A Combined QM/MM Study on the Reductive Half-Reaction of Xanthine Oxidase: Substrate Orientation and Mechanism

Quantum mechanical/molecular mechanical (QM/MM) methods were used to investigate the conversion of xanthine to uric acid in xanthine oxidase. Seven mechanistic variants were considered with different tautomeric forms of xanthine, different protonation states of the active-site residues, and different substrate orientations. The most favorable pathway (setup G) has a B3LYP/MM barrier of abcut 14 kcal mol(-1), consistent with the available experimental data. This multistep mechanism starts with Glu1261 deprotonating the xanthine at the N3 position followed by a proton transfer from the cofactor to the N9 atom of xanthine; the thus activated cofactor and substrate then react to form a tetrahedral intermediate, and a subsequent rate-limiting hydride transfer generates the product. The substrate orientation that has commonly been assumed in the literature leads to the most stable reactant complex, but the, opposite orientation ("upside down") is computed to be the most favorable one during the reaction (setup G). In the "upside down" conformation, the Arg880 residue can best stabilize the reactive xanthine species with the negatively charged N3 atom, especially the tetrahedral intermediate and the following transition state for hydride transfer which is generally the highest point on the computed energy profiles. QM-only calculations for a minimal gas-phase model and for larger cluster models are performed for comparison, in particular for establishing intrinsic reactivities and a common energy scale. An analysis of the computational results provides detailed insight into the essential mechanistic role of the active-site residues