382

Biochimica et Biophysica Acta, 5 8 0 ( 1 9 7 9 ) 3 8 2 - - 3 9 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 38298

PREPARATION AND P A R T I A L CHARACTERIZATION OF IRON-SULFUR, IRON-SELENIUM, AND IRON-TELLURIUM COMPLEXES OF BOVINE SERUM ALBUMIN *

SUMIO ARAKAWA and TOKUJI KIMURA

Department o f Chemistry, Wayne State University, Detroit, MI 48202 (U.S.A.) (Received February 20th, 1979)

Key words: Adrenodoxin; Ferredoxin; Iron-sulfur; Selenium: Serum albumin: Tellurium

Summary An artificial Fe-S* protein was prepared by the reaction of bovine serum albumin with FeSO4 and Na2S or with a synthetic Fe-S*-l,4-butanedithiol complex. These improved methods enabled us to characterize the derivatives from serum albumin. The Fe-S* albumin complex has about 20 iron ions and 14 labile sulfur atoms per molecule of the protein, whose absorption spectrum closely resembled that of 2Fe-2S* proteins. Its electron paramagnetic resonance spectrum exhibited signals different from those of ferredoxins. The addition of p-chloromercuriphenylsulfonate quenched the optical absorption in the visible region as well as the electron paramagnetic resonance signals. These properties of the albumin-iron complex are similar to those of iron-sulfur dithiothreitol and mercaptoethanol complexes, suggesting that the albuminiron complex has one or more protein ligands besides sulfur ligands. Presumably, the oxygen atom of the tyrosine residue, or other h y d r o x y a m i n o acids participates in the complex formation. In this context, the albumin polypeptide appears to be incapable of forming an iron-sulfur cluster identical to those of ferredoxins. Yet, from the albumin-iron derivative, the extrusion of the iron-sulfur core with benzenethiol provided products similar to those from ferredoxins. The iron-selenium and iron-tellurium derivatives of the bovine serum albumin were prepared and partially characterized by optical absorption and electron paramagnetic resonance spectroscopies. These results imply that both selenium and tellurium can be incorporated into the protein molecule as the respective labile components. * A part of this investigation was reported at the 6th International Biophysics Congress, September, 1978, Kyoto, Japan. Abbreviation: S*, labile sulfur.

383 Introduction Ferredoxins, which are widely distributed from anaerobic bacteria to mammals, contain either binuclear, [Fe2S*2(Cys)4] 2-, or tetranuclear [Fe4S*4(Cys)4] 2- clusters [1,2]. Rubredoxins, which occur among bacteria, contain a nearly tetrahedral ion-sulfur center, [Fe(Cys)4] 2- [2,3]. A fundamental question is how four cysteine residues of a polypeptide can choose a particular Fe-S* core among these three possible structures. We prepared a rebredoxintype iron-sulfur center by utilizing an apoadrenodoxin polypeptide [4]. This artificial derivative was so unstable at room temperature that it was easily converted to the native 2Fe-2S* complex upon the addition of Na2S. Later, we observed that a reconstituted adrenodoxin from its apoprotein, iron and Na2S had a 2Fe-2S* core, but there was no other iron-sulfur complex as judged from the extrusion m e t h o d with benzenethiol. Thus, the native proteins appear to have a selective mechanism for a particular cluster. In 1967, we reported the preparation and the properties of an iron-sulfur protein from bovine serum albumin [5]. The product displayed a similar optical absorption spectrum to those of ferredoxins. Further characterization of the derivative was subsequently published by Lovenberg and McCarthy [6,7] who distinguished the artificial complex from bacterial ferredoxins, adrenodoxin, and rubredoxin by comparison of their circular dichroism spectra. However, the crucial question of whether Or not the iron-sulfur core is identical to those of ferredoxins remains to be examined. Materials and Methods Bovine serum albumin (less than 0.005% fatty acid) and spinach ferredoxin were obtained from Sigma. Ferredoxin was further purified by DEAE-cellulose chromatography. The ratio of A420 to A276 was 0.46. Other chemicals were obtained from commercial sources.

Preparation of Fe-S* bu tanedith iolcomplex All procedures were performed under an Ar atmosphere. Trace oxygen in Ar gas was removed by the m e t h o d of Meites and Meites [8]. Fe-S* butanedithiol complex was synthesized according to the m e t h o d described by Averill et al. [9]. To a solution containing 3.0 ml of butanedithiol and 2.7 g of sodium methoxide in 50 ml of methanol, 0.4 g of anhydrous ferric chloride in 20 ml of methanol was slowly added. The solution turned reddish violet, and dark green precipitates were formed. 20 ml of a methanol solution, containing 0.16 g of sodium methoxide and 0.17 g of sodium hydrosulfide, were added to the suspension. The solution turned dark brown. It was allowed to stand for 16 h at room temperature and was filtered. The filtrate was used for the following experiments w i t h o u t isolation.

Preparation of Fe-S* albumin complex All procedures were carried out under an oxygen-free Ar atmosphere. To 10 ml of 0.02 M borate buffer solution (pH 8.8) containing 100 mg of bovine serum albumin, 0.09 ml of mercaptoethanol were added under stirring. After 1 h

384 at 22°C, 0.5 ml of 0.1 M FeSO4 and 0.5 ml of 0.1 M Na2S were added to the solution. The resulting solution was passed through a Sephadex G-75 column (coarse, 2.5 × 15 cm) equilibrated with 0.2 M borate buffer (pH 8.8). Two brownish bands were observed in the process of gel filtration. The protein fraction was eluted prior to the second brownish band which was iron-mercaptoethanol complex on the basis of molecular weight. The protein fraction showed clear dark brown color. This procedure is called 'Method I'. Fe-S* albumin complex was alternatively prepared (Method II). 0.5 ml of the methanol solution of Fe-S* butanedithiol complex was added to the albumin solution instead of FeSO4 and Na2S. The reaction mixture turned turbid and then clear dark brown. Gel filtration was performed by the procedure described in 'Method I'. Preparation o f Fe-Se and Fe-Te albumin complexes: These complexes were prepared to 'Method I' except H2SeO3 or K2TeO3 instead of Na2S. Tritation with p-chloromercuriphenyl sulfonate p-Chloromercuriphenyl sulfonate titration was performed in a quartz cuvette with 1.0 cm light path containing a Fe-S* albumin complex solution. To This, 10 p.l of 27 mM p~chloromercuriphenyl sulfonate solution was added by using a syringe under an Ar atmosphere. Absorption spectra were taken after each addition of p-chloromercuriphenyl sulfonate. Core ex trusion: Fe-S* core extrusion was carried o u t under an Ar atmosphere according to the m e t h o d described previously [10,11]. A solution containing 1.0 ml ol benzenethiol in 10 ml of dimethylformamide was deoxygenated by bubbling with Ar gas for 12 h. To a conical glass centrifuge tube containing 4.0 ml of the oxygen-free dimethylformamide solution, 1.0 ml of the Fe-S* albumin solution was anaerobically added. After 50 min, the mixture was centrifuged at 3000 rev./min for 15 min. The supernatant solution was anaerobically transferred to an Ar-filled optical cuvette. The optical spectrum was taken at room temperature. Other methods Protein concentrations were determined by the m e t h o d of Lowry et al. [12]. Iron and labile sulfur were determined by the 1,10-phenanthroline m e t h o d [13] and methylene blue m e t h o d [14], respectively. EPR and optical absorption spectroscopies were carried out using a Varian EPR spectrometer (model E4) and a Cary s p e c t r o p h o t o m e t e r (model 118), respectively. Results and Discussion

Iron and labile sulfur contents o f Fe-S* albumin complex Iron and labile sulfur analyses was performed in the three samples prepared

385 TABLE I ANALYTICAL

DATA OF Fe-S* ALBUMIN Sample No.

Protein Iron Labile sulfur S*/Fe (mol/mol) Fe/protein (mol/mol)

COMPLEX

PREPARED

BY M E T H O D

I

1

2

3

7.3" 10 -s M 1 2 . 7 • 1 0 -6 M 8.8 • 10-6 M 0.69 18

6.7 • 10 - s M 16.1 • 10-6 M 11.2 • 10-6 M 0.70 24

9.1 • 1 0 -S M 16.4 • 10-6 M 10.6 • 10-6 M 0.65 18

by 'Method I'. Molar ratios of S* to Fe and Fe to bovine serum albumin are presented in Table I. S * / F e was below unity, which indicates t hat the sample contains iron complexes w i t h o u t labile sulfur. The iron c o n t e n t was f o u n d to be ap p r o x imatel y 20 mol of iron per mol of protein, which is much higher than those prepared by McCarthy and Lovenberg [7].

Optical absorption spectra of Fe-S* albumin complex The Fe-S* albumin com pl e x synthesized by 'Method I' d e c o m p o s e d slowly u n d er aerobic conditions. Fig. 1 shows the spectral changes as a function of time. At 12 min after the reaction started, the protein eluate from a Sephadex co lu mn showed an absorption m a x i m u m at 415 nm with two broad shoulders at 455 and 590 rim. After 66.5 h of the reaction time, no absorption peak was f o u n d in the range between 350 and 600 nm. The absorbance at 415 nm

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Fig. 1. Optical absorption spectal changes of Fe-S* albumin complex in 0.2 M borate buffer ( p H 8.8) at 2 2 ° C under aerobic conditions. T h e spectra were recorded at (A) 12, (B) 20, (C) 36, (D) 64, (E) 117, and (F) 3 9 9 0 rain after gel filtration of the complex, which was synthesized by M e t h o d I. T h e concentration of iron was 58 p M . Fig. 2. Optical absorption spectra of (A) Fe-S* albumin c o m p l e x prepared by M e t h o d II (the iron concentration = 83 #M), (B) Fe-S* butanedithiol c o m p l e x in methanol (the iron concentration = 120/zM), and (C) aerobically degraded product of (A). T h e spectrum of (A) was recorded immediately after gel filtration. All spectra except (C) were recorded at 2 2 ° C under anaerobic conditions.

386 decreased as labile sulfur contents in this sample decreased. Compared with the stability of ferredoxins, the artificial iron-sulfur protein is much unstable under aerobic conditions. As shown in Fig. 2, the Fe-S* albumin complex prepared by 'Method lI' displays absorption maxima at 278, 315, and 417 nm with broad shoulders at 450 and 490 nm. These optical properties resemble those of the Fe-S* albumin complex prepared by 'Method I'. This similarity can be extended towards native 2Fe-2S proteins which have absorption bands at 320 -350, 410 -420, and 450--460 nm [1].

The titration of Fe-S* albumin with p-chloromercuriphenyl sulfonate In order to examine whether or not sulfur ligand participates in the albumin complex, the derivative was anaerobically titrated with p-chloromercuriphenyl sulfonate by following absorbance changes at 410 and 320 nm. As shown in Fig. 3, the decrease in absorbance were plotted against the amounts of p-chloromercuriphenyl sulfonate added. Linearity of the titration curve held until about I t~mol of p-chloromercuriphenyl sulfonate was added, but after this point a slight deviation from linearity was observed. The titration was completed with 1.46 t~mol of p-chloromercuriphenyl sulfonate per 25 nmol of albumin, containing 495 nmol of iron. The amounts of p-chloromercuriphenyl sulfonate consumed by S 2- was estimated to be 0.69 t~mol based on the mole concentration of labile sulfur in the sample. Thus, 0.77 t~mol of p-chloromercuriphenyl sulfonate (0.77 tLmol = 1.46--0.69 t~mol) should be consumed by cysteine residues of albumin. The number of the titratable cysteine residues per molecule of bovine serum albumin is calculated to be 31 (0.77 ~mol/0.025

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Fig. 3. T i t r a t i o n o f F e - S * a l b u m i n c o m p l e x (Method I ) w i t h p-chloromercu~phenyI s u l f o n a t e . T h e s p e c t r a were measured after each additions of 0.275 ~mol of p-chloromercuriphenyl sulfonate under anaerobic c o n d i t i o n s . S y m b o l s : s~ 3 2 0 n m , cz 4 1 0 n m . T h e a m o u n t o f t h e F e - S * a l b u m i n c o m p l e x u s e d w a s 2 . 5 • 1 0 -2 t ~ m o l o f p r o t e i n o r 0 . 4 9 5 t l m o l o f i r o n .

F i g . 4. E P R s p e c t r u m o f F e - S * a l b u m i n c o m p l e x ( M e t h o d I) a t 7 7 K . T h e c o n c e n t r a t i o n o f i r o n w a s 1 . 6 • 1 0 - 3 M; m i c r o w a v e p o w e r , 2 0 roW; m o d u l a t i o n a m p l i t u d e , 1 2 . 5 G ; t i m e c o n s t a n t , 1 s; f r e q u e n c y , 9 . 0 6 GHz.

387 ~ m o l = 30.8). This implies t h a t 31 o u t o f 35 h a l f - c y s t e i n e residues o f an a l b u m i n m o l e c u l e [15] are t i t r a t e d with p - c h l o r o m e r c u r i p h e n y l s u l f o n a t e . As s h o w n in T a b l e I, a p p r o x i m a t e l y 20 iron ions are b o u n d to the a l b u m i n m o l e c u l e . T h e r a t i o o f t i t r a t a b l e h a l f - c y s t e i n e residues t o iron ions was t h e n c a l c u l a t e d t o be 1.5. This r a t i o d o e s n o t c o i n c i d e with the ratios o f 4, 2 and 1, w h i c h are p r e d i c t e d f r o m t h e i r o n - s u l f u r cores o f Fe(Cys)~, Fe2S*2(Cys)4, and Fe4S*(Cys)4, r e s p e c t i v e l y . T h e s e results suggest a possibility t h a t the Fe-S* core consists o f a m i x t u r e o f t w o or m o r e t y p e s o f iron-sulfur c o m p l e x e s .

EPR spectrum of Fe-S* albumin complex As s h o w n in Fig. 4, Fe-S* c o m p l e x displays E P R signals at g = 4.3 a n d 2.0. T h e l a t t e r signal was splitted at g~ = 1.99 w i t h H~ = 21 gauss and gll = 1.95. Fig. 5 s h o w s a linear d e p e n d e n c e o f E P R signals on m i c r o w a v e p o w e r . U p o n r e d u c t i o n o f this c o m p l e x w i t h d i t h i o n i t e , b o t h signals d i s a p p e a r e d . T h e E P R s p e c t r u m o b s e r v e d d o e s n o t r e s e m b l e e i t h e r t h a t o f r e d u c e d a d r e n o d o x i n or o x i d i z e d h i g h - p o t e n t i a l iron p r o t e i n w h i c h e x h i b i t E P R signals at g; = 1.94 a n d gll = 2 . 0 2 [ 16 ] a n d gl -~ 2 . 0 3 7 a n d gtt = 2 . 1 1 5 [ 17 ], respectively. T h e r e s o n a n c e o b s e r v e d at g = 4.3 with H = 75 gauss m a y result f r o m a t e t r a h e d r a l high-spin ferric ion. T h e E P R s p e c t r u m at t h e region o f g = 2.0 has a close similarity to t h o s e o f i r o n - s u l f u r c o m p l e x e s w i t h thiol ligands such as m e r c a p t o e t h a n o l a n d d i t h i o t h r e i t o l o f which g values are r e p o r t e d t o be gl = 2.01 with H i = 18 gauss, gll = 1.96 [ 2 2 ] , a n d g~ = 2 . 0 0 a n d gll = 1.06 [ 2 3 ] , r e s p e c t i v e l y . It s e e m s t o be likely t h a t h y d r o x y l g r o u p as ligand m a y be r e s p o n s i b l e f o r the E P R characteristics. A l b u m i n m o l e c u l e c o n t a i n s the t o t a l s u m o f 70 residues o f serine, t h r e o n i n e a n d t y r o s i n e [ 24]. WAVELENGTH, 600 } >I- 24 (/) z t,d I-Z

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