EXAFS INVESTIGATIONS ON A N-TERMINAL FRAGMENT OF HUMAN TRANSFERRIN CONTAINING A SINGLE IRON BINDING SITE

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EXAFS INVESTIGATIONS ON A N-TERMINAL FRAGMENT OF HUMAN TRANSFERRIN CONTAINING A SINGLE IRON BINDING SITE

I. Bertini, S. Mangani, L. Messori, S. Mobillo, P. Orioli

To cite this version:

I. Bertini, S. Mangani, L. Messori, S. Mobillo, P. Orioli. EXAFS INVESTIGATIONS ON A N-TERMINAL FRAGMENT OF HUMAN TRANSFERRIN CONTAINING A SINGLE IRON BINDING SITE. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1193-C8-1196.

�10.1051/jphyscol:19868234�. �jpa-00226141�

Colloque C8, suppl6ment au n o 1 2 , Tome 47, dbcembre 1986

EXAFS INVESTIGATIONS O N A N-TERMINAL FRAGMENT OF HUMAN TRANSFERRIN CONTAINING A SINGLE IRON BINDING SITE

I. BERTINI, S. M A N G A N I * * , L. MESSORI, S. MOBILIO* and P.L. ORIOLI

Dipartirnento di Chemica, ~ n i v e r s i t s di Firenze, I-50125 Firenze, Italy

"Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Frascati, C P 13, 1-00044 Frascati, Italy

'"~ipartimento d i Chemica, ~ n i v e r s i t 5 d i Siena, I-53100 Siena, Italy

Transferrins are a group of glycopmteins from different sources, characterized by the a b i l i t y t o bind specifically and reversibly iron(II1) a t two d i s t i n c t s i t e s with somewhat different a f f i n i t i e s ; they zre l-esponsible f o r iron transport from s i t e s of absorption t o s i t e s of stoi-age and utilization.

'he rx2ture, the number and the geometry of the ligands around i m n centers have been the subject of much debate. Spectmscopic studies have shown that the two metal s i t e s are very similar and that the metal ligands are probably three oxygen atoms from two tyrosine residues and a water molecule, and two nitrogen atoms fmm histidine imidazole u s 1 . he presence of the "synergistic anion1' ( p h y s i o l q p a l l y HCO o r CO s a ) ' sixth ligand has been demonstrated by. C NIR stu8ies ( 2 ) 3 and by E W S measurements on ovotransferrin (3). A very recent UAFS investigation on the isolated C-terminal and Pi-teminal fr=i~ents of ovotransferrin has shown the metal atoms to have coordination numbers consistent with a six-coordinate e n v i r o m n t ( 4 ) .

By limited proteolysis with themolysin of iron saturated human serum tr-ansferrin (T4bJ 80,000), a single iron binding fragment of YlW 35,000 can be obtained, corresponding to the h1-terminal s i t e ( 5 ) . The possibility of investigating by EXMS a single iron binding s i t e and avoiding superposition of the contributions from both s i t e s t o the X-ray absorption s p e c t m , has p m q t e d t h i s investigation. Comparison with model compounds has been extensively used f o r spectral

interpretation.

Human serum transferrin was purchased from Sigma Chemical Company

and further purified according t o standard procedures ( 2 ) . The

14-terminal fragnent w a s obtained by the procedure reported by

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19868234

C8-1194 JOURNAL DE PHYSIQUE

Lineback-Zins and Brew ( 5) . 14g [ ~ e ( rac-ehpg) ] Fe ( acac ) and 2 '

[Fe(phen) ] (C10 ) were prepared according t o published procedures and 3 4 3

used as model compounds (6).

MAFS spectra of the protein have been recorded by the fluorescence technique a t room temperature a t the PULS X-ray bean l i n e a t the Frascati SR f a c i l i t y i n the energy range 6950-7950 eV. bin f i l t e r on the s c i n t i l l a t i o n detector has been used t o reject the scattering noise.

The s q l e w a s 1.5 i n transferrin N-terminal f r w e n t , buffer Tris-HC1, pI-I 8.5. Several spectra of the protein fragnent were run under the same conditions and averaged. EXAFS spectra of the rnodel compow~ds have been collected i n the absorption mode on finely ground powders.

A l l data have been elaborated with a s e t of computer programs written o r adapted by one of the authors ( 7 ) .

The following abbreviations have been used throu@-~out the text:

TF/ZJ

li-terminal fragnent of human s e m transferrin ehpg

ethylene-bis-(0-hydroxyphenylglycine)

acac

acetyilacetonate - phen

phenantroline RESULTS AND DISCUSSION

Fig. 1 shows the shape of the W S spectrum f o r the protein fragment and the model compound Fe(acac) Similar amplitudes would be expected i f approximately the sane scat2kring atoms were present i n the f i r s t coordination s h e l l of the iron atoms. Close resemblance is observed between the protein and the Fe(acac) EXAFS both i n the overall shape

3 .

and i n the phases s w e s t i n g similar Iron binding s i t e s f o r the two compounds. Hoever a difference exists i n the amplitude.

Fig. 1

Fourier transforms of the EXAFS spectra are reported i n f i g . 2. The radial distribution f o r the protein fr-nt shows two ~najor features.

The larger peak i s due t o baclcscattering from atoms i n the f i r s t

coo$ination s h e l l and the second a r i s e s from atoms up t o approximately

4.0 A fmm the iron ion.

can be determined by a backtransform i n the k space of the isolated corresponding peak i n the Fourier transform. Fig.3 shows the plot of ln(A(k)/A ( k ) ) versus k2 where A(k) and A (k) are the envelope

rn rn

rmctions of the backtransforms of the f i r s t s h e l l f o r the iron f ragnent and the I Fe ( rac-ehpg) 1 -

".

r7-- ^' ; model complex respectively (7).

1 ^{7% intercept} of the regression line i n the origin indicates that the iron coordination nunber is i n

i good approximation the same i n the

-

'x

\-I two systems. A more precise

analysis can be done f i t t i n g the backtransformed X (k) flnction using amplitudes and phases from

- 4

a model complex. This has been

I W . 3 , . ei 5 2 s 7 3 . 7 5 95.

c*.2 c P - - - 2 ,

accomplished backtransforming i n k

space the peak at 1.44 A ^{i n the}

Fourier transform of the protein fr-nt and f i t t i n g the h c t i o n obtained with s i m i l a r data fmm the model complex Fe(acac) i n which the iron is coordinated by s i x oxygen atoms i n a regular octahedral 3 arrwernent. The best f i t , s h m i n Fig.

4 was obtained with a coordination s h e l l of four oxyPgen(nitrogen) atoms at 1.86 A and two oxygen(nitrogen) atoms a t 1.98 A. A complete list of the f i n a l f i t t i n g parameters is reported i n Table I .

Table I I r ,

-

e . 1 5 - -,...be,: ,:s i

i J 3.8 o(F.1) 2.4 O(N)

l? (A) ^{1.86 1.98}

b a2 (i2) ^0.0035 -

0.0011

- -

Eo (eV) 0.04 9.2

F i t Index R = 0.037

- 0 . 3

2. 5 c. 375 6. 2 5 8. iQi :2.

I< <err-l,

Fig. 4

C8-1196 JOURNAL DE PHYSIQUE

In conclusion our analysis of the W S data based on the comparison with model compounds is consistent with the findings of Garratt e t 61. (4) on ovotransferrin. The i m n ckromophore appears t o be

~ u b s t ~ m t i a l l y the same; the different tranferrins showing always a s i x coordinate environment of low Z atoms.

1 Chasteen, N.D. i n "Advances in Inorganic Biochemistry", . ^{vol. 5,}

(Theil, E.C., Eichorn, G.L., and I l a r z i l l i , L.G., eds.), PP.

201-233, Elsevier, New York (1983).

2 Dertini,I., Luchinat, C., PlTessori, L., Scozzafava, A., Pellacani, G., and Sola, M. (1986) Inorg. Chem. 25, 1782-1786.

3 Schneider, D.J., Roe, A.L., Player, R . J . , andQue, J r . , L. (1984) J . Biol. Chem. 259, 9699-9703.

4 Ga-ratt, R.C., Evans, R.W., I-Iasnain, S.S., Lindley, P.F. (198G) Biochern. J . ^{233, 479-484.}

5 Linebaclc-Zins, J., and B r e w , K. (1980) J. Biol. Chern. 225, 708-713.

6 Roof, II.B.Jr. (1956) A c t a C r y s t . 9, 781-786; Baker, J., Engclhardt, L.I.I., Figgis, B.N., and White, A.II. (1975) J.C.S.

U2Jton 530-534; Bayley , P.J.A. , C u m i n s , D. , McKenzie, E.D., and

\:orthington, J.N. (1981) Inorg. kirn. Acta 50, 111-120.

7 IIobilio S., Comin, F., and Incoccia, L., (1982) INFN Internal

lieport.