Resonance Raman (RR) spectra of the acidic form of FeIII horseradish peroxidase (HRP) were obtained at room and low temperatures using B- and Q-band excitation. At 296 K, HRP exhibits two sets of porphyrin skeletal stretching frequencies which are attributed to a thermal mixture of 5- and 6-coordinate high-spin FeIII states. When the temperature is lowered, the observed bands shift to higher frequencies, and these are assigned to intermediate- and low-spin states. Addition of 40% glycerol has no effect on the spectra at 296 K, but at 20 K, all four frequency sets are observed corresponding to the two forms observed at room and low temperature in the absence of glycerol. The 296 K RR spectrum of the HRP-hydroquinone complex is similar to that of free HRP, but conversion to the intermediate- and low-spin states is complete at a higher temperature than in the free enzyme. Addition of benzohydroxamic acid (BHA) to HRP shifts the RR frequencies to those corresponding to a 6-coordinate high-spin species at both room and low temperature. Two upsilon (C = C) stretching modes are observed for HRP and its donor complexes, indicating that the vinyl groups are inequivalent. On BHA binding, one of the vinyl modes and upsilon 37 (Eu) are enhanced, suggesting symmetry lowering of the heme site.

The scheme of energy levels previously proposed to describe dual excitation and emission associated to excited state intramolecular proton transfer (ESIPT) of some hydroxyanthraquinones (HAQ’s) has been made more quantitative in the present paper. The zero-point energy and the frequency of the νOH mode for the HAQ’s have been calculated on the basis of the Lippincott–Schroeder double-minimum potential for the O–H⋯O hydrogen bond. The second derivative absorption (D2) spectra show that the vibrational structures of the electronic excited state of HAQ’s giving rise to ESIPT are characterized by the progression of the νOH stretching mode. The νOH mode in the ground state is observed as a very strong band in the vibrational structure of the short wavelength emission for HAQ’s showing ESIPT. The combined resonance Raman band assignment of four hydroxyanthraquinones and transform analysis show that the visible transition involves the hydrogen bonded cycle and induces proton transfer in the excited state in most cases. On the basis of the isotopic effects, some vibrations of the hydrogen bonded cycle, namely the νC=O, δC=O, νCOH, and δOH modes, have been identified. The transform method, including the combined analysis of the absorption and D2 spectra in terms of sum-over-states, was checked by directly deriving the displacement parameters (Franck–Condon factors) of 1,4-DHAQ from the high resolution free-jet spectrum. The values of the displacement parameters of the νOH mode are quite large for the HAQ’s showing ESIPT, while are negligible for 1,4-DHAQ. High values of the displacement parameters for the other vibrations of the hydrogen bonded cycle were found for all HAQ’s.

The absorption and second-derivative spectra of β-carotene in n.hexane, isopentane, and CS2, as well as the Raman excitation profiles for six modes in n.hexane and isopentane have been measured. The combined analysis of the absorption and second-derivative spectra in terms of model summing over the vibronic excited states furnished reliable values of the excited state displacement parameters. The input parameters for the calculation of the full spectra were taken from the Raman band relative intensities, while the line shape functions and their widths were determined by the second-derivative absorption spectra. The Raman excitation profiles were then calculated in terms of the transform theory, both the observed and calculated absorption spectra being transformed. Very good fits with the experimental profiles were found by including a small contribution of inhomogeneous broadening to the total width. It was concluded that the homogeneous broadening mainly derives from dephasing of nondegenerate low-frequency vibrations in the resonant electronic excited state. The low temperature profiles were easily calculated by decreasing this homogeneous contribution. The agreement between calculated and experimental results was improved by assuming that the vibrational frequencies of the carbon–carbon stretching modes increase at the excited state. The resulting nuclear diplacements and frequency shifts at the excited state as well as the Gaussian widths correlate very well with the absorption spectral properties of linear polyenes.

A spectroscopic investigation by resonance Raman has been carried out at pH 7.0 in 0.1 M phosphate buffer on the cooperative homodimeric myoglobin from Nassa mutabilis. The study has been performed on the unligated ferrous form, as well as on the ligated species MbO2 and MbC, and on the ferric form met-Mb. Two v(C = C) vinyl stretching modes have been observed in all the investigated forms, reflecting different degrees of vinyl conjugation with the porphyrin ring, as a consequence of a strongly asymmetric environment for the two side groups of the heme. Furthermore, the ferric form displays a hexacoordinate low-spin heme, which suggests the presence of an endogenous ligand bound to the Fe atom. The frequency of the v(Fe-Im) stretching mode of Mb from Nassa mutabilis shifts down by 4 cm-1 as compared with that of horse heart myoglobin, reflecting a protein-induced proximal strain as a result of heme-heme interaction due to the close proximity of the two hemes in the dimer. The lower frequency of the v(Fe-Im) stretching mode agrees well with the lower affinity for oxygen binding found for Nassa mutabilis Mb and with the slight heme core expansion with respect to horse heart Mb, suggesting a critical role for the Fe-His bond on the heme's function and structure.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mut#bilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH ('~ 3.0) and the consequent cleavage of the HisFgNE2-Fe proximal bond brings about a ~ 20-fold increase of the second.order rate constant for CO binding. This process displays a pK~ = 4.0 ± 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisFgNE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N, mutabilia' Mb, like that of the homodimerie hemoglobin (Hb) from Scapharca inaequh,alvis (Coletta, M., Boffi, A., Ascenzl, P., Brunori, M. and Chianeone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assumin~ a concerted proton.linked transition with n = 1.8 __. 0. i. Such a characteristic suggests, also on the basis of the amino acid sequenc~ homology between N. mutabilis Mb and S. inaequivah,is Hb in the region forming the subanit interface, that the interaction mechanism is similar for the two homodimerie proteins, and drastically different from that operative in otimr hemoproteins.