NMR Studies of the Dinuclear Iron Site in Reduced Uteroferrin and Its Oxoanion Complexes

The chemical shifts and relaxation behavior of paramagnetically shifted proton NMR resonances of reduced uteroferrin (Uf_r) and its XO₄^n- complexes (n = 2: X = Mo, W; n = 3: X = P, As) are reported. Integrations with respect to an internal standard show that downfield resonances (δ > 14 ppm vs TMS) arise from 10 protons on ligands to the paramagnetic dinuclear iron center of Uf_r, and upfield resonances (δ >-10 ppm vs TMS) arise from at least two protons. Two downfield and one upfield resonance are lost in D₂O. Binding of molybdate, tungstate, or arsenate of Uf_r at pH 4.9 causes minor changes in δ, whereas anaerobic addition of phosphate results in loss of interpretable NMR signals outside of the-3 to 15 ppm range. Uf_rPO₄ at pH 3 exhibits downfield-shifted resonances somewhat larger than those of the other oxoanion complexes at pH 4.9, which suggests weaker antiferromagnetic coupling in Uf_rPO₄. Primarily on the basis of δ and T₁, values, seven of the paramagnetically shifted resonances are assigned to a single tyrosine bound to iron(III) and to a histidine bound to each of the two iron atoms. Probable assignments are given for other paramagnetically shifted peaks.