Reductive Activation of Phenylalanine Hydroxylase and Its Effect on the Redox State of the Non-Heme Iron

Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (g_eff = 9.4-8.7, 4.3 and g_eff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the g_eff ⋍ 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the g_eff ⋍ 6.7-S.4 region correlates with the activity of the enzyme. The latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of o-phenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fe²⁺ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing Fe²⁺ that functions aerobically to produce tyrosine from added 6-methyltetrahydropterin in a 1/1 stoichiometry. Reductive titration of the hydroxylase by dithionite is consistent with the addition of one electron/subunit for coupled turnover. The implications of these findings for the mechanism of action of this enzyme are briefly discussed.