Low-Spin Ferriheme Models of the Cytochromes: Correlation of Molecular Structure with EPR and Mössbauer Spectral Parameters

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1 Hyperfine Interactions 156/157: , Kluwer Academic Publishers. Printed in the Netherlands. 285 Low-Spin Ferriheme Models of the Cytochromes: Correlation of Molecular Structure with EPR and Mössbauer Spectral Parameters T. TESCHNER 1,A.X.TRAUTWEIN 1,, V. SCHÜNEMANN 1, L. A. YATSUNYK 2 andf.a.walker 2 1 Institute of Physics, University of Lübeck, Lübeck, Germany 2 Department of Chemistry, University of Arizona, Tucson, AZ , USA Abstract. The magnetic Mössbauer spectra of a series of low-spin ferriheme complexes have been investigated and compared with their EPR spectral parameters and molecular structures. To date there has been little systematic analysis of either estimated or fitted values of the hyperfine coupling constants for low-spin ferriheme centers and no meaningful correlation has been established between the Mössbauer parameters and the axial ligands of such species. With the results of the present study, we have been able to find correlations of molecular structures with iron-orbital splittings, g-tensor values derived from EPR signals and magnetic hyperfine interaction components A zz obtained from magnetic Mössbauer spectra. These correlations should be useful to future workers in the field of heme-containing enzymes. Key words: heme model compounds, cytochromes, molecular structure, magnetic hyperfine interaction, g-tensor, iron d-orbital splitting. 1. Introduction The biological function of six-coordinated heme centers ranges from participation in electron transfer processes of cytochrome-containing systems in inner mitochondrial membranes (e.g. in cytochrome c552 of Nitrosomonas europaea) to enzymatic catalysis like in the case of the cytochromes P450, which play important roles in detoxification in a number of organisms. Bis-histidine-coordinated heme centers are involved in electron transfer in photosynthesis and cell respiration. EPR data for the cytochrome bc 1 complex of mitochondria and the related cytochrome b 6 f complex of chloroplasts show that both of the b hemes of each protein exhibit EPR signals known as the large g max or HALS type (highly anisotropic low-spin, with the g max value 3.2). Model systems have greatly aided the correlation of the structure of heme centers with their spectroscopic properties. Walker, Scheidt and their coworkers have shown that the large g max signal occurs for ferriheme complexes with (d xy ) 2 (d xz,d yz ) 3 electronic ground state when * Author for correspondence.

2 286 T. TESCHNER ET AL. the planar axial ligands are aligned in mutually perpendicular planes [1, 2], and likewise a normal rhombic signal occurs when the ligands are aligned in parallel planes. The arrangement of the planar axial ligands is of particular importance in defining the spectroscopic properties and may be also in estimating the reduction potentials of heme proteins. Several crystal structures of these protein complexes have such a low resolution that determination of the orientation of the axial ligands is not possible. Therefore low-spin ferriheme model complexes with different types and orientations of axial ligands are useful in correlating structural and spectroscopic parameters in heme proteins such as the electron-transferring cytochromes. In this work, three structurally-characterized model heme complexes [3] with 1-methylimidazole axial ligands have been studied by EPR and Mössbauer spectroscopy. 2. Materials and methods Crystals of the heme model complexes were grown by liquid diffusion methods as reported previously; [OMTPPFe(1-MeIm) 2 ] + crystallized in two forms, one with 90 (perpendicular) and the other with 19.5 (nearly parallel) axial ligand dihedral angles [3]. EPR spectra were recorded on a Bruker ESP-300E EPR spectrometer (operating at 9.4 GHz with 100 khz field modulation) equipped with an Oxford Instruments ESR 900 continuous flow helium cryostat. Spectra were obtained for crystalline samples at 4.2 K. Microwave frequencies were measured using a Systron-Donner frequency counter. Mössbauer spectra were recorded using a conventional spectrometer in the constant-acceleration mode. Isomer shifts are given relative to α-fe at room temperature. The spectra obtained at 20 mt were measured in a He bath cryostat (Oxford MD 306) equipped with a pair of permanent magnets. For the high-field spectra a cryostat equipped with a superconducting magnet was used (Oxford Instruments). Magnetically split spectra of paramagnetic samples were simulated in the spin-hamiltonian approximation, otherwise spectra were analysed by least-squares fits using Lorentzian line shapes. 3. Results and discussion In Figure 1 are shown field-dependent 4.2 K Mössbauer spectra of [OMTPPFe(1- MeIm) 2 ] +, the planar axial ligand planes of which are oriented perpendicular to each other [3]. Based upon the large g max (= g z ) value of 3.61, as obtained from EPR measurements, the spread of the magnetically split Mössbauer lines can be simulated with the following magnetic hyperfine coupling tensor elements: A zz =+90.1 T,A yy =+25.2 T,A xx = 16.9 T. For the magnetic splitting the effective hyperfine field, which is the sum of the external field and the internal field, must be considered. Nevertheless, the internal field dominates strongly, and therefore the magnetic splitting of this ferric low-spin species is effectively determined by the large and positive A zz component. The physical basis of both the large g z

3 FERRIHEME MODELS OF THE CYTOCHROMES 287 Figure 1. Mössbauer spectra of [OMTPPFe(1-MeIm) 2 ] + with axial ligand planes rotated by 90 relative to each other [3]. The spectra are simulated in the slow relaxation limit with parameters given in Table I (g-values) and in Table II. Figure 2. Mössbauer spectra of [OETPPFe(1-MeIm) 2 ] + with axial ligand planes rotated by 73 relative to each other [3]. The spectra are simulated in the slow relaxation limit with parameters given in Table I (g-values) and in Table II.

4 288 T. TESCHNER ET AL. Figure 3. Mössbauer spectra of [OMTPPFe(1-MeIm) 2 ] + with axial ligand planes rotated by 19.5 relative to each other [3]. The spectra are simulated in the fast relaxation limit with parameters given in Table I (g-values) and in Table II. value and the large and positive A zz component is the near energy degeneracy of iron d xz and d yz orbitals (V/λ = 0.64), which in turn is caused by the perpendicular arrangement of the axial ligand planes in [OMTPPFe(1-MeIm) 2 ] + [3]. As a consequence of the small orbital splitting, V = E(d yz ) E(d xz ), strong spin orbit coupling within the ferric low-spin (d xy ) 2 (d xz, d yz ) 3 configuration exists, which yields strong orbital contributions to g z and A zz. Opposite to the negative Fermicontact contribution to the internal field, the orbital contribution to A zz is positive which, in the present case, acquires the large value of +90 T. This qualitative line of arguments is supported by quantitative calculations on the basis of Oosterhuis and Lang s [4] crystal-field treatment which correlates g-values, quadrupole splitting E Q, orbital splittings V/λand /λ and A-components and which yields the theoretical value A zz =+91.9 T (Table II). Figure 2 presents field-dependent 4.2 K Mössbauer spectra of [OETPPFe(1- MeIm) 2 ] +, the planar axial ligand planes of which are oriented nearly perpendicular (73 ) to each other [3]. This species falls in the same category as the one described above, with the only difference being that its orbital contributions to g z and A zz are slightly smaller because of its somewhat larger rhombic orbital splitting V/λ(Table I). According to the arguments described above, ferric low-spin heme species with planar axial ligand planes being oriented parallel (0 ) or nearly parallel (19.5 )to each other are expected to exhibit larger rhombic orbital splitting V/λ compared to species with perpendicular (90 ) or nearly perpendicular (73 ) orientation of

5 FERRIHEME MODELS OF THE CYTOCHROMES 289 Table I. g-values and crystal-field parameters for the complexes of this study Complex (i) g-tensor (ii) V/λ (iii) /λ (iii) V/ parallel 0 g xx = [OEPFe(4-NMe 2 Py) 2 ] + [5] g yy = 2.30 g zz = 2.83 nearly parallel 19.5 g xx = [OMTPPFe(1-MeIm) 2 ] + [3] g yy = 2.51 g zz = 2.71 nearly perpendicular 73 (g xx = 1.14) [OETPPFe(1-MeIm) 2 ] + [3] (g yy = 2.00) g zz = 3.27 perpendicular 90 (g xx = 0.63) [OMTPPFe(1-MeIm) 2 ] + [3] (g yy = 1.53) g zz = 3.61 (i) Dihedral angle between the planes of the axial ligands. Reference to structure given in brackets. (ii) The g-values obtained result from EPR measurements for the largest g-value and from Mössbauer spectral fits for the two smaller g-values. (iii) V/λ = E(d yz ) E(d xz ) = g xx /(g zz + g yy ) + g yy /(g zz g xx ); /λ = E(d yz ) E(d xy ) 1/2V/λ = g xx /(g zz + g yy ) + g zz /(g yy g xx ) 1/2V/λ. axial ligand planes. This is indeed the case, as shown in Table I. Consequently, larger V/λ values correlate with smaller orbital contributions to g z and A zz (Tables I and II) and therefore also to considerably smaller overall magnetic hyperfine splitting. The field-dependent 4.2 K Mössbauer spectra (Figure 3)of [OMTPPFe(1- MeIm) 2 ] +, which has axial ligand planes being rotated by 19.5 to each other, indeed proves that this is the case. 4. Conclusions We have shown that investigations of the molecular structures, EPR, and Mössbauer spectra of well-defined low-spin ferriheme model compounds have provided conclusive proof that perpendicular or near perpendicular alignment of planar axial ligands correlates with near-degeneracy of iron d xz and d yz orbitals and with large orbital contributions to g z and A zz. Hence, these species are characterized by g z 3.2 anda zz T. Likewise, species with parallel or near parallel orientation of planar axial ligands are characterized by considerably smaller orbital contributions to g z and A zz. The sequence of increasing dihedral angles (0,19.5, 73,90 ) of axial ligand planes (1-methylimidazole), including literature data for [OEPFe(4-NMe 2 Py) 2 ] +, which has different planar axial ligands (4-NMe 2 Py) [5],

6 290 T. TESCHNER ET AL. Table II. Mössbauer data obtained from the simulation of the Mössbauer spectra for the complexes of this study Complex (i) δ [mm/s] EQ [mm/s] (ii) η A/gN µn [T] parallel [OEPFe(4-NMe2Py)2] + [5] nearly parallel ± ± ± ± 1.5 ( 42.0) (iii) [OMTPPFe(1-MeIm)2] + [3] (2.37) (iii) ( 1.46) (iii) 23.6 ± 0.5 (28.4) (iii) 50.2 ± 0.5 (38.2) (iii) nearly perpendicular ± ± ± ± 2.0( 35.8) (iii) [OETPPFe(1-MeIm) 2 ] + [3] (2.19) (iii) ( 1.07) (iii) 2.4 ± 1.0 (10.0) (iii) 71.4 ± 0.5(71.4) (iii) perpendicular ± ± 0.02 α = ± ± 75.0( 29.5) (iii) [OMTPPFe(1-MeIm)2] + [3] (1.98) (iii) ( 0.62) (iii) 25.2 ± 15.0 (14.9) (iii) 90.1 ± 0.5(91.9) (iii) (i) Dihedral angle between the planes of the axial ligands. Reference to structure given in brackets. (ii) Euler angles between the principle axes systems of the g- and efg-tensor. (iii) Values in brackets are theoretical values obtained from the crystal-field model of Oosterhuis und Lang [4].

7 FERRIHEME MODELS OF THE CYTOCHROMES 291 is directly reflected by the sequence of iron orbital splitting values, V/λ(2.15, 2.44, 1.16, 0.64), g z -values (2.83, 2.71, 3.27, 3.61) and A zz -values (+44.6 T, T, T, T). Thus, we are able to correlate for the first time a series of ferriheme complexes of known molecular structures [3, 5] simultaneously with both EPR and Mössbauer data. Acknowledgement FAW acknowledges the support by the Alexander von Humboldt-Stiftung. References 1. Walker, F. A., Huynh, B. H., Scheidt, W. R. and Osvath, S. R., J. Am. Chem. Soc. 108 (1986), Scheidt, W. R., Kirner, J. F., Hoard, J. L. and Reed, C. A., J. Am. Chem. Soc. 109 (1987), Yatsunyk, L., Carducci, M. D. Walker, F. A., J. Am. Chem. Soc. 125 (2003), Oosterhuis, W. T. and Lang, G., Phys. Rev. 178(2) (1969), Safo, M. K., Gupta, G. P., Walker, F. A. and Scheidt, W. R., J. Am. Chem. Soc. 113 (1991), 5497.