Electron spin-echo studies of the copper(II) binding sites in dopamine beta-hydroxylase.

The active site structure of Cu(II) in dopamine beta-hydroxylase, isolated from bovine adrenal medulla, was studied by pulsed electron paramagnetic resonance (EPR) spectroscopy. Fourier transformation of the stimulated electron spin-echo envelope revealed frequency components characteristic of Cu(II)-histidyl imidazole coordination. The three major lines in the spectrum at 0.7, 1.4, and 4.0 MHz are typical for Cu(II)-imidazole complexes where imidazole is protonated and equatorially coordinated. Quantitation of the number of imidazole ligands bound to Cu(II) in enzyme containing two, four, and eight Cu per protein tetramer, as well as characterization of the superhyperfine coupling parameters, was achieved by spectral simulation. In all cases, it was shown that there are three, or more likely four, imidazole ligands bound to Cu(II). Addition of deuteriated substrate analogues to the enzyme did not produce any observable deuterium modulation in the spin-echo envelopes, thus indicating that the distance between substrate deuterons and Cu(II) is greater than 5 A.