Physical characterization of two-iron uteroferrin. Evidence for a spin-coupled binuclear iron cluster.

Purple uteroferrin bearing two iron atoms exhibits the same distinctive g' = 1.74 EPR signal detected previously in one-iron preparations, but with an intensity accounting for only 0.13 spins (or 7% of the iron) per molecule of protein.A low, non-Curie magnetic susceptibility of the purple protein further indicates that the bulk of its EPR-silent iron is at most only weakly paramagnetic at low temperature.Conversion of the protein to its pink form intensifies the g' = 1.74 signal which then accounts for 0.67 spins (37% of the iron) per molecule.Together, these observations suggest that the g' = 1.74 EPR signal arises from a spincoupled binuclear iron cluster.Thus, the two iron atoms of fully oxidized purple uteroferrin would both be ferric with their spins coupled antiferromagnetically to give an EPR silent spin singlet ground state.In the pink protein, the paramagnetism arises from the same pair of iron atoms now present as a ferric-ferrous couple with an EPR-active Kramers doublet ground state.The Curie-law behavior exhibited by the intensity of the g' = 1.74 signal between 1.4 and 4.2 K supports this model.Addition of phosphate to the pink form of the protein virtually obliterates its g' = 1.74 signal, simultaneously shifting the protein's absorption maximum from 515 to 545 nm, suggesting that phosphate binding renders the ferrous atom of the binuclear pair susceptible to air oxidation.Changes in the protein's CD spectrum accompanying the pink-to-purple transition induced by phosphate are consistent with this hypothesis.Upon standing, the pink two-iron enzyme is gradually converted to a form with a predominantly axial EPR signal (al = 1.95 and gi = 1.55).This axial signal, but not its rhombic precursor, is insensitive to both phosphate and molybdate and becomes unobservedly broad between 20 and 25 K.Uteroferrin is a single chain, purple, iron-binding glycoprotein with acid phosphatase activity and a molecular weight of 35,000-40,000 (1-5).It may be isolated in large quantities from either the allantoic fluid of pregnant sows or the uterine flushings of pseudopregnant sows (3, 6, 7).The protein can exist in either of two interconvertible forms: purple, which is enzymatically inactive, or pink, which is active (4, 8).The purple-to-pink conversion is effected by mild reductants such as P-mercaptoethanol, ascorbate, or dithiothreitol (3, 4, 8).Resonance-enhanced Raman and CD spectroscopic studies indicate that uteroferrin is an iron-tyrosinate protein (9-10).