382 Abstracts

H23 XAS STUDIES OF H U M A N T Y R O S I N E H Y D R O X Y L A S E W. Meyer 1, J. Haavik 2, H. Winkler 1, A. X. Trautwein 1, H.-F. Nolting 3

lInstitut fiir Physik, Medizinische Universitat zu Ltibeck, Germany; 2Department of Biochemistry and Molecular Biology, University of Bergen, Norway; 3EMBL, Outstation at DESY, Hamburg, Germany

Tyrosine hydroxylase (TH) catalyses the rate-limiting reaction in the biosynthesis of catecholamines, using 02, tyrosine and tetrahydrobiopterin (BH4) as substrates. The enzyme isolated from adrenal medulla contains approximately one atom of tightly bound non-heme iron per subunit [ 1 ]. While the ligands to the iron, or its catalytic function are not known, the iron is considered to be important in oxygen activation, and 1H-NMR spectroscopy has indicated that BH 4 is binding close to the metal center [2]. Human TH is present as four isoforms (hTHI-hTH4). In order to study the function of the metal center, all hTH isoforms have been expressed in E. coli and purified to homogeneity as the apoenzymes (metal free). The apoenzymes are selectively activated by adding Fe(II). In the present study, the pure hTH apoenzymes have been reconstituted with Fe(II) and the iron environment has been studied by X-ray absorption spectroscopy (XAS).

XAS measurements were performed on both the Fe(II) and Fe(III) forms of hTH1, as well as on the BH 4 complex with the Fe(II) enzyme and the dopamine or tyramine complex of the Fe(III) enzyme. Data analysis was carried out using curved-wave calculations (EXCURVE) to account for multiple scattering for distances up to 4.5 ,~. From our investigation it is clear that the first coordination shell of iron consists of low-Z backscatterers (O, N or C but not S). An additional feature of the spectra, i.e. the camel-back structure at k ~ 45 nm 1, characteristic for multiple scattering of aromatic ligands such as imidazole, is along this line.

The addition of BH 4 does not significantly change the iron coordination of the Fe(II) enzyme. Likewise, the addition of dopamine or tyramine does not change the iron coordination of the Fe(III) enzyme. However, oxidation of the enzyme- bound Fe(II) to Fe(III) decreases the average iron-ligand bond lenght by about 2 %.

In summary we note that the analysis of X-ray absorption spectra of various forms of the Fe(II)- and Fe(III)-hTH1 enzymes does not allow to conclude whether the bound BH 4 coordinates directly to the metal or not. However, the analysis definitely shows that sulfur is not in its first coordination sphere; this finding is in agreement with our M6ssbauer results on 57Fe(II)-hTH1..

This study was supported by the German Ministery for Research and Technology and the Research Council of Norway.

[1] J. Haavik, K. K. Andersson, L. Petersson, T. Flatmark. Biochim. Biophys. Acta 953 (1988) 142; [2] A. Martinez, C. Abeygunawardana, J. Haavik, T. Flatmark, A. S. Mildvan, Adv. Exp. Medicine 338 (1993) 77.