Dopamine beta-monooxygenase. Binding to apoenzyme and rapid exchange in holoenzyme of 64Cu studied with high-performance size-exclusion gel chromatography.

The binding of 64Cu to the water-soluble form of dopamine beta-monooxygenase from bovine adrenal medulla was studied in reconstitution and exchange experiments using high-performance size-exclusion gel chromatography. The reconstitution experiments provide evidence for a specific binding of four copper atoms/enzyme tetramer using either Cu(I) or Cu(II), but some weaker copper-binding sites were observed in the presence of a large excess of copper. The exchanges of both Cu(I) and Cu(II) in this protein are so rapid that exact half-lives for the exchange reactions can not be obtained by the present method. The results indicate, however, that the half-life for the exchange of the enzyme-bound copper in the holoenzyme with a twofold excess of 64Cu(II) at pH 6.1 was about 1 min, whereas the exchange of Cu(I) measured at similar conditions with ascorbate present, was complete in 1 min. This is by far the most rapid exchange reported for any copper-protein, and the results points to a unique copper-binding site in this enzyme.