RESONANCE RAMAN INVESTIGATION OF IMIDAZOLE AND IMIDAZOLATE COMPLEXES OF MICROPEROXIDASE - CHARACTERIZATION OF THE BIS(HISTIDINE) AXIAL LIGATION IN C-TYPE CYTOCHROMES

In order to evaluate the steric and electronic influences of the heme axial ligands on the vibrational modes of heme c, various ferric and ferrous six-coordinate heme c compounds have been prepared from microperoxidase-8 (MP8) and different extrinsic ligands. In this paper, the absorption and Soret-excited resonance Raman (RR) spectra of imidazole, imidazolate, 1-methylimidazole, and histidine complexes of MP8 are presented. The absorption characteristics of the unligated forms, either aggregated or monomeric, as well as of the ligated forms of MP8(II) have been determined as a function of pH, the presence of a cationic detergent, and the ligand concentration. Spectrophotometric titrations have shown that MP8(III) and MP(II) can bind one or two molecules of exogenous ligand, forming monoligated or bisligated complexes. The latter form, observed with large excesses of ligand, results from the displacement of the intrinsic proximal His of MP8 by a exogenous ligand. Several structural marker bands have been detected in the high- and low-frequency regions of RR spectra. The high-frequency RR spectra of the ImH compounds of MP8(III) exhibit a nu(10) mode sensitive to ligand deprotonation(s). Moreover , the replacement of His by an exogenous ImH in MP8(III) complexes induces the upshift of the nu(10) mode frequency (1637-1641 cm(-1)), indicating that the porphyrin skeleton is less distorted when the internal coordination of proximal His to heme is broken. A similar dependence of the out-of-plane porphyrin distortion is suggested for the low-frequency mode nu(8) (343-347 cm(-1)). As far as the ferrous compounds are concerned, the mode most sensitive to the ImH deprotonation is nu(11), which is downshifted from 1539 to 1527 cm(-1). Comparisons of the low-frequency regions of the RR spectra of imidazole-type ligated MP8(III) and MP(II) complexes, as well as observations based on isotopic substitutions of the corresponding 1-methylimidazole complexes (MeIm --> MeIm-d(6)), allow the assignment of two bands in the 184-197 and 400-409 cm(-1) regions to modes involving the symmetric and asymmetric stretches of the axial ligands, respectively. Two other bands in the 343-347 and 359-362 cm(-1) regions, sensitive to the mass and/or deprotonation states of the axial ligands, have been tentatively assigned to nu(Fe-N(pyrrole)) modes coupled to either a deformation mode of axial bonds or an internal mode of the bound imidazole(s). All of the preceding spectroscopic data are used to extract information on the ionization states of axial imidazoles, as well as on the heme structure in bis(histidine)-ligated hemes of cytochromes c. They will also provide good references in a study to follow on the N-acetylmethionine and lysine complexes of MP8.