Mössbauer Studies of Some Complexes of the Undecapeptide of Cytochrome c

Abstract

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 ...

Insights into porphyrin chemistry provided by the microperoxidases, the haempeptides derived from cytochrome c

“…isomer shift of 0.35 mm s −1 at 80 K. 90 Magnetic susceptibility data collected to 6 K with a SQUID susceptometer are in accord with the M össbauer results. 83 M össbauer spectroscopy is useful in identifying the predominant spin state of the metal ion in the haempeptides but is of limited utility in characterising the coordination sphere of the metal ion.…”

“…The imidazole complex of MP11 has values of 0.40 and 2.09 mm −1 , respectively. 90 As expected, the quadrupolar splitting of the high spin component of [H 2 O-N-AcMP8] is smaller (1.08 mm s −1 at 4.2 K) although the isomer shift (0.30 mm s −1 ) is very similar. The M össbauer spectrum of the fluoride complex of MP11 (which is predominantly high spin) is reported to have a quadrupolar splitting of 0.63 mm s −1 and an Fig.…”

Microperoxidase-11: Molecular Dynamics and Q-Band Excited Resonance Raman of the Oxidized, Reduced and Carbonyl Forms

“…In the ferric species, the presence of 6-coordinate iron has been proposed with two possible spin states: at pH< 7, HS Fe3+ with his18 as 5th ligand and a water as 6th ligand, and at pH -7-11, LS Fe3+ with a stronger field 6th ligand, such as valll or lys13 of a second MP11 aggregated molecule (13). This LS Fe3+ assignment at neutral pH was confirmed by other workers (14) but is inconsistent with reports concluding that MPII is present in two spin states in the neutral pH range (15,16). Finally, the helical arrangement of the polypeptide has also been inconsistently described as a random coil ( 11) and as maintaining the helical structure which it has in the parent cytochrome (12,13).…”

“…Cys 14 and cys 17 provide the sulfurs for the thioether linkages and the imidazole ofhis18 is coordinated to the iron as a fifth ligand (10) (Figure 1). No xray structure has been determined but the undecapeptide has been characterized by visible optical spectroscopy, circular dichroism (8,9,11,12), NMR (13) and Moessbauer (14)(15)(16). These studies have shown that MPll is monomeric at acid pH and polymeric at alkaline pH, the state of aggregation being dispersed however, by coordination of the iron to a sixth ligand or by dilution to the mM range (8).…”

Resilience of the Iron Environment in Heme Proteins

“…Mössbauer spectroscopy may be used to measure the values of the electron density, the electric field gradient, and magnetic field of the iron atom in heme proteins, which can provide information about the symmetry and strength of the covalent bonds with the iron atom. (33,(73)(74)(75)(76)(77)(78)(79)(80)(81)(82) Previously published investigations (22,32,(83)(84)(85) also used Mössbauer spectroscopy to determine the Fe MSD…”