Characterization of pink and purple uteroferrin by resonance Raman and CD spectroscopy.

Changes effected in purple uteroferrin’s resonance Raman spectrum by mercaptoethanol reduction suggest that the protein’s single iron atom is coordinated to more than one tyrosyl residue. Detection of a conservative pair of visible CD bands, indicative of exciton splitting of a tyrosinate-to-iron charge transfer band, strongly supports this suggestion. Additional features in the protein’s visible and near-W CD spectra may result from iron coordination by a third tyrosyl residue. Resonance-enhanced Raman bands detected at 576 cm” in purple uteroferrin and 571 cm“ in the pink protein may arise from vibrations associated with ciscoordinated phenolates, again suggesting iron coordination by multiple tyrosyl residues. Changes in purple uteroferrin’s Raman spectrum which accompany the purple-to-pink conversion indicate that reduction perturbs the protein’s iron-binding site without disrupting the iron-tyrosyl coordination. Differences between the near-W and visible CD spectra of pink and purple uteroferrin are consistent with Raman data, suggesting that mercaptoethanol reduction induces a reorientation of tyrosyl residues coordinated to iron. In contrast, the virtual coincidence of the aromatic region of the CD spectra of pink and purple uteroferrin signifies that there is little difference between the environments of tryptophanyl and uncoordinated tyrosyl residues in the two forms of the protein. The decrease of uteroferrin’s low a-helical content by 40% upon conversion to its pink form suggests that the structural changes effected by mercaptoethanol reduction may be limited primarily to a particular region of the polypeptide chain. Thus, the purple-to-pink conversion probably results from cleavage of an accessible disulfide bond in the vicinity of the protein’s chromophoric metal-binding site. The unprecedentedly large splitting found for a previously assigned Fermi doublet of tyrosine can be explained by a doubling of the coupling strength between the v1 fundamental and 2vlGa overtone undergoing the Fermi resonance. The proposed increase in coupling strength may result from the highly covalent metalligand bonds of uteroferrin’s chromophoric iron.