NMR characterization of substrate binding in the phthalate dioxygenase system

The paramagnetic enhancements in the NMR relaxation rates for the fluorine in fluorophthalates have been used to determine the position of the phthala te with respect to the mononuclear metal ion in native and metal-substitute d derivatives of phthalate dioxygenase (PDO). These studies show directly t hat the substrate interacts with the mononuclear metal of PDO and provide t he first structural characterization of this interaction. With a molecular mass of 200 kDa, PDO is one of the largest proteins studied to date by para magnetic NMR. Two paramagnetically broadened F-19 lines were observed for m onofluorophthalates bound to CoPDO. This demonstrates that fluorophthalate binds to PDO with a handedness, i.e., with the fluorine label facing to the "right" or to the "left", relative to the hyperfine tensor of the Co(II). The relative affinities of the two orientations are slightly different, wit h a 2-fold and 5-fold excess of the preferred orientation for 4-fluorophtha late and 3-fluorophthalate, respectively. The longitudinal relaxation rate (T-1) and transverse relaxation rate (T-2) data give mutually consistent fl uorine to cobalt distances. These results are consistent with approximate b ilateral symmetry, with the Co to 3-fluorophthalate distances (similar to 5 .5 Angstrom) approximately 25% longer than the Co to 4-fluorophthalate dist ances (similar to 4.5 Angstrom). A detailed geometric model is derived from these data. This structural characterization of the mononuclear site provi des a framework to develop hypotheses for the mechanism of oxygenation by t he Fe(II)-containing aromatic dioxygenases.