Electron paramagnetic resonance of the copper in dopamine beta-monooxygenase. Rapid reduction by ascorbate, the steady-state redox level, chelation with EDTA, and reactivation of the apoenzyme by added copper.
The water-soluble form of dopamine beta-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 Mr 290 000. 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.
D000313:Adrenal Medulla / Q000201:enzymology; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001205:Ascorbic Acid / Q000494:pharmacology*; D002417:Cattle; D003300:Copper; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D010084:Oxidation-Reduction
D001051:Apoenzymes; D001059:Apoproteins; D003300:Copper; D004492:Edetic Acid; D004299:Dopamine beta-Hydroxylase; D001205:Ascorbic Acid