Cu(II) to Cu(I) a normal EPR spectrum 

In phosphate buffer pH 7.4 a (Co ,Co )-derivative of BESOD was obtained, while the addition of Co in acetate buffer pH 5.4 yielded the (—,Co )-derivative .  

The inactive (Ag ,Cu )- and (Ag, Co )-derivatives of BESOD have also been prepared. The EPR spectra of (Ag, Cu )- and (Cu ,Cu )-BESOD were very similar like the visible absorption spectra of (Ag ,Co )- and (Cu, Co )-BESOD. From model fitting to the electron-density map at 0.3-nra resolution by an interactive computer graphics system is was concluded that the bridging His-61 is slightly out of the plane of Cu and its three other histidine ligands .  

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