Phenylalanine hydroxylase from Chromobacterium violaceum (cPAH), which catalyzes phenylalanine oxidation to tyrosine, is homologous to the catalytic domain of eukaryotic PAHs. Previous crystallographic and spectroscopic studies on mammalian PAH conflict on whether O-2 binds to the open-coordination site or displaces the remaining water ligand to yield either a six-or a five-coordinate iron, respectively. The abilities of nitric oxide to behave as an oxygen mimic and a spectroscopic probe of ferrous iron are used to investigate the geometric and electronic effects of cofactor and substrate binding to cPAH by electron paramagnetic resonance (EPR) and UV-vis spectroscopies. A rhombic distortion observed for the ternary complex is due to two factors: a decrease in the Fe-NO angle and an alteration in the equatorial ligand geometry. Both factors are consistent with NO displacing the sole remaining water ligand to yield a five-coordinate iron center. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by O-17-enriched water is observed in the absence of substrates or presence of cofactor only ( binary complex), demonstrating that water is bound to the Fe-II. However, in the presence of substrate and cofactor ( ternary complex), the EPR resonances of the nitrosyl complex are not broadened by O-17-enriched water, indicating the displacement of water by NO to afford a five-coordinate iron. Furthermore, the increased intensity in the 500-600 nm range of the UV-vis spectrum of the ternary nitrosyl complex indicates an increased overlap between the in-plane NO 2 pi* and d(x2-y2) and d(xz) orbitals, which corroborates a five-coordinate iron.