The copper of dopamine β-Monooxygenase: High accessibility and rapid exchange

Abstract

Dopamine β-monooxygenase (dopamine β-hydroxylase: EC 1.14.17.1) catalyzes the reaction: Dopamine + ascorbate + O 2 → noradrenaline + dehydroascorbate + H 2O (RH + 2e − + 2H + + O 2 → ROH + H 2O) The purified water-soluble enzyme from bovine adrenal medulla contains 4 copper atoms per enzyme tetramer of 290,000 daltons. These copper atoms are essential for enzymic activity, and they most probably participate in both electron transfer and binding of O 2 [1]. The copper in dopamine β-monooxygenase can be classified as type 2 copper according to its EPR spectrum, with a large hyperfine splitting and the low absorption in the visible spectrum (ϵ = 40 M −1 Cu · cm −1 at 680 nm, the maximum of the Cu(II)-band) [1]. This enzyme-bound copper is, however, different from type 2 copper in the blue oxidases by other criteria, especially by showing a high accessibility of the copper sites. Thus, we have shown that the copper atoms of dopamine β-monooxygenase can be rapidly removed by chelators at nondenaturing conditions both in the reduced and oxidized states, and the inactive apoenzyme is reactivated in less than 2s by addition of CuSO 4 [2, 3]. We have now studied the binding of 64Cu 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 64Cu, and the amount of 64Cu bound to the protein was determined from the radioactivity eluting together with the protein. Experiments with binding of 64Cu(II) to the apoenzyme 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 64Cu(II) per tetramer, about 3.5 copper atoms were eluted with the protein indicating that the binding of Cu(II) is not extremely tight. Similar amounts of 64Cu were bound to the apoenzyme in the presence of ascorbate indicating the binding of Cu(II) is similar to that of Cu(II). The exchanges of both Cu(I) and Cu(II) in the holoenzyme are rapid and a half-life of about 1 min was estimated for the exchange of the enzyme bound Cu(II) in the presence of a two-fold excess of 64Cu(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 β-monooxygenase are thus much more rapid than reported for other copper proteins, and the present results point to unique copper-binding site in this protein.