The non-heme iron-dependent metalloenzyme, rat hepatic phenylalanine hydroxylase (EC 22.214.171.124; phenylalanine 4-monooxygenase (PAH)) was overexpressed in Escherichia coli and purified to homogeneity, allowing a detailed comparison of the kinetic, hydrodynamic, and spectroscopic properties of its allosteric states. The homotetrameric recombinant enzyme, which is highly active and contains 0.7-0.8 iron atoms per subunit, is identical to the native enzyme in several properties: Km, 6-methyltetrahydropterin = 61 μM and L-Phe = 170 μM; Vmax = 9 S-1 (compared to 45 μM, 180 μM, and 13 S-1 for the rat hepatic enzyme). L-Phe and lysolecithin treatment induce the rPAHT → rPAHR (where r is recombinant) allosteric transformation necessary for rPAH activity. Characteristic changes in the fluorescence spectra, increased hydrophobicity, a large activation energy barrier, and a 10% volume increase of the tetrameric structure are consistent with a significant reorganization of the protein following allosteric activation. However, optical and EPR spectroscopic data suggest that only minor changes occur in the primary coordination sphere (carboxylate/histidine/water) of the catalytic iron center. Detailed steady state kinetic investigations, using 6-methyltetrahydropterin as cofactor and lysolecithin as activator, indicate rPAH follows a sequential mechanism. A catalytic Arrhenius Eact of 14.6 ± 0.3 kcal/mol subunit was determined from temperature-dependent stopped-flow kinetics data. rPAH inactivates during L-Phe hydroxylation with a half-life of 4.3 min at 25 °C, corresponding to an Arrhenius Eact of 10 ± 1 kcal/ mol subunit for the inactivation process. Catechol binding (2.4 × 106 M-1) is shown to occur only at catalytically competent iron sites. Ferrous rPAH binds NO, giving rise to an ST = 3/2 spin system.