A quantum mechanical/molecular mechanical study of the hydroxylation of phenol and halogenated derivatives by phenol hydroxylase

A combined quantum mechanical and molecular mechanical (QM/MM) method (AM1/ CHARMM) was used to investigate the mechanism of the aromatic hydroxylation of phenol by a flavin dependent phenol hydroxylase (PH), an essential reac tion in the degradation of a wide range of aromatic compounds. The model fo r the reactive flavin intermediate (C4a-hydroperoxyflavin) bound to PH was constructed on the basis of the crystal structure of the enzyme-substrate c omplex. A potential energy surface (PES) was calculated as a function of th e reaction coordinates for hydroxylation of phenol (on C6) and for proton t ransfer from phenol (O1) to an active-site base Asp54 (OD1). The results su pport a reaction mechanism in which phenol is activated through deprotonati on by Asp54, after which the phenolate is hydroxylated through an electroph ilic aromatic substitution. Ab initio test calculations were performed to v erify these results of the QM/MM model. Furthermore, the variation in the c alculated QM/MM activation energies for hydroxylation of a series of substr ate derivatives was shown to correlate very well (R = 0.98) with the natura l logarithm of the experimental rate constants for their overall conversion by PH (25 degrees C, pH 7.6). This correlation validates the present QM/MM model and supports the proposal of an electrophilic aromatic substitution mechanism in which the electrophilic attack of the C4a-hydroperoxyflavin co factor on the activated (deprotonated) substrate is the rate-limiting step at 25 degrees C and pH 7.6. The correlation demonstrates the potential of t he QM/MM technique for predictions of catalytic activity on the basis of pr otein structure. Analysis of the residue contributions identifies a catalyt ic role for the backbone carbonyl of a conserved proline residue, Pro364, i n specific stabilization of the transition state for hydroxylation. A cryst al water appears to assist in the hydroxylation reaction by stabilizing the deprotonated C4a-hydroxyflavin product. Comparison of the present results with previous QM/MM results for the related p-hydroxybenzoate hydroxylase ( Ridder et al. J. Am. Chem. Sec. 1998, 120, 7641-7642) identifies common mec hanistic features, providing detailed insight into the relationship between these enzymes.