title: Resonance Raman studies of xanthine oxidase: The reduced enzyme-product complex with violapterin.
abstract: A study of the molecular, electronic, and vibrational characteristics of the molybdenum-containing enzyme complex xanthine oxidase with violapterin has been carried out using density functional theory calculations and resonance Raman spectroscopy. The electronic structure calculations were carried out on a model consisting of the enzyme molybdopterin cofactor [in the four-valent, reduced state; Mo(IV)O(SH)] covalently linked to violapterin (1H,3H,8H-pteridine-2,4,7-trione in the neutral form) via an oxygen bridge, Mo-O-C7. Full geometry optimizations were performed for all models using the SDD basis set and the three-parameter exchange functional of Becke combined with the Lee, Yang, and Parr correlational functional. Harmonic vibrational frequencies were determined for a variety of isotopes in an attempt to correlate experimentally observed shifts upon 18O-labeling of the Mo-OR bridge to bound product as well as shifts seen upon substitution of solvent-exchangeable protons in samples prepared in D2O. The theoretical vibrational frequencies compared favorably with experimentally observed vibrational modes in the resonance Raman spectra of the reduced xanthine oxidase-violapterin complex prepared in H2O and D2O and with 18O-labeled product. Correlating the isotopic shifts from the calculations with those from the resonance Raman experiments resulted in complete normal mode assignments for all modes observed in the 350-1750 cm(-1) range. The present work demonstrates that a model in which the violapterin is coordinated to the molybdenum of the active site in a simple end-on manner via the hydroxyl group introduced by an enzyme accurately predicts the observed resonance Raman spectrum of the complex. Given the numerous modes involving the bridging oxygen, a side-on binding mode can be eliminated.
authors.affiliation: Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Hemann
authors.forename: Predrag
authors.initials: P
authors.lastname: Ilich
authors.forename: Amy L
authors.initials: AL
authors.lastname: Stockert
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Choi
authors.forename: Russ
authors.initials: R
authors.lastname: Hille
mesh_terms: D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry; D017666:Deuterium Oxide / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D008982:Molybdenum / Q000737:chemistry; D000090006:Molybdenum Cofactors; D010100:Oxygen / Q000737:chemistry*; D011621:Pteridines / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry*
chemical_list: D003067:Coenzymes; D017666:Deuterium Oxide; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D010100:Oxygen; D011621:Pteridines; D012997:Solvents; D014867:Water; D014969:Xanthine Oxidase; C040826:violapterin; C022261:molybdopterin