Resonance Raman studies on xanthine oxidase: observation of Mo^VI-ligand vibrations

Abstract.

Resonance Raman spectra were investigated for the sulfo and desulfo forms of cow's milk xanthine oxidase, with various visible excitation lines between 400 and 650 nm, and Mo^VI-ligand vibrations were observed for the first time. The Mo^VI=S stretch was identified at 474 and 462 cm^–1 for the ³²S- and ³⁴S-sulfo forms, respectively, but was absent in the reduced state and in the desulfo form. The Mo^VI=O stretch was weakly observed at 899 cm^–1 for the sulfo form and shifted to 892 cm^–1 with very weak intensity for the dioxo desulfo form. In measurements of an excitation profile, the two bands at 474 and 899 cm^–1 showed maximum intensity at similar excitation wavelengths, suggesting that the Raman intensity of the metal-ligand modes is due to the Mo^VI←S charge transfer transition, and that this is the origin of the intrinsically weak features of the Mo^VI-ligand Raman bands. When the sulfo form was regenerated from the desulfo form, the 899 cm^–1 band reappeared. However, the band at 899 cm^–1 showed no frequency shift when regeneration was conducted in H₂ ¹⁸O, or after several turnovers in the presence of xanthine in H₂ ¹⁸O. When the sulfo form was reduced and reoxidized in H₂ ¹⁸O buffer, the 899 cm^–1 band reappeared without any frequency shift. These observations suggest that the oxo oxygen in the Mo center of xanthine oxidase is not labile. Low-frequency vibrations of the Mo center were observed together with those of the Fe₂S₂ center with some overlaps, while FAD modes were observed clearly. The absence of dithiolene modes in XO is in contrast to the Mo^VI centers of DMSO reductase and sulfite oxidase.