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Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔE(Q)| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 °C.

The non-heme iron enzyme phenylalanine hydroxylase from Chromobacterium violaceum has previously been shown to catalyze the hydroxylation of benzylic and aliphatic carbons in addition to the normal aromatic hydroxylation reaction. The intrinsic isotope effect for hydroxylation of 3-cyclochexylalanine by the enzyme was determined in order to gain insight into the reactivity of the iron center. With 3-[(2)H(11)-cyclohexyl]alanine as the substrate, the isotope effect on the k(cat) value was 1, consistent with an additional step in the overall reaction being significantly slower than hydroxylation. Consequently, the isotope effect was determined as an intramolecular effect by measuring the amount of deuterium lost in the hydroxylation of 3-[1,2,3,4,5,6-(2)H(6)-cyclohexyl]alanine. The ratio of 4-HO-cyclohexylalanine that retained deuterium to that which lost one deuterium atom was 2.8. This gave a calculated value of 12.6 for the ratio of the primary deuterium kinetic isotope effect to the secondary isotope effect. This value is consistent with hydrogen atom abstraction by an electrophilic Fe(O) center and a contribution of quantum-mechanical tunneling to the reaction.

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine. In this study, we used deuterium kinetic isotope effects to probe the chemical mechanisms of aromatic and benzylic hydroxylation to compare the reactivities of bacterial and eukaryotic aromatic amino acid hydroxylases. The (D) k cat value for the reaction of CvPheH with [(2)H 5]phenylalanine is 1.2 with 6-methyltetrahydropterin and 1.4 with 6,7-dimethyltetrahydropterin. With the mutant enzyme I234D, the (D) k cat value decreases to 0.9 with the latter pterin; this is likely to be the intrinsic effect for addition of oxygen to the amino acid. The isotope effect on the subsequent tautomerization of a dienone intermediate was determined to be 5.1 by measuring the retention of deuterium in tyrosine produced from partially deuterated phenylalanine; this large isotope effect is responsible for the normal effect on k cat. The isotope effect for hydroxylation of the methyl group of 4-CH 3-phenylalanine, obtained from the partitioning of benzylic and aromatic hydroxylation products, is 10. The temperature dependence of this isotope effect establishes the contribution of hydrogen tunneling to benzylic hydroxylation by this enzyme. The results presented here provide evidence that the reactivities of the prokaryotic and eukaryotic hydroxylases are similar and further define the reactivity of the iron center for the family of aromatic amino acid hydroxylases.