¹H NMR and NOE Studies of the Purple Acid Phosphatases from Porcine Uterus and Bovine Spleen

The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by ¹H NMR and NOE techniques. The paramagnetically shifted features of the ¹H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the >90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N_e-coordinated, while the Fe(II)-His is Hj-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R.C., Pyrz, J.W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well.