MÖSSBAUER STUDIES OF THE COFACTOR CENTERS OF NITROGENASE

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MÖSSBAUER STUDIES OF THE COFACTOR CENTERS OF NITROGENASE

B. Huynh, E. Münck, W. Orme-Johnsons

To cite this version:

B. Huynh, E. Münck, W. Orme-Johnsons. MÖSSBAUER STUDIES OF THE COFACTOR CEN- TERS OF NITROGENASE. Journal de Physique Colloques, 1979, 40 (C2), pp.C2-526-C2-527.

�10.1051/jphyscol:19792182�. �jpa-00218559�

JOURNAL DE PHYSIQUE Colloque C2, supplément au n° 3, Tome 40, mars 1979, page C2-526

MOSSBAUER STUDIES OF THE COFACTOR CENTERS OF NITROGENASE

B.H. Huynh, E Munck, and W.H. Orme-Johnson

Freshwater Biological Institute, Departement of Biochemistry, University of Minnesota, P.O. Box 100, Navarre, Minnesota 55392, U.S.A.

^Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

Résumé.-Les centres du cofacteur (S •= 3/2) de la protéine MoFe de la nitrogénase ont été étudiés par spectroscopie Mossbauer avec des champs magnétiques appliqués allant jusqu'à 50 kG. L'analyse des résultats montre la présence de six composantes, dont trois sont caractérisées par une cons- tante de couplage magnétique hyperfine positive. Les résultats suggèrent que chaque centre a de 5 à 7 et même plus probablement, 6 atomes de fer, ce qui confirme nos conclusions précédentes éta- blies par analyse qualitative des données de RPE et en supposant que la protéine MoFe possède 30 atomes de fer.

Abstract.- The Mossbauer spectra of the S « 3/2 cofactor centers of the MoFe protein of nitrogenase have been studied in magnetic fields up to 50 kG. Analysis of the data reveals six subsites, three of which are characterized by a positive magnetic hyperfine coupling constant. The results of this study suggest that the cofactor centers contain 5-7, most probably 6, Fe atoms, thus confirming our earlier conclusions which were based on the quantitation of EPR data and on the assumption that the MoFe protein contains 30 Fe atoms.

For the past few years we have investigated is characterized by a local hyperfine coupling cons- the molybdenum and iron containing protein (MoFe tant A (actually the assumption of an A-tensor with protein) of the biological nitrogen fixing system axial symmetry around the electronic z-axis is suf- with Mossbauer and EPR spectroscopy /l,2,3,4/. The ficient).

results obtained for the proteins from A. vinelandii and £. pasteurianum suggest that the MoFe protein contains (30 ± 2) Fe atoms /2/. The assumption that the Debye-Waller factor is the same for all iron sites has led to the conclusion that 12 Fe atoms belong to two identical centers (labeled M) with electronic spin S = 3/2 /3/. We have recently shown that the M-centers are a structural component of the cofactor of nitrogenase /3/.

In figures I and 2 we have displayed the low- temperature Mossbauer spectra of component M of the ! A. vinelandii protein. The spectra were prepared as ! 2 + i described previously by removing components D, Fe

and S /l/. The spectra in figure 1 result from the M = ±1/2 doublet of an electronic system with a cluster spin S = 3/2 /l/. The electronic properties, in particular the EPR data, can be described by H - D[s 2 - 5 / 4 + X(S 2 - S 2)1 + g e s . i l. (1)

e " - z x y -1 o VELOCITY IN nivs For the A.vinelandii protein, D = +5.5 cm- 1,

, „ „,_ , - , « / • / „ .. , _ • c Fig. 1 : Mossbauer spectra o.f component M (the co- X = 0.055, and g = 2.0 \ . For the evaluation of , 6 t . , * « TT - • c A • i

' 6o factor centers) of the MoFe protein from A. vinelan- the Mossbauer data equation (1) is augmented by dii. The data were taken at 4.2 K in a parallel (A) and transverse (B) magnetic field of 600 gauss. The

" •+ •+ zz p,T 2 _ T/T . i •> spectra were obtained from the raw data by subtrac- nf o 12 L. z ting the contributions of components D, Fe +, and S

. 2 _T2 \ ~ i _ O H T' f21 a s de s c riD ed previously /l/. The solid lines in fi- x y n n ' gures 1 and 2 are theoretical spectra generated with . . . , . t. i. • £ *i. u .. t n e parameter set listed in table I. In figure 1A We have assumed that each subsite of the M-centers . •_, . , ° . .

the theoretical spectra of the subcomponents Al (—) A2 ( ) , A3 ( ) , and B ( ) are indicated.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19792182

The electronic spin in equation (2) is the cluster

1 ^{spin S}

312; its expectation values can be computed

V E L O C I T Y IN nM/S

Pig. 2

~Essbauer spectra of component M taken in parallel applied magnetic fields. The spectrum in (A) was taken at 1.5 K while the data in (B) and (C) were recorded at 4.2 K. The contributions of the diamagnetic components D and Fe2+ were removed by simulating their high-field spectra. Component S was assumed to be diamagnetic; since it is a minori-

ty component (6% of total Fe) no appreciable error is made if this assumption is incorrect.

from equation

The features around +3 mm/s in figure I reveal two subcomponents (for a detailed discussion see B.H. Huynh, E.Mi;nck, and W.H. Orme- Johnson, Biochim. Biophys. Acta (1970), in ~ress).

These two subcomponents, in turn, account for about 30-35% of the total ~Gssbauer absorption of component M which leads to the conclusion that the cofactor centers contain 6 Fe atoms.

The solid lines in figures

and

are the re- sult of fitting the data set to equations (I) and

h h h

(21, H

He + Hhf. In figure 1A we have indicated the spectra of the subcomponents. The parameters used are listed in table I. The methodology of data reduction was similar as described earlier 121.

The major results are as follows

the decom- position of the data suggests that each M-center con- tains 6 Fe atoms, in agreement with our earlier, in- dependent, quantitations. 2) The observation of positive and negative hyperfine coupling constants demonstrates spin-coupling. 3) From the extent of level mixing in strong applied fields we find

D

+(6 +

cm-', in good agreement with our earlier EPR results,

5.5 cm-'.

Acknowledgements.- These studies were supported by National Science Foundation Grant PCM-08522 and Na- tional Institutes of Health Grant GM 17170, and Research Career Development Award KO4-GM 70683 (E.M.).

Table I

Spin Hamiltonian parameters used to generate the theoretical curves in figu- res I and 2 from equations (I) and (2). For all simulations, D

cm-', h

0.055, and r

0.30 mm/s (full width at half maximum) was used.

Spectral : ^{Number of} ieQVZ.

^{G(mm/s) b}

Component Fe atoms gg6,,

l 1 1 1 l

1 I l 1 1

I I 1 l 1

A

: ^-144 : ^{-0.73 0.5} ; ^+0.47

l l l l l

A2

: ^{-124 -0.76} 0 +0.47

l 1 l

I

l 1

1 I

j ^+0.38 j

A3

- ^{96 3 +0.47}

I

i

⁷⁶

B

: ^{-0.38 -3 +0.35}

a ~ h e EEG tensors are referred to the frame (x,y,z) defining the electronic zero-field splitting; Vzz is not necessarily the largest component; n

(Vxx - ^Vyy)/Vzz.

b~someric shift at 4.2 K with respect to Fe metal at room temperature.

References

/l/ ~ Z n c k , E., Rhodes, H., Orme-Johnson, W.H., Davis, L.C., Brill, W.J. and Shah, V.K., Biochim. Biophys. Acta 400 (1975) 32.

/2/ Zimermann. R., Mcnck, E., Brill. W.J., Shah, V.K., Henzl, M.T., Rawlings, J., and Orme-Johnson, W.H., Biochim. Biophys. Acta (1979) (in press).

/ 3 /

Rawlings,

Shah, V.K., Chisnell, J.R., Brill. W.J., Zimermann, R., ~ S n c k , E.

and Orme-Johnson, W.H.,

Biol. Chem. 253 (1978) 1001.

/ 4 /

~iinck, E. and Zimmermann, R., "Mgssbauer Effect Methodology", Vol.

(Plenum

Press) 1976, p. 119.