Spectroscopic studies on the interaction of phosphate with uteroferrin.
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.
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
D007527:Isoenzymes; D008667:Metalloproteins; D010710:Phosphates; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase