Studies on the reaction mechanism of the reductive half-reaction of xanthine oxidase.

摘要

Xanthine oxidase, a molybdenum containing hydroxylase, catalyzes the final two steps in purine metabolism, converting hypoxanthine to xanthine and xanthine to uric acid and is of fundamental physiological importance. The reductive half-reaction of the catalytic cycle is of intrinsic chemical interest due to its unique chemistry and several reaction mechanisms have been proposed in the literature. In the present study the two most probable mechanisms, a "hydroxyl transfer" as proposed by Huber and Hille and an "oxygen insertion" as proposed by Bray and coworkers, have been extensively evaluated by investigating two catalytic intermediates spectroscopically and examining the compatibility of the results with the proposed reaction mechanisms. These catalytic intermediates are the species giving rise to the "very rapid" Mo(V) EPR signal and the Mo(IV)-violapterin charge-transfer complex.;The "very rapid" species has been investigated by Electron Nuclear DOuble Resonance (ENDOR) and Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. To make the system suitable to investigation by these techniques, [8-13C]HMP was synthesized starting from 6-methyl uracil. The results yield a distance between active site molybdenum and C 8 of HMP to be 2.84∼3.02 A, indicating that the bound product is coordinated to molybdenum in a "simple" fashion with a bent Mo-O-R geometry.;The Mo(IV)-violapterin charge-transfer complex encountered in the course of the enzymatic hydroxylation of lumazine has also been studied by resonance Raman spectroscopy to reveal the chemical nature of C7-O bond in the complex. This has involved a comparative study which requires two standards, the neutral form of free violapterin and 7-methoxylumazine to obtain reference frequencies for C=O and for C-O, respectively. The results of the resonance Raman study show that C7-O bond is considerably weaker than the formal C=O in violapterin on the action of xanthine oxidase, consistent with a bridging C7-O bond to metal in the charge-transfer complex.;Since the information obtained from the present study is compatible with the Mo-O-C structure in both Mo(IV)-OR and Mo(V)-OR intermediates, it is concluded that the "hydroxyl transfer" mechanism by Huber and Hille is the most likely reaction mechanism for the reductive half-reaction of xanthine oxidase. (Abstract shortened by UMI.).