RESONANCE RAMAN STUDIES OF CATECHOLATE AND PHENOLATE COMPLEXES OF RECOMBINANT HUMAN TYROSINE-HYDROXYLASE

Human tyrosine hydroxylase isoform 1 (hTH1) was expressed in Escherich ia coil, purified as the apoenzyme, and reconstituted with iron. The r esonance Raman spectra of hTH1 complexed with dopamine, noradrenaline, tyramine, and catechol have been studied and compared to those obtain ed for TH isolated from bovine adrenal glands or rat phaeochromocytoma tissue. A TH-phenolate complex is reported for the first time. Using dopamine selectively O-18-labeled in the 3-position or both 3- and 4-h ydroxy positions, we have been able to assign unambiguously the origin of the low-frequency vibration bands: the band at 631 cm(-1) involves the oxygen in the 4-position; the band at 592 cm(-1) involves the oxy gen in the 3-position, and the band around 528 cm(-1) is shifted by bo th, suggesting a chelated mode vibration. A small shift of the 1275 cm (-1) band and no shift of the 1320 cm(-1) band were observed, showing that those two bands involve essentially ring vibrations of the catech olate moiety, rather than the C-O stretching vibration as previously s uggested. The spectrum of the catechol-d(6)-hTH1 complex confirms this assignment. The resonance Raman spectra of the Fe-54, Fe-56, or Fe-57 isotope-containing enzymes complexed with dopamine are virtually iden tical, showing that the component of the iron in the approximate to 60 0 cm(-1) vibrations is too small to be observed. These results provide a better understanding of the Raman properties of iron-catecholate co mplexes in this enzyme, as well as in other metalloproteins and model compounds.