page_title: Phenylalanine hydroxylase
section_title: Enzyme mechanism
hierarchical_section_title: Phenylalanine hydroxylase / Enzyme mechanism
caption_attribution_description: English: Formation and cleavage of BH4-O-O-Fe(II) bridge.
context_page_description: Phenylalanine hydroxylase is an enzyme that catalyzes the hydroxylation of the aromatic side-chain of phenylalanine to generate tyrosine. PAH is one of three members of the biopterin-dependent aromatic amino acid hydroxylases, a class of monooxygenase that uses tetrahydrobiopterin and a non-heme iron for catalysis. During the reaction, molecular oxygen is heterolytically cleaved with sequential incorporation of one oxygen atom into BH₄ and phenylalanine substrate.
  Phenylalanine hydroxylase is the rate-limiting enzyme of the metabolic pathway that degrades excess phenylalanine. Research on phenylalanine hydroxylase by Seymour Kaufman led to the discovery of tetrahydrobiopterin as a biological cofactor. The enzyme is also interesting from a human health perspective because mutations in PAH, the encoding gene, can lead to phenylketonuria, a severe metabolic disorder.
context_section_description: The reaction is thought to proceed through the following steps:
  formation of a Fe(II)-O-O-BH₄ bridge.
  heterolytic cleavage of the O-O bond to yield the ferryl oxo hydroxylating intermediate Fe(IV)=O
  attack on Fe(IV)=O to hydroxylate phenylalanine substrate to tyrosine.
  Formation and cleavage of the iron-peroxypterin bridge. Although evidence strongly supports Fe(IV)=O as the hydroxylating intermediate, the mechanistic details underlying the formation of the Fe(II)-O-O-BH₄ bridge prior to heterolytic cleavage remain controversial. Two pathways have been proposed based on models that differ in the proximity of the iron to the pterin cofactor and the number of water molecules assumed to be iron-coordinated during catalysis. According to one model, an iron dioxygen complex is initially formed and stabilized as a resonance hybrid of Fe²⁺O₂ and Fe³⁺O₂⁻. The activated O₂ then attacks BH₄, forming a transition state characterized by charge separation between the electron-deficient pterin ring and the electron-rich dioxygen species. The Fe(II)-O-O-BH4 bridge is subsequently formed. On the other hand, formation of this bridge has been modeled assuming that BH4 is located in iron's first coordination shell and that the iron is not coordinated to any water molecules. This model predicts a different mechanism involving a pterin radical and superoxide as critical intermediates. Once formed, the Fe(II)-O-O-BH₄ bridge is broken through heterolytic cleavage of the O-O bond to Fe(IV)=O and 4a-hydroxytetrahydrobiopterin; thus, molecular oxygen is the source of both oxygen atoms used to hydroxylate the pterin ring and phenylalanine.
  Hydroxylation of phenylalanine by ferryl oxo intermediate. Because the mechanism involves a Fe(IV)=O (as opposed to a peroxypterin) hydroxylating intermediate, oxidation of the BH₄ cofactor and hydroxylation of phenylalanine can be decoupled, resulting in unproductive consumption of BH4 and formation of H2O2.  When productive, though, the Fe(IV)=O intermediate is added to phenylalanine in an electrophilic aromatic substitution reaction that reduces iron from the ferryl to the ferrous state.  Although initially an arene oxide or radical intermediate was proposed, analyses of the related tryptophan and tyrosine hydroxylases have suggested that the reaction instead proceeds through a cationic intermediate that requires Fe(IV)=O to be coordinated to a water ligand rather than a hydroxo group. This cationic intermediate subsequently undergoes a 1,2-hydride NIH shift, yielding a dienone intermediate that then tautomerizes to form the tyrosine product. The pterin cofactor is regenerated by hydration of the carbinolamine product of PAH to quinonoid dihydrobiopterin (qBH₂), which is then reduced to BH₄.
caption: PAH mechanism, part I
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