X-RAY-ABSORPTION STUDIES ON CATECHOL 2,3-DIOXYGENASE FROM PSEUDOMONAS-PUTIDA MT2

X-ray absorption spectroscopy has been utilized to investigate the str ucture of the active site of iron(II) catechol 2,3-dioxygenase from Ps eudomonas putida mt2 both in the native and the 2-chlorophenol inhibit ed forms. XANES (X-ray absorption near edge structure) and EXAFS (exte nded X-ray absorption fine structure) results allow us to discuss the coordination number and geometry of the ferrous ion in the native enzy me. The metal geometry is not significantly affected by the binding of the inhibitor. The EXAFS spectrum is consistent with an iron(II) boun d to six N/O atoms at an average distance of 2.05 Angstrom or to five N/O at an average distance of 2.04 Angstrom. The simulation of the exp erimental data is greatly enhanced by considering the iron ligands div ided in two different shells. Analysis of the outer shells performed u sing multiple scattering theory shows that there are histidines in the coordination sphere. The best fitting is obtained assuming the presen ce of two of them. Similar results are obtained for the inhibited enzy me, which, however, are indicative of a slight shortening of the avera ge metal-donor bond distances. The direct binding of inhibitors to the metal center is confirmed by H-1 NMR data.