Electron Paramagnetic Resonance of the Copper in Dopamine β‐Monooxygenase

Abstract

The water-soluble form of dopamine β-monooxygenase from bovine adrenal medulla was studied. Addition of excess CuSO₄ to purified enzyme preparations followed by extensive ultrafiltration against copper-free buffer at pH 7.0, gave preparations with about four copper atoms per enzyme tetramer of M_r 290000. The enzyme-bound copper was shown by the rapid-freeze technique and electron paramagnetic resonance (EPR) to be reduced by ascorbate at a rate which was faster than the overall catalytic rate; about 10% of the copper was oxidized to Cu(II) during steady-state catalysis in the presence of excess ascorbate. These results support an electron-transfer function of the enzyme-bound copper during catalysis and indicate that the reduction by ascorbate is not the rate-limiting step. The enzyme-bound copper was rapidly chelated by EDTA at pH 7.0, and the apoenzyme thus obtained after dialysis revealed no EPR-detectable copper. Addition of CuSO₄ to the apoenzyme gave an EPR spectrum similar to that of the native enzyme, and the apoenzyme was fully reactivated by the optimal concentration of CuSO₄ in less than 2 s.