Complexes of cytochrome c and of an undecapeptide of the protein have been studied by means of Mossbauer spectroscopy in order to obtain information concerning the active heme site.The variation of the quadrupole splitting with the temperature permits an evaluation of the distortions from octahedral symmetry around the iron ion in the heme group, and also an estimation of the spin-orbit constant. The effect of the different ligands such as CN-, F-, imidazol, etc., is discussed.Complexes of cytochrome c have been studied in order to obtain a structural model and therefore to understand the activity of this protein. Peptides formed by the enzymatic digestion of the cytochrome c have been used as models for the protein since they retain a similar environment around the iron atom in the heme structure. The undecapeptide (Pep)ll has been extensively studied. This peptide retains the aminoacid residues 11 to 21 of the cytochrome c [l]. Mossbauer spectroscopy provides a probe of the iron atom in these molecules. It is sensitive to changes in the spin and oxidation state and the changes in the electronic environment around the iron atom [2]. The present communication deals with complexes of (Pep)ll withligands such as CN-, F-, imidazole and methionine, and the results are compared with those obtained with the parent protein cytochrome c. METHODS AND MATERIALS Preparation of Complexes(Pep)ll was prepared by the peptic digestion of horse heart ferri-cytochrome c (Calbiochem) and purification by column chromatography [4]. The complexes of (Pep)ll with CN-and F-were prepared following the procedures described by Butt and Keilin for the analogous complexes of cytochrome c [5]. The methionine complex was prepared according to the method of Harbury ef al. [6]. In all cases the ligand Abbreviation. (Pep)ll, undecapeptide of cytochrome c. ____-was added in small portions to the solution of (Pep)ll in the appropriate buffer and the visible spectrum recorded after each addition. Addition of the ligand continued until no further changes were observed in the visible spectrum.Attempts to prepare the CO complex met with failure, as expected, because CO generally coordinates to Fe(I1) but not to Fe(II1) in heme compounds. Mossbauer StudiesThe Mossbauer spectrometer was of the constant acceleration type with moving absorber geometry [7]. The absorber and source were maintained at the same temperature. A gas-flow proportional counter using 10 % methane and 90 % argon was used as the detector [8]. A cryostat described by Travis and Spijkerman [9] was used for low-temperature measurements and was modified for variable temperature operation [ 101. The source was 40mCi 57Co in palladium matrix. The absorbers were mounted in aluminium foil cups. The Mossbauer spectrum of the aluminium used revealed an absorption of about 0.02%, which is completely negligible as compared with the measured spectra. These were measured with lyophilized samples (approximately 50 mg) and frozen solutions (1 ml) in which the concentration of (Pep)ll was about 10 ...
Undecapeptide of Ferricytochrome c, Electron Spin Resonance, Mössbauer Spectroscopy, Visible Spectroscopy The ESR, Mössbauer, and visible spectra of the undecapeptide of ferricytochrome c (H P p) were measured for both the solid peptide and solutions of the peptide at pH 1.5, 7, and 10. At the low pH, the iron exists in the H Pp in the high-spin species. At neutral and alkaline pHs, the low-spin ferric species predominate. The results of these measurements support the previous suggestions concerning the ligands in the fifth and sixth position of HPp at the neutral and alkaline pHs. The spectra of lyophilized preparations of HPp show the presence of both high and low-spin ferric species. The study of the Mössbauer spectra of the lyohilized preparations as function of temerature shows that there exists a spin-spin equilibrium between the two spin states. The relative amounts of the two components vary with the preparation. In these preparations there exist two components, one of which exists in the equilibrium mixture and the second in the high-spin form.
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