Magnetic Resonance Studies of Cyanide and Fluoride Binding to Galactose Oxidase Copper(II): Evidence for Two Exogenous Ligand Sites

Evidence for a second, inner-sphere coordination site at the copper(II) center in galactose oxidase (GOase) has been obtained from an analysis of cyanide-fluoride competition manifested in 19F NMR relaxation rate changes. A quantitative description of the ¹⁹F NMR relaxation rate deenhancement produced by added CN^- is provided by the following model. (1) At equatorial sites CN^- and F^- (both detected by ESR superhyperfine splitting effects in the GOase ESR spectra) bind strongly to the copper(II) atom. (2) In addition, fluoride (detected by 19F NMR relaxation rate enhancement) binds more weakly to an axial coordination site. (3) The binding of axially coordinated F^- is weakened and/or the hyperfine coupling constant of this fluoride is diminished when equatorially coordinated F^- is displaced by CN^-. The effect of CN^- coordination on unpaired electron spin density near the copper(II) center is also shown by changes in 14N ESR hyperfine splittings attributed to endogenous GOase ligands. The model proposed above is consistent with previously reported magnetic resonance data and results of kinetic competition studies.