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 (BH4), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH4 and Phe. The addition of phenylalanine and BH4 to PheHmiddotFe increased the stability of this enzyme (delta T-m of 8.5 (+/- 0.7) degrees C with an associated delta delta H 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.