Phenylalanine Hydroxylase (PheH) is a liver enzyme that catalyzes the conversion of L- phenylalnine to L-tyrosine using tetrahydrobiopterin and molecular oxygen. Deficiencies in this enzyme cause phenylketonuria (PKU), an autosomal recessive metabolic disorder that occurs in ~1/10,000 live births. The general mechanism of PheH consists of two major steps: (1) formation of the ferryl-oxo species and (2) hydroxylation of the aromatic amino acid L-phenylalanine. Before the coupled hydoxylation can occur, PheH is activated by the presence of L-phe from the T-(unactivated) to R-(activated)-state. Here activation is marked by a distinct increase in the specific activity, which corresponds to a 2.2 and 8-fold increase in the specific activity for hPheH and rPheH, respectively. Accompanying the activation of PheH is a global conformational change, which results in a ligand rearrangement of the active site. Mixed signals about the presence of water in the literature have lead to multiple accounts concerning the role water plays in the formation of the ferryl-oxo species. This work provided the basis for studying water ligands in pterin dependent non-heme iron enzymes with an [FeNO]7 spin system using 1H ESEEM and HYSCORE. Based on qualitative argument with the 1H ESEEM spectra from the [FeNO]7-(N2O)(H2O)2 and [FeNO]7-(N2O2)(H2O)1 model complexes, it was determined that the activation of PheH, without BH4 complexed, lead to a one water dehydration of PheH's active site. The water ligands were then quantitatively analyzed to show characteristic dipolar distances (from the iron to the water protons) of 2.52 - 2.62 Å and β angles between 58° - 83° and 97° - 122°. The observed dipolar distances and angles are consitent with water ligands that are located cis to the binding site of NO. Overall, this analysis demonstrated the ability of using 1H ESEEM and HYSCORE to study water ligands bound to non-heme iron enzymes.
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