The Resonance Raman Spectrum of the Metalloprotein Ovotransferrin

Abstract

Using ovotransferrin it is shown that amino-acid side chains forming the metal binding site in coloured metalloproteins can be identified and monitored by resonance Raman spectroscopy. All major features above 800 cm−1 in the resonance Raman spectrum of iron–bicarbonate ovotransferrin could be assigned to histidine or tyrosine vibrational modes, by comparison with Raman and infrared data for these amino acids and related compounds. Comparison of the features due to ring modes of imidazole ligated to Fe3+ in ovotransferrin with those of aqueous histidine at pH 8.0 suggests that rearrangement of the ring π electrons takes place upon ligation. Although iron–bicarbonate and iron–oxalate ovotransferrin have similar visible absorption spectra, differences in their resonance Raman spectra are clearly distinguishable, but no evidence was found for the direct binding of bicarbonate or oxalate to the iron. Differences between the geometries of the two sites at which iron is bound in iron–bicarbonate ovotransferrin appeared to be small compared with the perturbation caused to these sites by replacing bicarbonate with oxalate.