title: Spectroscopic studies on the interaction of phosphate with uteroferrin.
abstract: The effect of phosphate on the binuclear iron center of pink (reduced) uteroferrin was examined by magnetic resonance and optical spectroscopy. The purple (oxidized) protein, which contains 1 mol of tightly bound phosphate per mol of enzyme at isolation, does not give rise to a 31P NMR signal. Phosphate binding to phosphate-stripped pink uteroferrin is indistinguishable from that in the native purple phosphoprotein. As measured by EPR and optical spectroscopy, the rate of reaction between phosphate and pink uteroferrin is pH-dependent, decreasing as the pH increases. Phosphate is capable of binding to the reduced protein between pH 3 and 7.8, resulting in formation of the purple uteroferrin-phosphate complex. Evans susceptibility measurements at pH 4.9 indicate that the EPR silent species with a maximum absorption at 535 nm, generated upon phosphate addition to pink uteroferrin, is diamagnetic. Moreover, phosphate causes disappearance of the hyperfine-shifted resonances in the 1H NMR spectra of the reduced protein. We therefore have not been able to identify the paramagnetic "purple reduced enzyme-phosphate complex" reported by Pyrz et al. (Pyrz, J. W., Sage, J. T., Debrunner, P. G., and Que, Jr., L. (1986) J. Biol Chem. 261, 11015-11020) using Mossbauer spectroscopy and dithionite-reduced 57Fe-reconstituted uteroferrin. Our present data with native unmodified enzyme are in accord with our earlier results (Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756) and with the results of Burman et al. (Burman, S., Davis, J. C., Weber, M. J., and Averill, B. A. (1986) Biochem. Biophys. Res. Commun. 136, 490-497) on bovine spleen phosphatase, suggesting that phosphate binding to reduced protein rapidly induces oxidation of the binuclear iron center.
mesh_terms: D000135:Acid Phosphatase; D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D011555:Pseudopregnancy / Q000378:metabolism; D013053:Spectrophotometry; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D007527:Isoenzymes; D008667:Metalloproteins; D010710:Phosphates; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase