ELSEVIER Physica B 208&209 (1995) 717 718

XAFS of human tyrosine hydroxylase

W. Meyer a'*, J. Haavik b, H. Winkler a, A.X. Trautwein a, H.-F. Nolting ~

alnstitut fdr Physik, Medizinische Universitiit zu Liibeck, Ratzeburger Allee 160, 23538 Liibeck, Germany bDepartment of Biochemistry and Mol. Biolog, University of Bergen, Norway

CEMBL, Outstation at DESY, Hamburg, Germany

Abstract

Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1].

Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

1. Experimental procedures

Samples were measured at beam line D2 at DORIS III at EMBL-Outstation Hamburg/DESY (4.6 GeV, lmax = 80mA). Harmonics were rejected by detuning the Si(1 1 l)-monochromator to about 70% of its peak inten- sity and using a segmented focusing Au-coated mirror with a cut-off energy of 21.5 keV [2]. Samples were measured as frozen glasses at 18 K in fluorescence mode using a 13-element Ge-detector perpendicular to the in- coming beam. For a continuous energy calibration a Si(2 2 0) single crystal was placed at the end of the beam line. Its energy-dependent reflections were detected by two scintillator-photomultiplier couples [3]. An energy resolution of better than 2 eV was reached.

2. Results

The XAFS-spectra (Fig. 1) show different edge struc- tures and beating patterns. Compared to the native

* Corresponding author.

Fe(II) enzyme the edge of the TH1 Fe(III) dopamine complex is shifted about 1.5 eV to higher energies corres- ponding to the valence change. The differences in the shape of the XANES of these two species indicate a change in the coordination geometry.

The jump was normalized and the pre-edge-peak areas calculated. For the native enzyme we obtained an area of 9.7 x 10- 2 eV. An analysis of pre-edge peaks of model compounds with four planar nitrogen atoms and one or two other ligands [4] shows that this is a typical value for 5 coordinated iron. The area of 5.4 x 10- 2 eV is typical for an Fe(III)-centre in an undisturbed octahedral coord- ination [5]. For the native Co-enzyme isoform 2 we obtained an area of 6.7 x 10 -2 eV.

The first coordination sphere was Fourier-filtered and simulations were performed for different combinations of N and O ligands. To reduce the number of parameters of the refinements the photoelectron lifetime parameter and the amplitude reduction factor were fixed at reference compounds with comparable F e - O / N distances. This was also a check of the theoretical phases we use. There- fore only the coordination number (allowing only integer steps), the Debye-Waller factor a 2, the first shell distance

0921-4526/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 1 - 4 5 2 6 ( 9 4 ) 0 0 8 9 3 - 0

718 W. Meyer et al./Physica B 208&209 (1995) 717-718

0.16

%

0.1 , , " ' , I

Fe(II)TH l Z'e(III)TH :o(II)TH 2

+ dop. -.~

0.0 ' ' l ~ 0.0 71150 i 41 0.0 ' L , t 7150 7475 7 75 7800 8125

E in eV E in eV E in eV

Fig. 1. XAFS of Fe(II)hTH1, Fe(III)hTH1 and Co(II)hTH2.

Table 1 EXAFS single shell fit parameters

AEo 0 N r, (A) ~ (~2) FI

Native 11.9 6 - - 2 . 1 0 0.020 0.94 Fe(II)-TH 1 13.3 - - 6 2.12 0.015 0.95

11.9 5 - - 2 . 1 0 0.016 0.98

Fe(III)-TH1 10.6 6 - - 2.05 0.021 0.21 + dopamine 12.2 6 2.06 0.016 0.64

Native 11.6 6 - - 2 . 0 8 0.015 0.55 Co(II)-TH2 12.9 - - 6 2.10 0.011 1.23

Note: The amplitude reduction factor was fixed to 0.85 and the constant imaginary potential, which describes the lifetime of the photoelectron, to - 3.0 eV.

3 10

k in ~-1

f , , , , , , , , , , , r ' , , ' , , , , , r , , , , , , , , ,

10 ~ - F e ( I I I ) T H 1 + d o p , 10 C o ( I I ) T H 2 f\

0 0

-5 -5

10 - 10

I0

k in ~-i

. . . . . . . . . , , , , , i 10

Fig. 2. EXAFS and simulations of Fe(II)hTH1, Fe(IH)hTH1 and Co(II)hTH2 spectra.

1 tyrosine and 1 imidazole for the native Fe species, 1 d. 1 t. 3 i. for the Fe(III)-dopamine complex and 2 d. 2 i. for the native Co species are shown.

3. Discussion

The analysis of the pre-edge-peak areas indicates that the native Fe(II) TH 1 is five coordinated, while the metal coordination in the other species seems to be octahedral. The oxidation of Fe(II) TH isoform 1 with its inhibitor dopamine results in a change of the metal coordination and bond lengths. An FeTH-dopamine complex is for- med. The multiple scattering calculations indicate the presence of dopamine and imidazole ligands. Because of the strong correlations between the different parameters no absolute numbers of ligands can be given. We will continue these investigation and analyse the other iso- forms and the effect of other inhibitors.

r~ and the magnitude of the photoelectron energy at zero wave vector AEo were varied in our simulations (Table 1). The first shell distances of about 2.1 A point to a non- heme structure of the catalytic centres. In the three spe- cies this shell consists mainly of low Z backscatterers (O, N or C). The oxidation of the enzyme with dopamine changes the structure and amplitude of the EXAFS- spectra. A TH 1-Fe(III)-dopamine complex is established.

In order to include the higher shells of such ligands multiple scattering was approached. By restrained refine- ment [61 the number of parameters being refined inde- pendently were reduced. While the overall coordination number was fixed at a value of six or five only integer numbers of ligands (dopamine, tyrosine and/or imidazole) were allowed during the refinement.

We found that one or two dopamine ligands are pres- ent in all samples. The number of imidazole ligands is always equal or greater than the number of tyrosine ligands. In Fig. 2 the best simulations with 2 dopamine

This project has been supported financially by the BMFT (grant 05 5FLAXI 4).

References

[1] J. Haavik, B. Le Bourdelles, A. Martinez, T. Flatmark and J. Mallet, Eur. J. Biochem. 199 (1991) 371.

1-2] C. Hermes, E. Gilberg and M.H.J. Koch, Nucl. Instr. and Meth. 222 (1984) 207.

13] R.F. Pettifer and C. Hermes, J. Appl. Crystallogr. 18 (1985) 404.

14] U. Knof, T. Weyermiiller, T. Wolter, K. Wieghardt, E. Bill, C. Butzlaff and A.X. Trautwein, Angew. Chem. 105 (11) (1993) 1701.

[5] A.L. Roe, D.J. Schneider, R.J. Mayer, J.W. Pryz, J. Widom and L. Que Jr., J. Am. Chem. Soc. 106 (1984) 1676.

[6] S.S. Hasnain and R.W. Strange, in: Synchrotron Radiation and Biophysics, ed. S.S. Hasnain (Ellis Horwood, Chiches- ter, 1990) pp. 104.