PHENYLALANINE-HYDROXYLASE FROM CHROMOBACTERIUM-VIOLACEUM - UNCOUPLED OXIDATION OF TETRAHYDROPTERIN AND THE ROLE OF IRON IN HYROXYLATION

A gene encoding phenylalanine hydroxylase has been cloned from Chromob acterium violaceum and expressed in Escherichia coli. The purified phe nylalanine hydroxylase contains copper, which does not support enzymat ic activity, Upon removal of copper by dithiothreitol (DTT), the enzym e contains substoichiometric amounts of calcium and zinc but Little or no redox-active metal ions. The copper-depleted hydroxylase catalyzes the phenylalanine-dependent oxidation of 6,7-dimethyltetrahydropterin (DMPH4) by O-2 in a reaction in which phenylalanine is not hydroxylat ed and does not appear to undergo a chemical change, and hydrogen pero xide is produced. Analogs of phenylalanine also activate the oxidation of DMPH4. Both the copper-phenylalanine hydroxylase and the copper-de pleted hydroxylase catalyze the hydroxylation of phenylalanine in the presence of DTT and FeSO4 in a reaction in which hydrogen peroxide is not produced. The apparent values of K-m for Fe2+ and DTT are 0.28 mu M and 1.1 mM, respectively, at 1.0 mM phenylalanine, 120 mu M DMPH4 an d pH 7.4 and 23 degrees C. The apparent value of k(cat) is 14.3 s(-1) under these conditions. Glutathione, mercaptoethanol, and dihydrolipoa te support the hydroxylation of phenylalanine essentially as well as D TT, Incubation of copper-depleted hydroxylase with FeSO4, phenylalanin e, and DTT followed by gel permeation chromatography leads to an iron- hydroxylase containing approximately 1 molecule of iron per molecule o f enzyme. The iron-hydroxylase displays an optical absorption band ext ending from 300 to 600 nm, and it catalyzes the hydroxylation of pheny lalanine at the same maximum rate as the iron-activated hydroxylase bu t does not require added Fe2+. We conclude that iron participates in t he hydroxylation of phenylalanine. Iron is not required for the oxidat ion of DMPH4, although it may exert a modest acceleration effect. A hy pothetical mechanism is presented wherein the reaction of iron with th e putative 4a-hydroperoxy-DMPH4 leads to 4a-hydroxy-DMPH4 and a high v alent iron-oxy species. The iron-oxy species is postulated to react wi th phenylalanine in the hydroxylation process.