Spectroscopic Characterization of the Catalytically Competent Ferrous Site of the Resting, Activated, and Substrate-Bound Forms of Phenylalanine Hydroxylase

Abstract

The geometric structure of the catalytically relevant ferrous active site of phenylalanine hydroxylase (PAH) has been investigated using magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies. From the excited state ligand field transitions in the MCD spectrum (10Dq ) 9400 cm -1 , ∆ 5 E g ) 1800 cm -1 ), the temperature and field dependence of these transitions (∆ 5 T 2g ) 590 cm -1 ), and the XAS pre-edge shapes and intensities, the resting ferrous site of the "tense" form of PAH is six-coordinate distorted octahedral. The low ligand field strength observed in the MCD spectrum results from significant oxygen ligation and longer Fe-O/N bond distances relative to model complexes as determined from an EXAFS analysis. Nonallosteric activation using N-ethylmaleimide does not notably affect the band positions in the MCD spectrum and therefore does not perturb the structure of the iron center. However, substrate addition without allosteric activation results in a different six-coordinate distorted octahedral structure as determined by MCD (10Dq ) 10 000 cm -1 , ∆ 5 E g ) 1450 cm -1 ) and XAS with a greater d π -orbital splitting (∆ 5 T 2g ) 1050 cm -1 ). EXAFS analysis indicates a shift in the relative number of ligands from the outer to the inner subshell of the first coordination sphere in the substrate-bound form of the enzyme relative to the resting site, consistent with the increased ligand field strength observed directly from the MCD spectrum. Substrate-induced allosteric activation (∼34 kcal/mol) does not alter the structure of the iron site in the "relaxed" form of PAH compared to the substrate-bound "tense" state. Thus, while activation is necessary for the enzyme to achieve complete catalytic competence, it does not appear to affect the geometry of the catalytically relevant six-coordinate ferrous active site and only directly influences the surrounding protein conformation. In contrast, substrate addition results in a geometric and electronic structural change at the iron center which may help orient the substrate for completely coupled hydroxylation.

Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases

“…This sequential mechanistic model is in accord with observations made in CD and XAS studies of rPAH, in that both substrates must be bound to the active site of PAH prior to oxygen activation [39,40]. Crystallographic [16,38] and spectroscopic [39,41,42] studies have shown independently that an open coordination position on iron becomes available only in the presence of cofactor (BH 4 ) and substrate (L-Phe). This implies the involvement of iron in the initial oxygen binding and activation.…”

Mechanism of Aromatic Hydroxylation by an Activated FeIVO Core in Tetrahydrobiopterin‐Dependent Hydroxylases

“…The detection of the density of only one water ligand in the first coordination shell means that at least one of the three water ligands in the PAH-BH 4 complex is lost when both the substrate and the cofactor are bound in the active site. [41] The change to a distorted square-pyramidal five-coordinate environment of the metal upon substrate binding agrees with CD and MCD spectroscopies, [42,43] which indicated substantial perturbations in the ligand field when both the substrate and the cofactor are bound to the ferrous PAH. The five-coordinate metal might thus offer an open coordination site for oxygen activation.…”

Mechanism of Aromatic Amino Acid Hydroxylation

“…Spectroscopic studies of rat PheH with phenylalanine and 6-methyl-5-deazatetrahydropterin bound confirm the decrease in the number of metal ligands in the ternary complex (48). It appears that both substrates must be bound before the change in the arrangement of the metal ligands occurs since spectroscopic studies of the ferrous iron in rat PheH do not show a change to a pentacoordinate site when phenylalanine alone binds (49).…”