Substrate Hydroxylation by the Oxido–Iron Intermediate in Aromatic Amino Acid Hydroxylases: A DFT Mechanistic Study

Substrate hydroxylation by an Fe IV =O cluster model of the active center in aromatic amino acid hydroxylases (AAHs) has been investigated by means of DFT calculations. Whereas benzene was used as a model for the aromatic amino acid substrate, the water‐free Fe IV =O cluster model has been used in previous studies of enzyme activation and formation of the hydroxylating intermediate. This cluster model also has the pterin cofactor placed in the first coordination sphere of the iron atom and differs substantially from models used in previous computational studies of AAH‐catalyzed hydroxylations. The formation of the (Fe)O–C(benzene) bond is associated with a free‐energy barrier (12.6 kcal mol –1 ) that is slightly lower than that calculated previously for the formation of the Fe IV =O species. The subsequent steps, the NIH shift and the tautomerization leading to the phenol product are both associated with lower energy barriers. The substrate hydroxylation is followed by protonation of the oxidized iron‐bound cofactor to give the pterin‐4a‐carbinolamine product. The latter is subsequently dissociated upon rebinding of water molecules to produce the hexacoordinate iron complex of the enzyme resting state. Consequently, no product is released before the oxidation of both the substrate and the cofactor has been completed. Finally, the current study completes the catalytic cycle and regenerates the catalyst, with a barrier energy comparable to that of the (Fe)O–C(benzene) bond formation.