Pulsed Electron Paramagnetic Resonance Studies of the Copper(II) Site in Galactose Oxidase

Two-pulse and three-pulse electron spin-echo decay envelopes for the nonblue copper protein galactose oxidase have been studied. Analysis of the modulation patterns indicates that the Cu(II) is coordinated to at least one and possibly two protein histidine imidazoles. Fourier cosine transforms of three-pulse data yield superhyperfine frequencies 0.55, 1.0, 1.53, and 4.1 MHz at a magnetic field H₀= 3170 G. Computer simulations of the superhyperfine spectrum for a ¹⁴N nucleus coupled to an electron spin indicate that these frequencies are due to ¹⁴N in a quadrupolar field characterized by frequencies v₊, v_-, and v₀= 1.54, 1.0, and 0.54 MHz and coupled to the electron spin by a term AI-S where A/h = 1.8 MHz. Comparison of the two-pulse echo envelope for the native enzyme with the envelopes for CN^--, F^-, and imidazole coordinated derivatives suggests that galactose oxidase contains a ligand other than imidazole which is readily displaced by any of these three. Linear electric field effect (LEFE) measurements were also made in order to compare the behavior of galactose oxidase with that of blue copper proteins. The form of the LEFE curves and the magnitude of the shifts were similar to those observed for nonblue copper centers and for a number of complexes where Cu(II) is coordinated by N or O. There was no resemblance to LEFE results obtained for blue copper proteins.