Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode

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Abstract
Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 17000 cm-1 indicate two distinct sites in Ni Hb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoprophyrin IX [Ni(ProtoP)] in noncoordinating solvents. The wavelength for the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted. A decrease of almost 7 cm-1 indicates a high degree of Ni-ligand character for the assigned mode. The frequency of the Ni-ligand stretch, falling as it does in the range of Fe-histidine stretching frequencies points to histidine as the axial fifth ligand. The frequency of the Ni-histidine mode is slightly higher than that for the Fe-hisitidine mode in hemoglobin but lower than that observed for horseradish peroxidase. We find that the Ni-histidine mode is 5 cm-1 higher in NiMb (R-like) than in NiHb (T structure). A similar increase in the Fe-histidine frequency is noted in the comparison of the native deoxy proteins. The Raman results show that the local structural changes associated with a change in quaternary structure are the same at the metal site for both Ni-reconstituted and Fe hemoglobins.
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