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 AN≈ 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||and g⊥components. Complete determination of the hyperfine and quadrupole tensor 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.
ASJC Scopus subject areas
- Colloid and Surface Chemistry