INVESTIGATION OF BACTERIAL FERREDOXIN BY MÖSSBAUER SPECTROSCOPY

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INVESTIGATION OF BACTERIAL FERREDOXIN BY MÖSSBAUER SPECTROSCOPY

H. Eicher, F. Parak, L. Bogner, D. Bade, G. Kalvius, K. Gersonde, H. Schlaak

To cite this version:

H. Eicher, F. Parak, L. Bogner, D. Bade, G. Kalvius, et al.. INVESTIGATION OF BACTERIAL FERREDOXIN BY MÖSSBAUER SPECTROSCOPY. Journal de Physique Colloques, 1974, 35 (C6), pp.C6-367-C6-370. �10.1051/jphyscol:1974666�. �jpa-00215823�

JOURNAL DE PHYSIQUE Colloque C6, supplément au no 12, Tome 35, Décembre 1974, page C6-367

INVESTIGATION OF BACTERIAL FERREDOXIN BY MOSSBAUER SPECTROSCOPY

H. EICHER, F. PARAK, L. BOGNER, D. BADE and G. M. KALVIUS Physik Department der Technischen Universitat München

D-8046 Garching, James-Franckstr., Germany K. GERSONDE and H. E. SCHLAAK

Abtlg. Physiologische Chemie der Technischen Hochschule Aachen D-51, Aachen, Melatener Str. 211-213, Germany

Résumé. - On a interprété théoriquement les spectres Mossbauer des ferredoxines bactériennes réduites mesurés dans des champs magnétiques externes et à basse température. Les lissages des données sont obtenus à l'aide d'un modèle théorique de l'interaction hyperfine dans lequel chacun des deux groupements fer-soufre de la molécule de ferredoxine réduite a pris un électron dans une orbite du groupe, mais avec l'interaction groupement-groupement totalement supprimée. Les spectres hyperfins ne sont pas d'un type simple à champ effectif et il faut deux paramètres p et ^x pour décrire l'interaction magnétique de l'électron du groupement avec le noyau de fer. On présente en outre des données pour un échantillon cristallin de ferredoxine oxydée.

Abstract. - Mossbauer spectra of reduced bacterial ferredoxin in external magnetic fields and at low temperatures are theoretically interpreted. Fits to the data are obtained with the help of a theoretical mode1 of the hyperfine interaction on which each of the two iron-sulfur clusters in the molecule of reduced ferredoxin has taken up one electron in a cluster-orbit, but with the cluster- cluster interaction completely broken. The hyperfine spectra are not of a simple effective field type and two parameters p and IC are needed to describe the magnetic interaction of the cluster electron with the iron nuclei. In addition data for a crystalline sample of oxidized ferredoxin are presented.

1. Introduction. - In recent years the electronic structure of iron-sulfur proteins have been a major target for investigation by Mossbauer spectroscopy.

Besides an interesting study of a synthetic analog of iron-sulfur proteins [l], the magnetic behaviour of the ferredoxin obtained from the bacteria Clostridium Pasteurianum has lately been reported independently by two groups [2, 31. Their results on oxidized ferre- doxin (Fd,,) and on reduced ferredoxin (Fd,,,) obtained over the temperature range between 250 K and 4.2 K and with external magnetic fields between O and 2 T applied at low temperatures are in good agree- ment. Only qualitative interpretations of the hyper- fine (hf) interaction are given. Especially, the complex spectra of Fd,,, in the presence of external magnetic fields and at low temperatures have not been fitted quantitatively to a proper theoretical Hamiltonian.

I t is the purpose of the present communication to present such a fit for the Mossbauer spectra of Fd,,, taken at 4.2 K in a field of 2 T parallel to the gamma ray direction [3]. In addition some new experimental data will be presented dealing with the measurement of Fd,, in a polycristalline sample at various tempe- ratures. The data proof that the behaviour of frozen samples [2, 31 is essentially the same as that of crys-

talline materials which can be investigated also a room temperature.

2. Magnetic behaviour of Fd,,,. - The active cen- ters of Clostridial ferredoxin contains two clusters of 4 iron and 4 sulfur atoms each. The structure [4] of one such cluster is shown in Fig. 1. ESR and Mossbauer data [3] led to the conclusion that the iron in Fd,, behaves non-magnetically. The cluster as a whole can be described by a common molecular wave function without Kramers degeneracy. In Fd,,, each iron-sulfur cluster takes up one electron which is not located at any particular iron atom, yielding a molecular wave function of the cluster with Kramers degeneracy.

That is to say an effective Spin of S = 112 can be assigned to each cluster. In the absence of an external magnetic field the two clusters couple to a state not having Kramers degeneracy. Applying rather small magnetic fields will break the cluster-cluster interaction.

The data under discussion here were obtained with an applied field of 2 T and the cluster-cluster interaction can be considered to be fully absent. A completely random orientation of the molecules with respect to the direction of the external field is further assumed.

In a forthcoming publication [5] the general theory

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1974666

C6-364 H. EICHER, F. PARAK, L. BOGNER, D . BADE, G. M. KALVIUS AND COLL.

FIG. 1. - Structure of the iron-sulfur cluster. The iron atoms are represented by full circles. The open circles are inorganic sulfur atoms, the circles with cross are sulfur atoms of the

cysteine residues.

of the hf-interaction of the cluster electron will be discussed in detail. Rather than giving the precise mathematical formulation we shall discuss here the physical piinciples involved and the resultant fit to the Mossbauer spectrum. The energy states of the cluster electrons are primarily given by the Coulomb and the spin-orbit interactions. The direct interaction with the external field and the hf-interaction are treated as perturbations. The cluster wave function, centered in the middle of the Fe& cluster has in good approxi- mation T, symmetry. The external field (He,) lifts the Kramers degeneracy and two singletts with a separation of typically AE = 2.6 K (at 4.2 K and He, = 2 T) will result. No relaxation processes shall take place between these two levels. Each singlett then will separately produce a static hf-interaction with the iron nucleus. The contribution of each level has to be weighted by its Boltzmann population. It is not possible to estimate a priori the magnetic hf-field originating from the cluster electron since details of the cluster wave function are not known. One therefore has to use an interaction Hamiltonian derived according to the principles outlined above and then to fit its parameter to the experimentally obtained Mossbauer spectra.

From Fig. 1 one sees that each iron site has in first approximation a C, point symmetry. The C, axis of one particular Fe site forms an angle 0 with respect to the direction of He,. The effective cluster spin will be aligned by the external field. One may then describe the hf-interaction of the cluster electron with the iron nucleus in terms of :

and

where H" and HL are the parallel and perpendicular components (with respect to the C, axis) of the effective magnetic hf-field produced by the two singlett cluster:

wave functions 1 1

>

>.

The C, axis defines the 2 directions. The Mossbauer spectrum observed is thus a function of the three parameters p, IC, and O. Keeping these parameters at a fixed value allows the calculation of the resulting spectrum as a sum of Lorentzians for each hf-transition between the excited and groundstate levels of the iron nuclei. It is assumed that al1 Lorentzians have the same linewidth. Polarization effects due to the fixed direction between He, and the gamma ray wave vector are taken into account on calculating the intensities of the various hf-transitions. As stated earlier, the molecular axes in Our absorber will be randomly distributed with respect to He,. This calls for the summation of the theoretical spectra for O < 8

<

It has to be least squares fitted to the experimental data at 4.2 K and He, = 2 T. Examples are seen in figure 2.

In the experiments a source of 57Co in Rh mounted directly above the absorber had been used. In such a source the interaction with the applied field leads only to a moderate broadening of the emission line. This has been taken into account by assuming a width of

re, ⁼ 1.1 mm/s for each Lorentzian in calculating the theoretical spectrum for any set of parameters. At higher fields, however, such a simple procedure is no longer allowed (e. g. because of polarization effects in the source), and we have thus obtained from fitting high field data at present. It will be necessary to obtain experimental data with the source in a much smaller field. Measurements in this direction are under way.

The electric quadrupole interaction is taken from zero field measurements as 1 3 e2 qQ 1 ⁼ 1.54 mm/s.

Although a small spread in q for the different iron sites in the cluster is clearly noticeable 12, 31, this effect has been considered here only as another source for the increased effective line-width. In addition, the zero field data give no information on the sign of q and fits were thus produced for q > O (Fig. 2a) and q < O (Fig. 2b). Both choices gave satisfactorily agreement to the data. The fit of figure 2a is slightly more favorable.

It leads to the following values for the magnetic parameters

IC = - 6.5 T and p = 12.1 T.

r -

100-

1

absorber consisting of a large number of small crystals

1 with diameters between and mm. Data were

FIG. 2. - Least squares fits to the Mossbauer spectrum of reduced ferredoxin taken at 4.2 K and Hex = 2 T. The fits a ) and b) are obtained for choices of opposite sign of the electric

field gradient (see text).

1 l

The other fit (Fig. 2b) gives K = - 9.2 T and l p = 10.6 T. It should be emphasized that the present

L ^{L L --A-_ A-}

i

fitting routine uses sums of Lorentzians to generate the - ¹² -8 - 4 _{a virn61si}

A

12

theoretical spectra. Since each hf-transition is actually

an overlay of rnany resonance lines at close separation, ^{FIG. 3. -} Mossbauer spectra of a crystalline absorber of

this procedure might be tao crude. It is presently oxidized ferredoxin at 273 K a), 263 K b), 2 5 7 ~ c), 253 K d),

attempted to improve this situation by using a fitting ^{243 K e), 233 K} f ) and 86 K g). The fit to spectrum g is a pure quadrupole interaction (see ref. [3]).

routine based on transmission integrals [6]. This will also allow us to take polarization effects in the source into account.

3. Crystalline Fd,, absorbers. - Mossbauer spec- troscopy on proteins is commonly performed on frozen solutions. Such a procedure always rises the question whether the results obtained are typical for the molecule under physiological conditions. Besides coarse effects like de-naturation which usually can be detected by a large series of measurements on samples of different origin, there may also be more subtle effects present like distortions of the molecule on account of the phase transition water-ice. The latter problem may be investigated by performing Mossbauer spectroscopy on crystalline samples of the protein near room tem- perature. Although the f-factor of protein crystals is usually rather low [7] such experiments can be per- formed with sufficient accuracy with the strong 57Co sources now available.

obtained in the temperature range between 273 and 86 K. The mother liquid of the crystals freezes at 253 K. The spectra of figure 3 show no substantial change upon the transition liquid-ice, indicating that severe distortions in the molecule will not occur at this point. The accuracy of the data is somewhat limited by the fact that only very small crystals could be obtained. Thus some Doppler broadening of the resonance lines arising from the Brownian motion of the small crystals in the mother liquid is clearly visible above the freezing point. A further reduction of the water content in the samples was not possible since this favoured denaturation effects. Attempts are continued to produce large crystals of both Fd,, and Fd,,,.

This work was supported by the Deutsche Forschungs Gemeinschaft.

H. EICHER, F. PARAK, L. BOGNER, D. BADE, G. M. KALVIUS AND COLL.

References

[l] FRANKEL, R. B., HERSKOVITZ, T., AVERILL, B. A., HOLM, [4] ADMAN, E. T., SIEKER, L. C. and JENSEN, L. H., 3. Biol.

R. H., KRUSIC, P. J. and PHILLIPS, W. D., Biochem. Chem. 248 (1973) 3987-3996.

Biophys. Res. Comm. (1974), in press. [5] _EICHER, H., PARAK, F., BOGNER, L. and GERSONDE, K.

121 THOMPSON, C. L., JOHNSON, C. E., DICKSON, D. P. E., (1974) to be published.

CAMMACK, R., HALL, D. O., WESER, U. and RAO, K. K.,

Biochem. J. 139 (1974) 97-103. [6] SHENOY, G. K., FRIEDT, J. M., MALETTA, H., RUBY, S. L., 131 GERSONDE, K., SCHLAAK, H. E., BREITENBACH, M., PARAK, F., in Mossbauer E m t Methodology 1974 (to be published).

EICHER, H., ZWRZALLA, W., KALVIUS, G. M. and [7] PARAK, F. and FORMANEK, H., Acta Cryst. A 27 (1971) MAYER, A., Eur. J. Biochem. 43 (1974) 307-317. 573-578