Raman and luminescence studies on phase transition of EuNbO4 under high pressure

Raman and luminescence studies on the phase transition of europium orthoniobates(EuNbO4) under high pressure were performed. The pressure dependent Raman spectra revealed that an irreversible phase transition from monoclinic phase to M'-fergusonite phase of EuNbO4 occurred at 7.3 GPa, and the two phases coexisted over a pressure range from 7.3 to 13.7 GPa. An obvious discontinuity on luminescence intensity ratio between 5D0 →7F2 and 5D0→7F1 transitions was observed with increasing pressure, indicating also that a phase transition occurred at 7.3 GPa, which was in agreement with the high pressure Raman spectra data. Meanwhile, the Raman and luminescence spectra in the temperature range of 20–300 K showed the structure stability at low temperatures.     

Raman difference studies of GDP and GTP binding to c-Harvey ras

The vibrational spectra of phosphate modes for GDP and GTP bound to the c-Harvey p21(ras) protein have been determined using 18O isotope edited Raman difference spectroscopy. A number of the phosphate stretch frequencies are changed upon GDP/GTP binding to ras, and the results are analyzed by ab initio calculations and through the use of empirical relationships that relate bond orders and bond lengths to vibrational frequencies. Bound GDP is found to be strongly stabilized by its interactions, mostly electrostatic, with the active site Mg2+. Bound GTP also interacts with the active site Mg2+ via its beta-phosphate group, as expected on the basis of crystallographic studies of bound GppNp. The angle between the nonbridging P&bondDot;O bonds of the gamma-phosphate of bound GTP increase by about 1-2 degrees compared to its solution value, thus bringing about a geometry that is closer to planar for these bonds as expected for the putative pentacoordinated transition state geometry of the phosphotransfer reaction. Modeling of the interactions at the nucleotide binding site suggests that the water molecule in-line with the P-O bond is positioned to bring about the change in bond angle. Moreover, a weak fifth bond (about 0.03 vu) appears to be formed between it and the gamma-phosphorus atom of bound GTP with a concomitant weakening of the O-P bond between the GDP leaving group and the gamma-phosphorus atom. Hence, an important role of the active site structure appears to be the strategic positioning of this in-line water. These structural results are consistent with a reaction pathway for GTP hydrolysis in ras of synchronous bond formation between the gamma-phosphorus of GTP and the attacking nucleophile and bond breaking between the gamma-phosphorus and the GDP leaving group.     

Resonance Raman studies of the protocatechuate 3,4-dioxygenase from Brevibacterium fuscum

Resonance Raman studies of the protocatechuate 3,4-dioxygenase (PCD) from Brevibacterium fuscum have been carried out to take advantage of the high iron-site homogeneity of this enzyme. Native uncomplexed PCD exhibits individual resonance-enhanced nu CO and delta CH vibrations for the two tyrosinates coordinated to the active site iron center, which can be assigned to a particular residue by their excitation profiles. Of the two nu CO features observed at 1254 and 1266 cm-1, only the latter is upshifted (to 1272 cm-1) when H2O is replaced by D2O. Similarly the 1254-cm-1 feature is unaffected, while the 1266-cm-1 feature is shifted to approximately 1290 cm-1 when inhibitors such as phenolates or terephthalate bind to the active site. These observed shifts can be rationalized by the presence of hydrogen-bonding interactions with solvent in the active site cavity, which are modulated by D2O and eliminated upon inhibitor binding. Examination of the PCD crystal structure suggests that the axial tyrosine can be hydrogen bonded in the uncomplexed enzyme to water molecules present in the substrate binding pocket. The equatorial tyrosine may also be hydrogen bonded but to solvent molecules which are trapped in a pocket inaccessible to bulk solvent. These studies allow for the first time the association of particular Raman spectroscopic features, i.e., the nu CO's at 1254 and 1266 cm-1, with the equatorial and axial tyrosine residues in the PCD active site, respectively; they lay the groundwork for further Raman studies on catalytically important species to determine the roles these tyrosine residues may play in the PCD reaction cycle.