Evidence for a spin-coupled binuclear iron unit at the active site of the purple acid phosphatase from beef spleen.

Abstract

The purple acid phosphatase from beef spleen, which contains 2 Fe atoms/molecule, is EPR silent in its native (oxidized) purple form. Treatment with mild reducing agents results in conversion to a pink, enzymatically active form, which exhibits an unusual EPR signal centered at g .apprxeq. 1.77; double integration of the EPR spectrum gives 1 spin/2 iron atoms. A similar EPR spectrum is observed for enzyme reduced anaerobically by 1 electrom, using sodium dithionite. Variable-temperature magnetic susceptibility measurements show that the oxidized and reduced proteins are both antiferromagnetically coupled systems, with S = 0 and 1/2 ground states, respectively. Replacement of one of the Fe atoms by Zn produces an FeZn enzyme with full catalytic activity. The FeZn enzyme exhibits a highly temperature dependent g = 4.3 EPR signal, and magnetic susceptibility data are consistent with an S = 5/2 paramagnet. Treatment of the FeZn enzyme with phosphate, a competitive inhibitor, results in sharpening of the EPR spectrum; double integration at 77 K gives 1 spin/Fe. These results strongly suggest the presence of a spin-coupled bimetallic unit at the active site of the enzyme.