Hemann Craig, Ilich Predrag, Stockert Amy L, Choi Eun-Young, Hille Russ
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
J Phys Chem B. 2005 Feb 24;109(7):3023-31. doi: 10.1021/jp046636k.
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.
利用密度泛函理论计算和共振拉曼光谱，对含钼酶复合物黄嘌呤氧化酶与紫蝶呤的分子、电子和振动特性进行了研究。电子结构计算是在一个模型上进行的，该模型由酶钼蝶呤辅因子[四价还原态；Mo(IV)O(SH)]通过氧桥Mo-O-C7与紫蝶呤（中性形式的1H,3H,8H-蝶啶-2,4,7-三酮）共价连接而成。使用SDD基组以及Becke的三参数交换泛函与Lee、Yang和Parr相关泛函，对所有模型进行了全几何优化。测定了多种同位素的谐波振动频率，试图将实验观察到的Mo-OR桥18O标记后与结合产物的位移以及在D2O中制备的样品中溶剂可交换质子取代后观察到的位移联系起来。理论振动频率与在H2O和D2O中制备的还原型黄嘌呤氧化酶-紫蝶呤复合物的共振拉曼光谱中实验观察到的振动模式以及18O标记产物的振动模式吻合良好。将计算得到的同位素位移与共振拉曼实验得到的同位素位移进行关联，得到了在350 - 1750 cm(-1)范围内观察到的所有模式的完整简正模式归属。目前的工作表明，紫蝶呤通过酶引入的羟基以简单的端基方式与活性位点的钼配位的模型，能够准确预测复合物观察到的共振拉曼光谱。鉴于涉及桥连氧的模式众多，可以排除侧基结合模式。
