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 (ES EEM) 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 harm onics in two-pulse ESEEM spectra of the g(parallel to) and g(perpendic ular to) components. Complete determination of the hyperfine and quadr upole tenser indicates equatorial coordination of the imine nitrogen o f the histidine residue. The presence of Cd2+ ion in the second, low-a ffinity metal-binding site does not affect the nitrogen couplings. The protons surrounding the VO2+ ion have been examined via the proton su m combinations in four-pulse ESEEM. They demonstrate the contribution of two protons probably belonging to the histidine ligand. These inves tigations strongly support the further application of VO2+ as a spin p robe in conjunction with ESEEM spectroscopy for detailed investigation of nitrogen ligands in the active metal sites of proteins.