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

The purple acid phosphatase from beef spleen, which contains two iron atoms per 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 approximately equal to 1.77; double integration of the EPR spectrum gives one spin per two iron atoms. A similar EPR spectrum is observed for enzyme reduced anaerobically by one electron, 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 iron atoms by zinc 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 one spin per iron. These results strongly suggest the presence of a spin-coupled bimetallic unit at the active site of the enzyme.