CHARACTERIZATION OF HISTIDINE COORDINATION IN VO2+-SUBSTITUTED D-XYLOSE ISOMERASE BY ORIENTATIONALLY-SELECTED ELECTRON SPIN-ECHO ENVELOPE MODULATION SPECTROSCOPY

An orientationally-selected electron spin-echo envelope modulation (ESEEM) spectroscopy investigation was performed on VO2+ introduced into the high-affinity metal-binding site of D-xylose isomerase. The ESEEM spectra clearly reveal the presence of nitrogen ligands with hyperfine coupling A(N) approximate to 6 MHz. Detailed analysis includes first- and second-order treatment of the nitrogen basic and combination harmonics in two-pulse ESEEM spectra of the g(parallel to) and g(perpendicular to) components. Complete determination of the hyperfine and quadrupole tenser indicates equatorial coordination of the imine nitrogen of the histidine residue. The presence of Cd2+ ion in the second, low-affinity metal-binding site does not affect the nitrogen couplings. The protons surrounding the VO2+ ion have been examined via the proton sum combinations in four-pulse ESEEM. They demonstrate the contribution of two protons probably belonging to the histidine ligand. These investigations strongly support the further application of VO2+ as a spin probe in conjunction with ESEEM spectroscopy for detailed investigation of nitrogen ligands in the active metal sites of proteins.