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 f errous six-coordinate heme c compounds have been prepared from micrope roxidase-8 (MP8) and different extrinsic ligands. In this paper, the a bsorption and Soret-excited resonance Raman (RR) spectra of imidazole, imidazolate, 1-methylimidazole, and histidine complexes of MP8 are pr esented. The absorption characteristics of the unligated forms, either aggregated or monomeric, as well as of the ligated forms of MP8(II) h ave been determined as a function of pH, the presence of a cationic de tergent, and the ligand concentration. Spectrophotometric titrations h ave shown that MP8(III) and MP(II) can bind one or two molecules of ex ogenous ligand, forming monoligated or bisligated complexes. The latte r form, observed with large excesses of ligand, results from the displ acement of the intrinsic proximal His of MP8 by a exogenous ligand. Se veral 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 de protonation(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 di storted when the internal coordination of proximal His to heme is brok en. A similar dependence of the out-of-plane porphyrin distortion is s uggested 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 Im H deprotonation is nu(11), which is downshifted from 1539 to 1527 cm(- 1). Comparisons of the low-frequency regions of the RR spectra of imid azole-type ligated MP8(III) and MP(II) complexes, as well as observati ons based on isotopic substitutions of the corresponding 1-methylimida zole 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 symm etric and asymmetric stretches of the axial ligands, respectively. Two other bands in the 343-347 and 359-362 cm(-1) regions, sensitive to t he mass and/or deprotonation states of the axial ligands, have been te ntatively assigned to nu(Fe-N(pyrrole)) modes coupled to either a defo rmation mode of axial bonds or an internal mode of the bound imidazole (s). All of the preceding spectroscopic data are used to extract infor mation 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-acety lmethionine and lysine complexes of MP8.