The Activity of Oxidized Bovine Spleen Purple Acid Phosphatase Is Due to an Fe(III)Zn(II) ‘Impurity'

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Abstract
Bovine spleen purple acid phosphatase (BSPAP) is a dinuclear iron protein with two stable redox states. The Fe3+Fe2+ state is the active state, while the fully oxidized protein (BSPAPox) has been reported to retain 5−10% activity, corresponding to a kcat of ca. 150 s-1 [Dietrich, M., Münstermann, D., Suerbaum, H., and Witzel, H. (1991) Eur. J. Biochem. 199, 105−113]. Here we show that this activity does not originate from Fe3+Fe3+-BSPAP, but rather from an ‘impurity' of FeZn-BSPAP. The FeZn form of BSPAP was prepared from apo-BSPAP following a new procedure, and its kinetic properties were carefully determined for comparison to those of BSPAPox. For the hydrolysis of p-NPP at pH 6.00, both kcat and KM were affected by the Fe2+-to-Zn2+-substitution [Fe3+Fe2+-BSPAP, kcat = (1.8 ± 0.1) × 103 s-1 and KM = 1.2 ± 0.2 mM; Fe3+Zn2+-BSPAP; kcat = (2.8 ± 0.2) × 103 s-1 and KM = 3.3 ± 0.4 mM]. The KM of BSPAPox was the same as that of FeZn-BSPAP. pH profiles of BSPAPox and FeZn-BSPAP were both shifted to lower pH compared to that of BSPAPred. FeZn-BSPAP, FeZn-BSPAP·PO4, and FeZn-BSPAP·MoO4 all showed characteristic EPR spectra similar to the corresponding complexes of FeZn-Uf. The same species could also be observed in concentrated samples of native BSPAP. Spin integration of these spectra showed a quantitative relation between the spin concentration of the FeZn-BSPAP ‘impurity' and the residual phosphatase activity after oxidation. Since all activity found after oxidation of BSPAP could be attributed to FeZn-BSPAP, there is no direct evidence that Fe3+Fe3+-BSPAP is catalytically active. These results set an upper limit to the possible catalytic activity of the Fe3+Fe3+ form of ≤1% of that of the Fe3+Fe2+ form, a finding that is important for understanding the fundamental chemistry by which these dinuclear enzymes catalyze the hydrolysis of phosphate esters.

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