OBSERVATION OF IRON-STORAGE PROTEIN IN E. COLI BY MÖSSBAUER SPECTROSCOPY

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OBSERVATION OF IRON-STORAGE PROTEIN IN E.

COLI BY MÖSSBAUER SPECTROSCOPY

E. Bauminger, S. Cohen, D. Dickson, A. Levy, S. Ofer, J. Yariv

To cite this version:

E. Bauminger, S. Cohen, D. Dickson, A. Levy, S. Ofer, et al.. OBSERVATION OF IRON-STORAGE

PROTEIN IN E. COLI BY MÖSSBAUER SPECTROSCOPY. Journal de Physique Colloques, 1979,

40 (C2), pp.C2-523-C2-525. �10.1051/jphyscol:19792181�. �jpa-00218558�

JOURNAL DE PHYSIQUE Colloque C2, supplément au n° 3, Tome 40, mars 1979, page C2-523

OBSERVATION OF IRON-STORAGE PROTEIN I N E. COLI BY MOSSBAUER SPECTROSCOPY

E.R. Bauminger, S.G. Cohen, D . P . E . D i c k s o n * , A. Levy, S. Ofer and J . Y a r i v

Raoah Institute of Physios, The Hebrew University, Jerusalem, Israel

"^Department of Biophysics, The Weizmann Institute, Rehovoth, Israel

Résumé.- Des mesures Mossbauer ont été faites à des températures allant jusqu'à 0,08 K sur des échantillons de E. Coli contenant 10 - 1000 ug de57Fe par gramme de cellules. Les résultats indiquent la présence d'un composé dominant de fer magnétiquement ordonné ayant une température de transition de 2,6 ± 0,2 K. Cela suggère l'existence d'un type nouveau de composé de stockage du fer.

Une protéine d'environ 500.000 daltons possédant un noyau de haute densité électronique, probablement constitué par le composé de fer magnétiquement ordonné, a été isolée à partir des bactéries.

Abstract.- 57Fe Mossbauer spectra have been measured at temperatures down to 0.08 K in E. Coli cells containing 10 - 1000 yg Fe/1 gram of packed cells. The results indicate the presence of a dominant magnetically ordered iron compound having a transition temperature of 2.6 ± 0.2 K. This provides evidence for the presence of a new type of an iron-storage compound in E. Coli. A protein of approxi- mately 500,000 daltons containing an electron dense core, which is probably the magnetically ordered iron compound, has been isolated from the bacteria.

Haem proteins and iron-sulphur proteins have been identified in E. Coli. However, no iron storage proteins have been found in this organism or other bacteria. In this study we present evidence based mainly on Mossbauer measurements for the presence of an iron storage protein, containing macroscopic magne- gnetically ordered iron, in E. Coli.

In a previous study IM Mossbauer spectra we- re obtained from whole cells of E. Coli grown in 57Fe £ enriched media. The 82 K spectra consist mainly of "•

an F e3 + doublet with broad lines. The spectra from g unfrozen cells at 3°C show large motional broadenings t- of up to 25mm/s, indicating diffusion of the domi- o

o nant iron component. In the present work these raea- surements have been extended to very low temperatures 2 using a pumped "He cryostat and a 'He/^He dilution <

refrigerator, in order to g a m more information on oc the nature and relative abundance of the iron com-

pounds in E. Coli.

Bacteria were grown and enriched with Fe as already described, but with glycerol as the carbon source rather than succinate /l/. The spectra change very little between 82 and 3 K but below the latter

temperature the lines begin to broaden and below 2 K there is clear evidence for a six-line magnetically split component (Fig. 1). This component has lines which become broader on going out from the center of the spectrum suggesting that the hyperfine fields at

, . Fig. I :5 Fe Mossbauer spectra of a sample of whole the various iron nuclei are not identical but cover „-ii_ „t v /.„>.• •„,•„.•„„ „ cn n „/ i , j

cells of b. LoLv containing "v 500 mg/cm packed a range of values. cells with ^ 620 ug 57Fe per gram of bacteria, ta-

ken in a range of temperatures. The solid line is

*„ , , . . , , a computer fit to the dominant magnetic component On leave from the University of Liverpool, England. u s i n g a distribution of hyperfine fields about a

mean value.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19792181

c2-524

I n a d d i t i o n , t h e r e i s a n F e 3 + d o u b l e t and an c i a t e d w i t h an i r o n - s t o r a g e compound.

Fe2+ d o u b l e t which have n e g l i g i b l e i n t e n s i t i e s i n

samples w i t h h i g h i r o n c o n c e n t r a t i o n s b u t become ap- _500, 1

p r e c i a b l e i n samples w i t h lower i r o n c o n c e n t r a t i o n s . The s p e c t r a were computer f i t t e d t o t h e s e t h r e e com- p o n e n t s i n o r d e r t o d e t e r m i n e t h e i r h y p e r f i n e p a r a - m e t e r s ( T a b l e I ) and r e l a t i v e i n t e n s i t i e s ( T a b l e 1 1 ) .

T a b l e I

Msssbauer chemical s h i f t s ( r e l a t i v e t o i r o n m e t a l ) and q u a d r u p o l e s p l i t t i n g s of t h e components of t h e E. C o l i s p e c t r a .

T a b l e 11

The r e l a t i v e i n t e n s i t i e s ( 2 ) of t h e components of t h e 0.1 K H s s s b a u e r s p e c t r a of t h r e e samples of E. C o l i w i t h d i f f e r e n t l e v e l s of i r o n i n c o r p o r a t i o n

(pg " ~ e p e r gram of b a c t e r i a ) . Fe '+(xagne t i c a l l y

s p l i t ) F e 3 + ( u n s p l i t ) Fe2+

An i m p o r t a n t f e a t u r e o f t h e s e s p e c t r a i s t h a t t h e r e l a t i v e i n t e n s i t i e s of t h e m a g n e t i c a l l y s p l i t and u n s p l i t p a r t s of t h e s p e c t r a do n o t v a r y w i t h t e m p e r a t u r e , i n d i c a t i n g t h a t t h e y a r i s e from d i f f e - r e n t i r o n com?ounds. The m a g n e t i c component h a s a mean h y p e r f i n e f i e l d which v a r i e s w i t h t e m p e r a t u r e

( F i g . 2) i n a way which c o r r e s p o n d s t o m a g n e t i c orde- r i n g w i t h a t r a n s i t i o n t e m p e r a t u r e o f 2.6 f 0 . 2 K and a s a t u r a t e d h y p e r f i n e f i e l d of 43 2 1 T. Where t h e r e may b e some r e l a x a t i o n e f f e c t s n e a r t o t h e t r a n s i t i o n t h e g e n e r a l b e h a v i o u r c a n n o t b e e x p l a i n e d by s l o w p a r a m a g n e t i c r e l a x a t i o n .

I t c a n b e s e e n from t a b l e I1 t h a t t h e main e f - f e c t o f v a r y i n g t h e i r o n c o n c e n t r a t i o n i n t h e growth medium (and hence t h e i r o n i n c o r p o r a t i o n i n t o t h e b a c t e r i a ) i s t o change t h e r e l a t i v e i n t e n s i t i e s of t h e m a g n e t i c a l l y s p l i t and u n s p l i t components. The m a g n e t i c component i s always p r e s e n t b u t d o m i n a t e s w i t h samples grown u n d e r c o n d i t i o n s o f i r o n e x c e s s . T h i s s t r o n g l y s u g g e s t s t h a t t h i s component i s a s s o -

Temperature (K) 4.1 0 . 0 8

1 . O 4.1

F e 3 + ( ~ a g n e t i c a l l y s p l i t ) Fe 3 + ( u n s p l i t )

2

+

Fe

. .

g

-

TEMPERATURE ( K ) Chemical

s h i f t ( m / ? ) 0 . 5 1 ( 1 ) 0.51 ( 3 ) 0 . 4 3 ( 4 ) 1 . 3 0 ( 1 )

I r o n i n c o r p o r a t i o n

F i g . 2 : The t e m p e r a t u r e v a r i a t i o n o f t h e mean h y p e r f i n e f i e l d f o r t h e m a g n e t i c component of t h e Mossbauer s p e c t r a of E. C o l i .

Quadrupole s p l i t t i n g

(mu/s) +0.70(2) -0. lO(2) C1.12(6) +3.00(2)

2, 620 98 2 I

< 1.5

2 2 1

The o b s e r v a t i o n o f m a g n e t i c o r d e r i n g i m p l i e s t h a t i n t h i s compound t h e i r o n atoms must be r e l a t i - v e l y c l o s e t o e a c h o t h e r and grouped i n c l u s t e r s con- t a i n i n g a s i g n i f i c a n t number of atoms. However, t h e m a g n e t i c a l l y s p l i t component i s q u i t e d i f f e r e n t , b o t h i n terms of i t s t e m p e r a t u r e dependence (no su- p e r p a r a m a g n e t i c b e h a v i o u r ) , and i t s h y p e r f i n e p a r a - m e t e r s , from t h e s p e c t r a of t h e two c l o s e l y r e l a t e d i r o n s t o r a g e p r o t e i n s , f e r r i t i n and h a e m o s i d e r i n , which a r e found i n f u n g i , p l a n t s and h i g h e r a n i m a l s

/ 2 , 3 , 4 / .

The low t e m p e r a t u r e ~ g s s b a u e r s p e c t r a of E.

C o l i p r o v i d e s e v i d e n c e f o r t h e p r e s e n c e of an i r o n - s t o r a g e compound. Mcssbauer measurements c a r r i e d o u t on s e p a r a t e d f r a c t i o n s from E. C o l i show t h a t t h i s compound i s i n s o l u t i o n w i t h i n t h e b a c t e r i a .

A p r o t e i n of l a r g e m o l e c u l a r w e i g h t (%500,000 d a l t o n s ) c h a r a c t e r i z e d by an e l e c t r o n d e n s e c o r e was i s o l a t e d by u s i n p u r e form from a n aqueous e x t r a c t of E. C o l i c o n t a i n i n g 800 mg Fe/ l gram packed c e l l s . I t seems v e r y p r o b a b l e t h a t t h i s i s t h e i r o n - s t o r a g e p r o t e i n d i s c o v e r e d i n t h e M&.sbauer measurements.

The p r o t e i n m o l e c u l e i s b i g enough t o s u g g e s t t h a t t h e d i f f u s i o n broadened s p e c t r u m o b s e r v e d by u s i n E. C o Z i c e l l s k e p t above O'C, which was t h e t o p i c of a p r e v i o u s r e p o r t / I / , o r i g i n a t e s i n t h e aqueous c e l l i n t e r i o r .

Acknowledgements.- The J e r u s a l e m group w i s h e s t o thank t h e S t i f t u n g Volkswagenwerk f o r p a r t i a l f i n a n - c i a l s u p p o r t of t h i s work. D.P.E. Dickson g r a t e f u l l y acknowledges f i n a n c i a l s u p p o r t from t h e Lady Davis F e l l o w s h i p T r u s t and t h e Royal S o c i e t y - I s r a e l Aca- demy Programme. We a r e i n d e b t e d t o U. Schmid f o r

2, 2 7 5 8 2 3

1 5 2 3 27 2 2

?. 14 35 2 6

3 6 2 6 29 2 3

i n v a l u a b l e t e c h n i c a l a s s i s t a n c e .

R e f e r e n c e s

/ l / Bauminger, E . R . , Cohen. S.G., Giberman, E . , Kowik, S . , O f e r , S . , Y a r i v , J . , W e r b e r , M.M. and Mevarech. M . , J.

P h y s i q u e C o l l o q .

21

/ 2 / Boas, J . F . and Window, B., Aust. J . P h y s .

2

1 3 1 F i s h b a c h , F.A., G r e g o r y , D . W . , H a r r i s o n , P.M., J o y , I . G . and W i l l i a r n s . J . M . , J. U l t r a s t r u c t . Res

21

1 4 1 S p a r t a l i a n , K., S m a r r a , N. and O o s t e r h u i s , W.T., A I P Conf. P r o c . ^{N O} 18 (1974) 1326.