title: Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy.
abstract: Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6 x histidine) N-terminal tags. After reconstitution with 57Fe(II), the Mössbauer 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 Mössbauer parameters typical for high-spin Fe(II) in a six-coordinate environment [isomer shift delta (1.8-77 K) = 1.26-1.24 mm s-1 and quadrupole splitting delta EQ = 2.68 mm s-1]. After dehydration, the Mössbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (delta = 1.07 mm s-1 and delta EQ = 2.89 mm s-1 at 77 K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (delta = 1.24 mm s-1 and delta EQ = 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 Mössbauer 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.
authors.affiliation: Institut für Physik, Medizinische Universität zu Lübeck, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Schünemann
authors.forename: C
authors.initials: C
authors.lastname: Meier
authors.forename: W
authors.initials: W
authors.lastname: Meyer-Klaucke
authors.forename: H
authors.initials: H
authors.lastname: Winkler
authors.forename: A X
authors.initials: AX
authors.lastname: Trautwein
authors.forename: P M
authors.initials: PM
authors.lastname: Knappskog
authors.forename: K
authors.initials: K
authors.lastname: Toska
authors.forename: J
authors.initials: J
authors.lastname: Haavik
mesh_terms: D015502:Absorptiometry, Photon; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007501:Iron; D011487:Protein Conformation; D051381:Rats; D013054:Spectrophotometry, Atomic; D015204:Spectroscopy, Mossbauer; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry*
chemical_list: D007501:Iron; D014446:Tyrosine 3-Monooxygenase