Observation of an isotope-sensitive low-frequency Raman band specific to metmyoglobin.

A resonance Raman band involving significantly the iron(III)-histidine stretching (upsilonFe-His) character is identified for metmyoglobin (metMb) through isotope sensitivity of its low-frequency resonance Raman bands, but the identification was not successful for methemoglobin (metHb) and its isolated alpha and beta subunits. A band at 218 cm-1 of natural abundance metMb exhibited a low-frequency shift for 15N-His-labeled metMb (-1.4 cm-1 shift), while the strong porphyrin bands at 248 and 271 cm-1 did not shift significantly. The frequency of the 218-cm-1 band of metMb decreased by 1.6 cm-1 in D2O, probably due to Ndelta-deuteration of the proximal His, in a similar manner to that of the upsilonFe-His band of deoxyMb in D2O. This 218-cm-1 band shifted slightly to a lower frequency in H2(18)O, whereas it did little upon 54Fe isotopic substitution (<0.3 cm-1), presumably because of the six-coordinate structure. The lack of the 54Fe-isotope shift shows that the 218-cm-1 band is specific to metMb and not due to the deoxy species. The intensity of this band decreased for hydroxymetMb and was indiscernible for cyanometMb. For metHb and its alpha and beta subunits, however, the frequencies of the band around 220 cm-1 were not D2O sensitive. These results suggest an assignment of the band around 220 cm-1 to a pyrrole tilting mode, which significantly contains the Fe-His stretching character for metMb but scarcely for metHb and its subunits. The differences in the isotope sensitivity of this band in different proteins are considered to reflect the heme distortion from the planarity and the Fe-His geometry specific to individual proteins.