Resonance Raman studies on the ligand-iron interactions in hemoproteins and metallo-porphyrins

The resonance Raman spectra (RRS) were measured for various hemoproteins, as well as for free hemes, and analysed in terms of the interactions between the porphyrin ring and the heme iron and between the axial ligand and the heme iron. To establish vibrational assignments, the RRS of metallo-octae-thylporphyrin (M(OEP)), its 15N substituted and meso-deuterated derivatives were measured. It was found that the Raman line corresponding to the oxidation state marker of hemoproteins involves appreciable displacement of the pyrrolic nitrogen atoms toward metal ion but not that of methine bridges and that the frequencies of methine-bridge CC stretching vibrations are altered by the conjugation interaction between the porphyrin ring and the metal ion present. The prominent Raman lines of Ni(OEP) were assigned on the basis of the normal coordinate analysis. The Raman spectra of heme a bis-imidazole complex (Fe(A)(Im)2) in the presence of cyanide changed when pH was reduced to neutral. It implied formation of cyanhydrin at the peripheral formyl group of heme a. The Fe(A)(Im)2 formed an addition compound with NaHSO3 in a neutral solution but the added NaHSO3 was eliminated when the heme iron was reduced. The Raman spectra of hemoproteins were classified into four groups on the basis of the relative intensities and frequencies of the four key bands. It demonstrated the existence of two kinds of ferrous low-spin states. The difference between the two species was attributed to the difference in the nature of chemical bond between the heme iron and its sixth ligand (L). The Fe-L bonds of ferrous low-spin molecules in Groups A and C are mainly associated with the dz2(Fe)-lone pair and dz(Fe)-II(L) interactions, respectively. This interpretation of the Raman spectra appears consistent with the experimental facts that the ferrous low-spin molecules in Group C photodissociate upon illumination of light at the Soret band and also that the internal stretching frequencies of the sixth ligand are shifted to lower frequency upon coordination to the heme iron. The frequency of the oxidation state marker of reduced cytochrome P-450 (P-450cam) was unusually low in comparison with those of other hemoproteins. It was ascribed to delocalization of electrons from the thiolate anion to the porphyrin II*(ring) orbital through the pi-type molecular orbital (Eq.3). The RRS of reduced P-450cam.metyrapone adduct was quite close to that of reduced cytochrome b5. In conclusion, the Raman frequencies of hemoproteins may depend primarily upon the amount of electrons delocalized to the II* orbital of the porphyrin ring. The coordination of lone-pair electrons of the axial ligand to the iron dz2 orbital yields bond energy but scarcely affects the Raman frequencies. If the pi orbital of the axial ligand interacts with the iron dpi orbital, it perturbs the delocalization of the dpi(Fe) electrons to the II*(ring) orbital of porphyrin ring and thus affects the Raman frequencies.