ENDOR study of Gd 3+ complexes in frozen solutions

Abstract

The coordination environment of Gd 3+ in frozen solutions is investigated by application of electron nuclear double-resonance spectroscopy. Proton ENDOR spectra of the Gd 3+ ion in frozen methanol-water cosolvent mixtures obtained with the static laboratory magnetic field (Ha) at the turning point of the electron paramagnetic resonance absorption exhibit single-crystal-type line pairs. With use of selectively deuterated materials, the ligand origin of each pair of ENDOR lines has been assigned. For GdCl 3 there are two distinguishable types of protons due to the HO groups of metal-coordinated solvent molecules, and there is one set belonging to the methyl group of metal-coordinated methanol. Similarly, for Gd(CH 3000) 3 and Gd(CH 3CH 2000) 3, the set of ENDOR lines belonging to the methyl group of acetate and to the methylene group of propionate ligands have been identified. By analysis of the dependence of the ENDOR spectra on Ho, we have determined the values of the principal hyperfine coupling (hfc) components of each of the metal-bound ligands. The hfc components of methyl protons of Gd 3+-bound acetate and of the methylene protons of Gd 3+-bound propionate exhibit axial symmetry. Under the point-dipole approximation, they yield correspondingly calculated metal-proton distances of 4.53 and 4.42 Å in good agreement with the value of 4.73 A deduced from crystallographic data for inner sphere coordinated ligands. The hfc components of the HO protons of metal-bound solvent molecules do not exhibit axial symmetry. One set is assigned to inner sphere coordinated H2O while the other is assigned to outer sphere bound CH 30H. The metal-proton distances, calculated on the basis of the largest anisotropic hfc components as lower limit estimates, support these structural assignments. Application of ENDOR spectroscopy is made to identify the primary lanthanide binding site in a-chymotrypsin and to demonstrate the accuracy with which this method of analyzing ENDOR can be employed for structural characterization of metal complexes in frozen solutions. Comparison of the proton ENDOR spectrum of Gd 3+ bound to a-chymotrypsin in frozen D20 solution to those of Gd(CH 3000) 3 and Gd(CH 3CH 2000) 3 indicates that the lanthanide binding site in the protein includes a glutamate residue rather than an asparlate residue. Identification of this protein residue by ENDOR spectroscopy resolves discussions in the literature about the primary lanthanide binding site of a-chymotrypsin.