Ab Initio QM/MM Modeling of the Hydroxylation Step in p-Hydroxybenzoate Hydroxylase

Abstract

p-Hydroxybenzoate hydroxylase (PHBH) is the model enzyme for the microbial flavin-dependent monooxygenases. The aromatic hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in PHBH has been studied by a combined ab initio quantum mechanics and molecular mechanics (QM/MM) method. Starting from a model of the C4a-hydroperoxyflavin intermediate in the PHBH reaction cycle, built on the basis of the crystal structure of the enzyme−substrate complex, a pathway for the hydroxylation step was calculated by imposing a reaction coordinate involving cleavage of the peroxide oxygen−oxygen bond and bond formation between the C3 atom of the substrate and the distal oxygen of the peroxide moiety of the cofactor. A QM/MM potential was used in which the QM region (49 atoms) was treated at the ab initio HF level with the 3-21G(d) or 6-31G(d) basis sets. The accuracy of various aspects of the QM/MM method for this system has been tested by comparison to higher-level calculations. I...