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(III) and 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 MP8(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 an 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 v10 mode sensitive to ligand deprotonation(s). Moreover, the replacement of His by an exogenous ImH in MP8(III) complexes induces the upshift of the v10 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 v8 (343-347 cm-1). As far as the ferrous compounds are concerned, the mode most sensitive to the ImH deprotonation is v11, 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 MP8(II) complexes, as well as observations based on isotopic substitutions of the corresponding 1-methylimidazole complexes (MeIm-->MeIm-d6), 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 v(Fe-N(pyrrole)) modes coupled to either a deformation mode of axial bonds or an internal mode of the bound imidazole(s).(ABSTRACT TRUNCATED AT 400 WORDS)

The low-frequency regions (150--700 cm-1) of resonance Raman (RR) spectra of various complexes of oxidized and reduced horse heart myoglobin were examined by use of 441.6-nm excitation. In this frequency range, RR spectra show 10 bands common to all myoglobin derivatives (numbered here for convenience from I to X). Relative intensities of bands IV, V, and X constitute good indicators of the doming state of the heme and, consequently, of the spin state of the iron atom. An additional band is present for several complexes (fluorometmyoglobin, hydroxymetmyoglobin, azidometmyoglobin, and oxymyoglobin). Isotopic substitutions on the exogenous ligands and of the iron atom (56Fe leads to 54Fe) allow us to assign these additional lines to the stretching vibrations of the Fe-sixth ligand bond. Similarly, bands II are assigned to stretching vibrations of the Fe-N-(pyrrole) bonds. An assignment of bands VI to stretching vibrations of the Fe-Nepsilon(proximal histidine) bonds is also proposed. Mechanisms for the resonance enhancement of the main low-frequency bands are discussed on the basis of the excitation profiles and of the dispersion curves for depolarization ratios obtained for fluorometmyoglobin and hydroxymetmyoglobin.

The visible absorption and Soret-excited resonance Raman spectra of ferrous microperoxidase-8 [MP8(II)], an octapeptide containing a heme c, are reported. These spectroscopies indicate that MP8(II), dissolved in aqueous buffered solutions, forms low-spin six-coordinated complexes in the 7-14 pH range. Intermolecular bonding interactions of MP8(II) in water account for this behavior. On the contrary, when the hemopeptide is dispersed in aqueous solutions containing detergent or an alcohol, the spectroscopic data show that the iron atom of MP8(II) is essentially high-spin five-coordinated in accordance with a monomeric structure of MP8(II). In addition to a high-spin signature to the heme skeletal modes, the high-frequency regions of resonance Raman spectra characterize an electronic influence of the thioether bridges on the frequency of stretching modes of C beta-C beta bonds (nu 2, nu 11, and nu 29). On the other hand, the low-frequency Raman spectra of monomeric MP8(II) at pH 7.5 present significant differences in the 150-250-cm-1 regions depending upon the solvent composition (pH, presence or absence of detergent, alcohol). These effects are attributed to frequency variations of the Fe-N(His)-involving mode which indicate changes in the H-bonding interactions of the axial His and therefore solvent-dependent changes of the octapeptide conformation. Our resonance Raman data further show that the axial His of monomeric MP8(II) could be totally deprotonated in aqueous cetyltrimethylammonium bromide solution at very alkaline pH (pKa = 13.3). The vibrational data (100-1700 cm-1) obtained for the various monomeric forms of MP8(II) are expected to be useful for determining the heme structure and environment in reduced c'-type cytochromes. Comparisons of resonance Raman data with X-ray crystallographic data available for different hemoproteins allow us to evaluate the ionization and H-bonding states of the His bound to the high-spin five-coordinated hemes. These data are discussed in terms of proximal influence of protein-His-heme interactions on the determination and the regulation of a particular biological function.

Abstract. Optical absorption spectra and resonance Raman (RR) spectra, obtained with Soret excitation, are reported for bis(imidazole) and bis(imidazolate) complexes of iron(II)-and iron(III)-protoporphyriri IX, prepared in aqueous conditions. Perdeuteration experiments on the axial ligands permitted the assignment of the symmetric Fe-(ligand)2 stretching mode of Fe[x]PP(L)2 to RR bands at 203 (x=II; L=ImH), 212 (x=II; L=Im-), 201 (x=III; L=ImH) and 226 cm -1 (x = III; L = Im-). These frequency differences indicate a strengthening of the axial bonds when the imidazole deprotonations occur. The larger difference observed for the ferric derivatives reflects the stronger a-donor capability of the Ira-anion for iron(III) over iron(II). For the ferrous derivatives, the frequencies of several skeletal porphyrin modes (v4, Vlo, vll and V3s ) are downshifted by 2-10 cm-J upon deprotonation of the ligands. This effect corresponds to an increased back-bonding from the metal atom to the porphyrin ring when the axial ligand decreases its n-acid strength. Bringing further support to this interpretation, an inverse linear relationship is established between the frequencies of v (Fe(II)-L2) and v~t. This correlation is expected to monitor the overall Hbonding state of histidine ligands of reduced cytochromes b. On the other hand, absorption measurements have characterized large pK~ differences for the sequential imidazole ionizations of Fe Abbreviations: RR, resonance Raman; EPR, electron paramagnetic resonance; PP, protoporphyrin IX; ImH, imidazole; Ira-, imidazolate; Im*, imidazole or imidazolate; 1Melm, t-methylimidazole; HisH, histidine; His-, histidinate; CTABr, cetyltrimethylammonium bromide; NaDS, sodium dodecylsulphate; VLP, very low potential; LP, low potential; HP, high potential good proton-acceptor and proton-donor, respectively, and suggest a model by which heme, located in a favorable environment inside a cytochrome, could couple a cycle of electron transfer with a proton transfer. Based on sequence data and structural models, it is further proposed that, in several membrane cytochromes b (b, b6, b559) , a positively charged amino acid residue and an imidazolate ligand of the ferriheme could form an ion pair involved in a redox control of proton transfer.

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