Electron Spin-Echo Studies of the Copper Binding Site in Phenylalanine Hydroxylase from Chromobacterium violaceum

The active-site structure of the Cu(II)-containing phenylalanine hydroxylase from Chromobacterium violaceum was studied by electron spin-echo spectroscopy. Fourier transformation of the stimulated electron spin-echo envelope for the copper protein revealed frequency components characteristic of copper-histidyl imidazole coordination. The observed nuclear quadrupole frequencies at 0.55, 1.0, and 1.55 MHz are slightly different from those observed for model Cu(II)-imidazole complexes, 0.68, 0.72, and 1.40 MHz, but can be observed in Cu(II) complexes with 2-methylimidazole. A method of analysis by spectral simulation is presented for the quantitation of the number of equatorial imidazoles coordinated to Cu(II). In phenylalanine hydroxylase, two are bound.