Thermodynamics of iron, tetrahydrobiopterin, and phenylalanine binding to phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O ₂ to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O ₂ -activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O ₂ activation. Exploring the energetics of each step before O ₂ activation can provide mechanistic insight into the initial steps that support the highly specific O ₂ activation pathway carried out by this metalloenzyme. Here the thermal stability of PheH and its substrate complexes were investigated under an anaerobic environment by using differential scanning calorimetry. In context with known binding constants for PheH, a thermodynamic cycle associated with iron(II), tetrahydrobiopterin (BH ₄ ), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH ₄ and Phe. The addition of phenylalanine and BH ₄ to PheH·Fe increased the stability of this enzyme (ΔT _m of 8.5 (±0.7) °C with an associated δΔH of 43.0 (±2.9) kcal/mol). The thermodynamic data presented here gives insight into the complicated interactions between metal center, cofactor, and substrate, and how this interplay sets the stage for highly specific, oxidative C-H activation in this enzyme.
Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2022 Elsevier Inc. All rights reserved.)