No abstract

We have designed andassembled a sensitive vidicon Raman spectrometer which is shown to be well suited for the studies of heme proteins in both Soret (near-ultraviolet) and Q (visible) band regions. This system, employing a dry ice-cooled silicon intensified target (SIT) detector and an additive dispersion double monochromator (two 600 g mm-' gratings blazed at 400 nm), has excellent stray light rejection and optimum bandpass and resolution (5-12 cm-') for biological applications. It is demonstrated that a 600 cm-' wide section of the low frequency Raman spectrum of ferrocytochrome c excited at 413.1 nm (70 mW) can be obtained in 30 ms, which is -2 x lo4 times faster than that required by the conventional scanning techniques. Our multichannel Raman system has an enhanced detection capability which enables us to identify at least 41 weak Raman lines below 850 cm-' in the spectrum of ferrocytochrome c. Using the SIT detection system, the Raman excitation profile of a depolarized line at 750 cm-' in Fe(II) cytochrome c has been constructed and compared with that reported for Ni etioporphyrin. Instead of strong 0-0 Raman intensity, the cytochrome c profile exhibits weaker 0-0 intensity, indicative of the absence of the Jahn-Teller effect. It is further demonstrated that the Soret-excited Raman spectrum of oxyhemoglobin can be readily obtained without photodissociation.

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).

No abstract

No abstract