1st. Internat. Cant on Bioinorganic Chemistry -Session 0
We have now studied the binding of 's"Cu to this enzyme in reconstitution and exchange experiments. High performance size-exclusion gel chromatography with the protein analysis column I-125 from Waters was used to separate the enzyme-bound and free e"Cu, and the amount of @"Cu bound to the protein was determined from the radioactivity eluting together with the protein. Experiments with binding of 64Cu(II) to the ap oenzyme give further evidence for a specific binding of 4 copper atoms per tetramer, but some weaker copper-binding sites were observed in the presence of an excess of copper. When the apoenzyme was incubated with 4 atoms of @'Cu(II) per tetramer, about 3.5 copper atoms were eluted with the protein indicating that the binding of Cu(I1) is not extremely tight. Similar amounts of @'Cu were bound to the apoenzyme in the presence of ascorbate indicating the binding of Cu(I) is similar to that of Cu(I1).
The exchanges of both Cu(I) and Cu(I1) in the holoenzyme are rapid and a half-life of about 1 min was estimated for the exchange of the enzymebound Cu(I1) in the presence of a two-fold excess of @'Cu(II) at pH 6.1. Experiments in the presence of ascorbate revealed that the exchange of Cu(I) was complete in 1 min at similar conditions. The exchange of the copper atoms in dopamine p-monooxygenase are thus much more rapid than reported for other copper proteins, and the present results point to a unique copper-binding site in this protein.