Formation of the iron-oxo hydroxylating species in the catalytic cycle of aromatic amino acid hydroxylases.

The first part of the catalytic cycle of the pterin-dependent, dioxygen-using nonheme-iron aromatic amino acid hydroxylases, leading to the Fe(IV)=O hydroxylating intermediate, has been investigated by means of density functional theory. The starting structure in the present investigation is the water-free Fe-O(2) complex cluster model that represents the catalytically competent form of the enzymes. A model for this structure was obtained in a previous study of water-ligand dissociation from the hexacoordinate model complex of the X-ray crystal structure of the catalytic domain of phenylalanine hydroxylase in complex with the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) (PAH-Fe(II)-BH(4)). The O-O bond rupture and two-electron oxidation of the cofactor are found to take place via a Fe-O-O-BH(4) bridge structure that is formed in consecutive radical reactions involving a superoxide ion, O(2)(-). The overall effective free-energy barrier to formation of the Fe(IV)=O species is calculated to be 13.9 kcal  mol(-1), less than 2 kcal  mol(-1) lower than that derived from experiment. The rate-limiting step is associated with a one-electron transfer from the cofactor to dioxygen, whereas the spin inversion needed to arrive at the quintet state in which the O-O bond cleavage is finalized, essentially proceeds without activation.