MOSSBAUER, 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 associat ed with an immediate and dramatic (40-fold) increase in specific activ ity. For X-ray absorption fine structure (XAFS) and electron paramagne tic resonance (EPR) measurements the apoenzyme was reconstituted with Fe-56 and for Mossbauer measurements with Fe-57. XAFS measurements at the Fe-K edge of human TH were performed on the native form [Fe(II)-hu man TH], as well as after addition of stoichiometric amounts of the su bstrate tetrahydropterin, the inhibitor dopamine and of H2O2. The addi tion of dopamine or H2O2 oxidizes the ferrous iron of the native human TH to the ferric state. In both redox slates 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) i midazols. Addition of tetrahydropterin does not significantly change t he iron coordination of the Fe(II) enzyme. The Mossbauer results confi rm the valence stares and the octahedral coordination of iran as well as the exclusion of sulfur ligation. Both the EPR spectra and the Moss bauer magnetic hyperfine pattern of dopamine- and H2O2-treated native human TH, were analyzed with the spin-Hamiltonian formalism, This anal ysis provides significantly different features for the two forms af hu man TH: the ferric iron (S = 5/2) of the H2O2-treated form exhibits a rhombic environment while that of the dopamine-treated form exhibits n ear-axial symmetry. The specific spectroscopic signature of dopamine-t reated human TH, including that of an earlier resonance-Raman study [M ichaud-Soret, I., Andersson, K. K., Que, L. Jr & Haavik, J. (1995) Bio chemistry 34, 5504-5510] is most likely due to the bidentate. binding of dopamine to iron.