Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coil and purified either with or without (6 x histidine) N-terminal tags. After reconstitution with 57Fe(II), the Mossbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization. Before dehydration, >90% of the iron in hTH1 had Mossbauer parameters typical for highspin Fe(II) in a six-coordinate environment [isomer shift δ(1.8- 77K)=1.26-1.24mm s-1 and quadrupole splitting ΔE(Q)=2.68mm s-1]. After dehydration, the Mossbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (δ=1.07mm s-1 and ΔE(Q)=2.89 mm s-1 at 77K), whereas 28% of the iron remained as six- coordinate high-spin Fe(II) (δ=l.24 mm s-1 and ΔE(Q)=2.87 mm s-1 at 77 K). Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag. After rehydration of hTH1 the spectroscopic changes were completely reversed. The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mossbauer spectra. The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five- coordinate iron center. Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron. The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center.