X-ray absorption spectroscopic studies of the FeZn derivative of uteroferrin.

The FeZn derivative of purple acid phosphatase from porcine uterus (FeZnUf) and its phosphate complex (FeZnUf.PO4) have been characterized by X-ray absorption spectroscopy at both the iron and zinc K-edges, to gain insight into the nature of the FeZn active site as well as the phosphate binding mode. Pre-edge data show that both FeZnUf and FeZnUf.PO4 have a 6-coordinate iron site. The iron site has an average Fe-O/N bond length of 2.01-2.02 A, which can be resolved into subshells of 1.92 and 2.11 A for FeZnUf.PO4. On the other hand, the zinc site has a shell of scatterers at 2.02-2.03 A plus one scatterer at ca. 2.4 A. These metal-ligand bond lengths are consistent with the nature of the ligands deduced from spectroscopic studies or identified in the crystal structure of the related kidney bean purple acid phosphatase (KBPAP). The outer-sphere analysis indicates an Fe-Zn separation of approximately 3.3 A in both FeZnUf and FeZnUf.PO4, consistent with the presence of an M2(mu-OR)2 diamond core as found in the crystal structures of KBPAP, calcineurin, and protein phosphatase 1. The Fe-P and Zn-P bond distances in FeZnUf.PO4 are determined to be 3.23 and 3.18 A, respectively, indicating that phosphate binds to the dinuclear center in a bidentate mode; such a mode has been observed in oxoanion complexes of KBPAP, calcineurin, and alkaline phosphatase, as well as in a number of synthetic FeFe and FeZn complexes. The implications of these structural results on the mechanism of phosphatase action are discussed.