Characterization of Histidine Coordination in VO2+-Substituted D-Xylose Isomerase by Orientationally-Selected Electron Spin-Echo Envelope Modulation Spectroscopy

Abstract

An orientationally-selected electron spin-echo envelope modulation (ESEEM) spectroscopy investigation was performed on VO{sup 2+} 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{sup N} {approx} 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{sub {parallel}} and g{sub {perpendicular}} components. Complete determination of the hyperfine and quadrupole tensor indicates equatorial coordination of the imine nitrogen of the histidine residue. The presence of Cd{sup 2+} ion in the second, low-affinity metal-binding site does not affect the nitrogen couplings. The protons surrounding the VO{sup 2+} 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 VO{sup 2+} as a spin probe in conjunction with ESEEM spectroscopy for detailed investigation of nitrogen ligands in the active metal sites of proteins. 41 refs., 8 figs., 2 tabs.