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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.