NMRD STUDIES ON PHTHALATE DIOXYGENASE - EVIDENCE FOR DISPLACEMENT OF WATER ON BINDING SUBSTRATE

Water proton T-1(-1) measurements at magnetic fields between 0.01 and 50 MHz [nuclear magnetic relaxation dispersion (NMRD) measurements] ha ve been performed on solutions of phthalate dioxygenase (PDO) reconsti tuted at the catalytic iron site with copper(II) or manganese(II). The data show evidence of a weakly coordinated water molecule in CuPDO; i n the presence of the substrate, phthalate, this water appears to beco me even less tightly bound, and an additional tightly coordinated wate r can be detected. In PDO reconstituted with manganese, one tightly co ordinated water is detected in the presence and in the absence of phth alate. An attempt is made to reconcile these data with low-temperature near-IR magnetic circular dichroism and X-ray absorption data, which show that PDO reconstituted with iron or cobalt is six-coordinate in t he absence of substrate and five-coordinate in the presence of substra te.