Phenylalanine hydroxylase (PAH), a non-heme iron enzyme, is responsible for the phenylalanine conversion to tyrosine. Its malfunction causes phenylketonuria (PKU). To better understand how protein structure and folding profiles are affected by the metal cofactor, we investigated the chemical (un)folding of apo- and holo-PAH from Chromobacterium violaceum (cPAH) using circular dichroism (CD) and analytical ultracentrifugation (AUC). Holo-cPAH shows a two-state unfolding transition. In contrast, the unfolding profile for apo-cPAH reveals a three-state (un)folding pathway and accumulation of an intermediate (apo-cPAH(I)). This intermediate is also observed in refolding experiments. Fluorescence studies are consistent with the CD findings. The intermediate apo-cPAH(I) and unfolded state(s) of apo- and holo-cPAH(U) have been characterized by analytical ultracentrifugation (AUC). At 2.4 and 2.8 M GuHCl, 90% of the signal for apo-cPAH has a weight average sedimentation coefficient in water at 20°C (s20,w) of about 48 S, representing multiple aggregate species made of multiple monomers of cPAH. Aggregate formation for apo-cPAH is also confirmed by dynamic light scattering and electron microscopy giving a hydrodynamic radius (R(H)) of 41 nm for apo-cPAH(I) versus 3.5 nm for the native protein.