The Fell of the binuclear (FeFeIII)-Fe-II active site of pig purple acid phosphatase (uteroferrin) has been replaced in turn by five M-II ions (Mn-II, Co-II, Ni-II, Cu-II, and Zn-II). An uptake of 1 equiv of M-II is observed in all cases except that of Cu-II, when a second more loosely bound Cu-II is removed by treatment with edta. The products have been characterized by different analytical procedures and by UV-vis spectrophotometry. At 25degreesC, 1 = 0.100 M (NaCl), the nonenzymatic reactions with H2PO4- give the mu-phosphato product, and formation constants K/M-1 show an 8-fold spread at pH 4.9 of 740 (Mn), 165 (Fe), 190 (Co), 90 (Ni), 800 (Cu), 380 (Zn). The variations in K correlate well with stability constants for the complexing of H2PO4- and (CH3O)HPO3- with M-II hexaaqua ions. At pH 4.9 with [H2PO4-] greater than or equal to 3.5 mM rate constants k(obs) decrease, and an inhibition process in which a second [H2PO4-] coordinates to the dinuclear center is proposed. The mechanism considered accounts for most but not all of the features displayed. Thus K, values for the coordination of phosphate to M-II are in the range 10-60 M-1, whereas K-2 values for the bridging of the phosphate to Fe-III are in the narrower range 7.8-12.4. From the fits described K similar to 103 M-1 for the inhibition step, which is independent of the identity of M-II. Values of kobs decrease with increasing pH, giving pK(a) values which are close to 3.8 and independent of M-II (Fe-II, Zn-II, Mn-II). The acid dissociation process is assigned to Fe-III-OH2 to Fe-III-OH-, where OH- is less readily displaced by phosphate.