Uteroferrin (Uf) is a purple acid phosphatase for which the reduced [(FeFeIII)-Fe-II] form of the enzyme is catalytically active in the hydrolysis of phosphate esters. The binuclear non-heme iron active site of the native reduced enzyme (1/2metUf) and its molybdate-and phosphate-bound forms have been studied with absorption (ABS), circular dichorism (CD), and magnetic circular dichroism (MCD) spectroscopies to probe their geometric and electronic structure. CD and MCD in the UV-Vis region give the tyrosine (phenolate) to Fe-III charge transfer bands which probe the ferric center. CD and MCD in the NIR region probe the d-d transitions of both the Fe-II and the Fe-III centers. variable-temperature variable-field (VTVH) MCD combined with EPR data are analyzed to determine the g values and energies of the ground state and the excited sublevels. These parameters are further interpreted in terms of a spin Hamiltonian model, which includes the zero-field splitting (ZFS) of Fe-II and Fe-III centers and the exchange coupling (J) between the irons due to bridging ligation. These ground and excited-state results confirm that both irons are six-coordinate with a mu-OH bridge. Anions bind to the active site in a bridging mode, which perturbs the ground and excited states of both iron centers. In particular, the exchange coupling decreases, mu-OH bridge --> Fe-II bonding strength increases, and the Tyr --> Fe-II donor interaction increases upon the phosphate binding. These results are correlated with the recent X-ray crystal structure of the kidney bean purple acid phosphatase, which contains an (FeZnII)-Zn-III active site. These studies provide geometric and electronic structure insight into the hydrolysis reaction mechanism of this enzyme.