J. Biol. Chem., Vol. 262, Issue 15, 6982-6985, 05, 1987
Spectroscopic studies on the interaction of phosphate with uteroferrin
K Doi, R Gupta and P Aisen
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.
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