Mössbauer, electron-paramagnetic-resonance and X-ray-absorption fine-structure studies of the iron environment in recombinant human tyrosine hydroxylase.
Isoforms (1-4) of human tyrosine hydroxylase (TH) have been expressed in Escherichia coli and purified as apoenzymes (metal-free). Apo-human TH binds 1.0 atom Fe(II)/enzyme subunit, and iron binding is associated with an immediate and dramatic (40-fold) increase in specific activity. For X-ray absorption fine structure (XAFS) and electron paramagnetic resonance (EPR) measurements the apoenzyme was reconstituted with 56Fe and for Mössbauer measurements with 57Fe. XAFS measurements at the Fe-K edge of human TH were performed on the native form [Fe(II)-human TH], as well as after addition of stoichiometric amounts of the substrate tetrahydropterin, the inhibitor dopamine and of H2O2. The addition of dopamine or H2O2 oxidizes the ferrous iron of the native human TH to the ferric state. In both redox states the iron is octahedrally coordinated by low-Z backscatterers, thus sulfur coordination can be excluded. From the multiple scattering analysis of the EXAFS region is was surmised that part of the iron coordination is due to (3 +/- 1) imidazols. Addition of tetrahydropterin does not significantly change the iron coordination of the Fe(II) enzyme. The Mössbauer results confirm the valence states and the octahedral coordination of iron as well as the exclusion of sulfur ligation. Both the EPR spectra and the Mössbauer magnetic hyperfine pattern of dopamine- and H2O2-treated native human TH, were analyzed with the spin-Hamiltonian formalism. This analysis provides significantly different features for the two forms of human TH: the ferric iron (S = 5/2) of the H2O2-treated form exhibits a rhombic environment while that of the dopamine-treated form exhibits near-axial symmetry. The specific spectroscopic signature of dopamine-treated human TH, including that of an earlier resonance-Raman study [Michaud-Soret, I., Andersson, K. K., Que, L. Jr & Haavik, J. (1995) Biochemistry 34, 5504-5510] is most likely due to the bidentate binding of dopamine to iron.
Institut für Physik, Medizinische Universität zu Lübeck, Germany.
W
W
Meyer-Klaucke
H
H
Winkler
V
V
Schünemann
A X
AX
Trautwein
H F
HF
Nolting
J
J
Haavik
D001665:Binding Sites; D002396:Catechols; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D011622:Pterins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015204:Spectroscopy, Mossbauer; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics; D014965:X-Rays
D002396:Catechols; D008024:Ligands; D011622:Pterins; D011994:Recombinant Proteins; C011552:tetrahydropterin; D007501:Iron; D014446:Tyrosine 3-Monooxygenase