Temperature dependence of resonance Raman spectra of metmyoglobin and methemoglobin azide. Detection of resonance-enhanced bound azide vibrations and iron-azide stretch.

Resonance Raman spectroscopy has been employed to study the thermal spin equilibria in metmyoglobin azide [Fe(III)Mb-N3] and methemoglobin azide [Fe(III)-Hb-N3]. The effect of temperature on Raman intensities permits us to assign lines to either high- or low-spin species. With excitation at 647.1 nm the intensity of an 15N3 isotope-sensitive mode at approximately 411 cm-1 was found to increase with decreasing temperature, indicating that its origin may not be the high-spin charge-transfer band at approximately 640 nm as suggested by Asher & Schuster [Asher, S. A. & Schuster, T. M. (1979) Biochemistry 18, 5377]. Instead, it may be enhanced via the weaker low-spin z-polarized charge-transfer band at approximately 650 nm which was identified by Eaton & Hochstrasser [Eaton, W. A., & Hochstrasser, R. M. (1968) J. Chem. Phys. 49, 985]. Our normal coordinate analysis on the model azide-Fe-imidazole and the polarized nature of the line allow us to establish that the approximate 411-cm-1 mode in Fe(III)Mb-N3 and Fe(III)Hb-N3 is assignable to the Fe-N3 stretch of low-spin species. Furthermore, we assign the out of plane azide mode (low spin) to the depolarized line at 573 cm-1 (15N3 isotope sensitive), which was previously assigned as the Fe-N3 stretch by Desbois et al. [Desbois, A., Lutz, M., & Banerjee, R. (1979) Biochemistry 18, 1510]. No internal vibrations of bound azide excitation at 406.7 nm, we have observed the enhancement of the antisymmetric azide stretch (both high and low spin), out of plane bending (low spin), and Fe-N3 stretch (low spin), indicating the existence of at least two charge-transfer transitions underlying the strong Soret band. The following four types of charge transfer are discussed in the light of our present resonance Raman data: (1) porphyrin (pi) leads to high-spin Fe (d pi), (2) azide (n) leads to low-spin iron (dz2), (3) azide (pi) leads to low-spin iron (dz2), and (4) azide (pi) leads to porphyrin (pi) (high spin).