The interaction of phosphate with uteroferrin. Characterization of a reduced uteroferrin-phosphate complex.

The interaction of phosphate with reduced uteroferrin has been re-examined in light of disagreements on the oxidation state of the binuclear iron cluster (Keough, D. T., Beck, J. L., de Jersey, J., and Zerner, B. (1982) Biochem. Biophys. Res. Commun. 108, 1643-1648; Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756). Our results based on Mossbauer observations and the kinetics of spectral change and activity loss show clearly that phosphate binds to reduced uteroferrin to form a reduced uteroferrin-phosphate complex. This complex exhibits a pair of quadrupole doublets at 119 K with parameters typical of a high spin ferric and a high spin ferrous center, respectively, but distinct from those of the native reduced enzyme. The reduced phosphate complex exhibits a pH-dependent visible absorption maximum ranging from 530 to 561 nm. In air, the reduced phosphate complex converts to the oxidized phosphate complex with a first order rate constant of 4 X 10(-3) min-1, as monitored by spectral changes and loss of enzyme activity.