Investigation of the active site structure of non-heme iron dependent phenylalanine hydroxylase: Spectroscopic studies and their implications for reaction mechanism.

摘要

Classic phenylketonuria (PKU) is an autosomal recessive human genetic disease caused by a deficiency of the hepatic non-heme iron dependent metalloenzyme phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase, E.C. 1.14.16.1). PAH is a mixed function oxidase that catalyzes the hydroxylation of L-phe to L-tyr in the presence of the reduced cofactor tetrahydrobiopterin and dioxygen.; A variety of spectroscopic (electronic, EPR, CD, MCD, ESEEM, XAS) and kinetic (stopped-flow and UV/vis) techniques were applied to investigate the geometric and electronic structure of the mononuclear ferrous active site of PAH and its interactions with substrate and pterin cofactor analogues. At 1.4 K, the lower Kramers doublet is fully populated and the protein gives rise to well resolved ESEEM spectra indicating electron-nuclear coupling to two 14N populations and not to NO. The 14N couplings arise from the two histidines coordinated to the iron center. In the presence of per-deuterated phenylalanine, ESEEM spectra show strong coupling to 2H, indicating that substrate binds in close proximity to the Fe-NO moiety. The preliminary studies indicate that the activation process might be responsible for causing the pterin to bind closer to the iron and allowing the enzyme to perform the tightly coupled hydroxylation of substrate. The ESEEM studies of enzyme in both resting T state and allosterically activated R state are consistent with the existence of one exchangeable water coordination site on the iron center. Addition of both L-phe and 5-deaza-6-MPH₄ to the resting state six-coordinate active site results in a five-coordinate iron site, leaving an open position that directly implicates iron in the coupled hydroxylation of its substrates. On the other hand, the iron center of E280K mutant form of PAH remains six-coordinate. This is the first direct evidence of an open coordination position on the iron of PAH prior to binding and activation of dioxygen, which suggests that a highly reactive oxygen intermediate can be generated only when both substrate and cofactor are present.