Open AccessMössbauer, Electron‐Paramagnetic‐Resonance and X‐ray‐Absorption Fine‐Structure Studies of the Iron Environment in Recombinant Human Tyrosine Hydroxylase
Author(s) -
MeyerKlaucke Wolfram,
Winkler Heiner,
Schünemann Volker,
Trautwein Alfred X.,
Nolting HansF.,
Haavik Jan
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00432.x
Subject(s) - chemistry , electron paramagnetic resonance , ferric , crystallography , coordination sphere , extended x ray absorption fine structure , ferrous , x ray absorption fine structure , inorganic chemistry , absorption spectroscopy , nuclear magnetic resonance , spectroscopy , crystal structure , organic chemistry , physics , quantum mechanics
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 56 Fe and for Mossbauer measurements with 57 Fe. 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 H 2 O 2 . The addition of dopamine or H 2 O 2 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 Mossbauer magnetic hyperfine pattern of dopamine‐ and H 2 O 2 ‐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 H 2 O 2 ‐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 Biolentate binding of dopamine to iron.