Mechanism of the Dinuclear Iron Enzyme p-Aminobenzoate N-oxygenase from Density Functional Calculations

AurF is a diiron enzyme that utilizes two dioxygen molecules as the oxidant to catalyze the oxidation of p-aminobenzoate to p-nitrobenzoate. Density functional calculations were performed to elucidate the reaction mechanism of this enzyme. Two different models were considered, with the oxygenated intermediate being a diferric peroxo species or a diferric hydroperoxo species. The calculations strongly favor the model with a diferric peroxo species and support the mechanism proposed by Bollinger and co-workers. The reaction starts with the binding of a dioxygen molecule to the diferrous center to generate a diferric peroxide complex. This is followed by the cleavage of the O-O bond, concertedly with the formation of the first N-O bond, which has a barrier of only 9.2kcal/mol. Subsequently, the first-shell ligand Glu227 abstracts a proton from the substrate. After the delivery of two electrons from the external reductant and two protons from solution, a water molecule and the experimentally suggested intermediate p-hydroxylaminobenzoate are produced and the diferrous center is regenerated. The oxidation of the p-hydroxylaminobenzoate intermediate requires the binding of a second dioxygen molecule to the diferrous center to generate the diferric peroxide complex. Similarly to the oxidation of p-aminobenzoate, the O-O bond cleavage and the formation of the second N-O bond take place in a concerted step. The p-nitrobenzoate product is formed after the release of two protons and two electrons from the substrate. The model with a hydroperoxo species gave a much high barrier of 28.7kcal/mol for the substrate oxidation due to the large energy penalty for the generation of the active hydroperoxo species.