Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein

462

Biochimica et Biophysica Acta, 578 (1979) 462--475 © Elsevier/North-Holland Biomedical Press

BBA 38200

OXIDATION-REDUCTION REACTIONS OF COPPER-THIOLATE CENTRES IN Cu-THIONEIN HEINZ RUPP a, RICHARD CAMMACK a, HANS-JURGEN HARTMANN b and ULRICH WESER b

a University o f London, King's College, School o f Biological Sciences, 68 Half Moon Lane, London S.E. 24 9JF (U.K.) and b Anorganische Biochemie, Physiologisch-chemisches Institut der UniversiM't Tiibingen, Hoppe-Seyler-Str. 1, D~7400 Tiibingen 1 (F.R.G.) (Received November 27th, 1978)

Key words: Redox reaction; Copper thiolate center; Cu-thionein; Metallothionein; ESR

Summary Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents. At pH 0.2 the copper was released, b u t no EPR signal from Cu(II) was observed, unless air was present. Optical experiments did n o t detect any disulphide groups which might have been formed during anaerobic release of copper. The mercurial, p-hydroxymercuribenzoate caused the release of EPRdetectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating. No reduction of the copper-thiolate units in Cu-thionein by ascorhate was detected. Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation. The former oxidizing agent required a lower oxidationreduction potential (+850 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible. The EPR signal from Cu(II) oxidized b y hexachloroiridate(IV) resembled that produced by p-hydroxymercuribenzoate in air, suggesting that the copper was released from its thiolate ligands. It is concluded that the EPR non-detectable copper in the native protein is Cu(I). Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper.

Introduction In the metal-sulphur proteins, the metal ions are co-ordinated to eysteine residues, or to inorganic sulphur. At present, the iron-sulphur proteins are the * Address for correspondence.

463 only ones whose structure is well characterized. Their biological function is in electron transfer [1]. The known iron-sulphur centres consist either of the [1Fe] type (one iron atom bound to four cysteine-sulphur atoms), the [2Fe2S] type (two iron atoms attached to two labile sulphide and four cysteinesulphur atoms) or the [4Fe-4S] type (cube-like arrangement of four iron atoms bound to four labile sulphide and four cysteine-sulphur atoms) [2]. In contrast, little is known about the class of sulphur-rich proteins which can bind zinc and copper [3--11]. The corresponding proteins from yeast were shown to bind copper specifically [3--7], whereas the proteins from higher organisms contain copper and/or zinc under physiological conditions [8--11]. Metal ions with a high affinity for thiol groups such as cadmium and mercury can compete for the binding sites in the protein. The affinity of chicken metallothionein [12-14] for metal ions increases in the order Zn-* Cd -* Cu [15--17]. The heavy metal ion cadmium was actually the clue to the discovery of this group of sulphur-rich proteins [18]. The name metallothionein was chosen reflecting the high content of metal and thiolate sulphur [19,20]. As an example of the Zn, Cu-sulphur proteins, we studied yeast Cu-thionein. The copper atoms in this protein appear to be in a different environment from those involved in electron-transferring copper proteins, as judged from the known examples containing diamagnetic copper [21]. In all the blue copper proteins, cysteine residues are most probably involved in the co-ordination of copper, but additional ligands such as histidine and methionine are thought to be essential for the catalytic mechanism [22]. Cu-thionein contains no aromatic amino acid residues. Based on the stoichiometry of one copper to two cysteine residues, a structure has been proposed consisting of tetrahedral Cu(I) sites with bridging cysteine sulphurs [17]. Since the iron-sulphur proteins are involved in electron transport reactions, a similar oxidation-reduction reactivity might be expected for the coppersulphur proteins. In this paper the oxidation-reduction properties of yeast Cuthionein [4] are further characterized. Changes in the oxidation state of the copper-sulphur system result in different magnetic properties and were therefore monitored by EPR spectroscopy. As normally prepared the protein shows only small signals due to Cu(II). The remainder of the copper may be either spin-coupled Cu(II) pairs, an interpretation favoured by Winge et al. [23], or diamagnetic Cu(I), as proposed by Rupp and Weser [15]. Experimental

Chemicals. Sephadex G-25, G-50 and QAE A-25 were from Pharmacia (Uppsala, Sweden), DE-23 DEAE-cellulose from Whatman (Maidstone, U.K.), Bio-Gel P-10 from Bio-Rad (Richmond, CA, U.S.A.) and Katalysator R3-11 from BASF (Ludwigshafen, F.R.G.). p-Benzoquinone and 1,2-naphthoquinone were from Koch-Light (Colnbrook, U.K.) and 2,5-dimethyl-p-benzoquinone from Eastman Chemicals (New York, U.S.A.). Hexachloroiridic(IV) acid 6-hydrate was a gift from Degussa (Hanau, F.R.G.). All other chemicals were of analytical grade. Quartz-distilled water (conductivity less 0.5 uS) and polyacrylic ware were used. Preparation of yeast Cu-thionein. Saccharomyces cerevisiae was grown as

464 previously described [3]. After fermentation, the cells (250 g) were ruptured in a Gaulin high-pressure homogenizer at 600 bar. The homogenate was centrifuged at 4000 X g for 1 h. Following filtration, the supernatant was heated to 60°C for 3 min. The heat treatment was carried o u t in a reaction vessel equipped with a thermometer, stirrer, and an argon flow-through system. The argon was purified b y passing through a column loaded with Katalysator R3-11. All further steps were carried o u t at 4°C under argon. Following centrifugation at 4000 X g for 30 min the supernatant was applied to a Bio-Gel P-6 column (8 X 60 cm) equilibrated with 50 mM Tris-HC1 buffer, pH 8.0. The coppercontaining fraction of apparent molecular weight of 10 000 was adsorbed onto a Sephadex QAE A-25 column (3 X 20 cm) and eluted with a linear gradient (5--400 mM NaC1 in 50 mM Tris-HC1 buffer, pH 8.0). The fraction which was eluted with 100 mM NaC1 was concentrated by ultrafiltration (Amicon UM-2 membrane) and rechromatographed on a Bio-Gel P-10 column (4 X 100 cm). Cu-thionein remained homogeneous during polyacrylamide gel electrophoresis and gel filtration. Further analytical data were given in [3,6,17]. Analytical procedures. Protein concentration was determined gravimetrically [13]. Copper, cadmium and zinc were quantified b y atomic absorption spectroscopy using a Perkin Elmer 400 S unit equipped with a HGA-76-B graphite cuvette. Absorption spectra were recorded on a Unicam SP 1800 spectrophotometer. Circular dichroic spectra were measured using a JASCO 20A recording spectropolarimeter (Japan Spectroscopic Company, Tokyo, Japan). EPR spectra. EPR spectra were recorded on a Varian E4 spectrometer (Varian Associates, Palo Alto, CA, U.S.A.). Low temperature studies were performed using either an insert liquid nitrogen dewar or a liquid helium transfer system (Oxford Instruments, Oxford, U.K.). For quantitative measurements the samples were prepared in quartz tubes matched for internal diameter. Copper-EDTA was used as a standard. EPR spectra were recorded digitally on a Nicolet 1020A oscilloscope (Nicolet Instrument Corp., Madison, WI, U.S.A.) interfaced to a HP 9830 calculator (Hewlett Packard, Inc., Palo Alto, CA, U.S.A.). The double integration was carried o u t numerically with a correction for a slope in the baseline. A correction for the dependence of transition probability on g value was carried o u t [24]. Anaerobic experiments were carried o u t under argon which was purified by passing it through a Nilox apparatus (Jencons Scientific Ltd., Hemel Hempstead, U.K.) containing alkaline sodium dithionite solution. Stainless steel tubing was used for all gas lines. Oxidation-reduction potential titration. Oxidation-reduction reactions of Cu-thionein under conditions of controlled oxidation-reduction potential were followed b y measuring the intensity of the EPR signal at 77 K as a function of the potential. A vessel containing a platinum and a calomel electrode was used [25,26]. The potential was adjusted with small additions of hexacyanoferrate(III) or hexachloroiridic(IV) acid and ascorbic acid or dithionite solution. Mediators present were: p-benzoquinone (E~ = +286 mV), 2,5-dimethyl-pbenzoquinone (E~ = +180 mV) and 1,2-naphthoquinone (E~) = +145 mV). Following equilibration for 3 min at a particular potential, the sample was withdrawn under argon, and frozen and stored in liquid N2. Similar results were

465

obtained after equilibration for I min. All titrations were carried o u t in 50 mM Tris-HC1, pH 8.7, at 25°C. The concentration of Cu-thionein in the titrations was 30--40/~M and the concentration of each of the mediators was 50 p M. Potentials are expressed relative to the standard hydrogen electrode. Results

Optical properties Cu-thionein from yeast (13 gatoms C u l l 0 000 g) exhibits a shoulder in the electronic absorption spectrum at 270 nm, tailing o f f into the visible region w i t h o u t further absorption maxima. The corresponding circular dichroic spectrum shows a positive Cotton band at 245 nm, a negative band at 283 nm, a shoulder at 302 nm, and t w o overlapping positive Cotton extrema at approximately 328 nm and 359 nm. In the circular dichroic (Fig. l d ) and electronic spectra (Fig. l b ) of the apoprotein prepared b y displacing copper with protons, no transitions due to disulphide chromophores could be detected. By contrast, the oxidized apoproteins prepared using either dialysis against a solution of diethyldithiocarbamate [3] or boiling in the presence of 10 mM EDTA [23] exhibited absorption maxima near 260 nm and 265 nm, respectively. For oxidized chicken thionein (cystine-thionein), a maximum was also observed near 255 nm [13] and circular dichroic bands at 282.5 nm and 260 nm [13,17]. We can, therefore, conclude that the H*-induced dissociation of copper results in cysteine-thionein. There is no evidence that the dissociation is

32

200 10o C

r'C",24 i

0

A0C M ×

-200'-'-'

8 J

t

250

"

.....

300 V~bvelength (nm)

350

400

Fig. 1. (a, b) e l e c t r o n i c s p e c t r a , a n d (c, d) c i r c u l a r dichroic spectra of Cu-thionein; (a, c) n a t i v e protein in 50 m M Tris-HCI, p H 8.7; (b) in 1.6 N HC1 a n d (d) a t p H 0.2, a d j u s t e d w i t h c o n c e n t r a t e d HC1; s a m p l e s (a) and ( b ) w e r e k e p t u n d e r argon; protein c o n c e n t r a t i o n was 0 . 0 3 rag/m1' for (a, b), a n d 0 . 1 4 m g / m l ' for (c, d). M e a s u r e m e n t s w e r e m a d e i n q u a r t z cells o f l i g h t - p a t h 0.2 c m for (a, b), s ~ d 1.0 c m a n d 0.1 c m for (c, d), respectively.

466

accompanied by the following possible intramolecular electron transfer: [(RS-).Cu(II)] ~

n

~ RSSR

+

Cu(I)

The dissociation of half of the copper ions by H ÷ occurs at pH 0.44, an unusually low pH value compared to other known copper or iron proteins. The high stability of metal binding in Cu-thionein is also shown by the unchanged circular dichroic properties of Cu-thionein in 5 M urea or 30 mM sodium dodecylsulphate. Heating under anaerobic conditions at 80°C for 5 min in the presence of these agents did not destroy the metal clusters. However, in the presence of oxygen the dichroic amplitudes of the extrinsic Cotton effects decreased and a shoulder near 260 nm appeared, possibly due to disulphide chromophores. It is noteworthy that an equimolar concentration of ascorbate did not affect the circular dichroic spectrum of the protein, as prepared. The optical data, therefore, show that in Cu-thionein, both metal and ligand atoms are in the reduced form. g - value 2.4

i

22

2.0

r

(c)

(b)

Ca)

o' 6

o'28

o'3o

Magnetic f i e l d

&2

J34

(T)

Fig. 2. E P R spectra of Cu-thionein under anaerobic conditions (a) in 3 N HCI, (b) at p H 12.5. After admission of air, (c) in 3 N HCI, (d) at p H 12.5; spectrum (c) corresponds to 9 5 % and spectrum (d) to 7 9 % of total copper present. Conditions of measurement: temperature 77 K, modulation amplitude I roT, microwave frequency 9.15 GHz, microwave p o w e r 20 roW.

467

EPR properties of Cu-thionein, as prepared In various preparations of Cu-thionein from yeast obtained by either heat treatment under anaerobic conditions or (NH4)2SO4 fractionation [3], the amount of EPR-detectable copper at 77 K varied from 1% to 12% of total copper present. At lower temperature the signal intensity decreased owing to power saturation, and no additional features, as might be expected from Cu(II) undergoing fast electron spin relaxation, were observed.

Chemical displacement of copper A way of probing the oxidation state of copper in Cu-thionein is by releasing it with acid under anaerobic conditions. This method [27] revealed the presence of cupric copper in tyrosinase and cuprous copper in haemocyanin, both being in full agreement with magnetic susceptibility data. Under strictly anaerobic conditioris, Cu-thionein in 3 N HC1 did not show any EPR signal (Fig. 2a) attributable to possibly released cupric copper. On aeration of the sample a signal appeared vchich corresponded to 95% of the total copper (Fig. 2c). Alkaline

26

g -value 22

24

2.0

(d)1 (b) (e) QI 6

I

Oi

0.28 3(3 Magnetic field ( T )

i

I

032

Q34

Fig. 3. E P R s p e c t r a o f C u - t h i o n e i n in 5 0 m M T r i s - H C l , p H 8.7 (a) u n d e r a r g o n ; (b) in t h e p r e s e n c e o f air; (c) a f t e r a d d i t i o n o f p - h y d r o x y m e r c u r i b e n z o a t e u n d e r a r g o n ; a n d ( d ) a f t e r a d m i s s i o n o f air t o s a m p l e (e); t h e m o l a r r a t i o o~ p - h y c t r o x y m e r c u L r i b e n z o a t e t o c o p p e r o f C u - t h i o n e i n w a s 5; s a m p l e s (b) a n d (d) w e r e k e p t f o r i d e n t i c a l t i m e s u n d e r a e r o b i c c o n d i t i o n s b e f o r e ~ e e z L n g . C o n d i t i o n s o f m e a s u r e m e n t w e r e as f o r

Fig. 2.

468

pH values had a similar effect to HC1 on Cu-thionein. Thus, copper was not detectable b y EPR at pH 11--13 under anaerobic conditions. Aeration of the sample resulted in the slow formation of an EPR signal resembling that of a copper-biuret complex in alkaline solution (Fig. 2d). Since an oxidation of thiolate to disulphide groups is not involved in the release of copper, as indicated by the optical data, we conclude that the EPR non-detectable form of copper is due to Cu(I) b o u n d to thiolate ligands. This was supported by the observation that p-hydroxymercuribenzoate did not displace EPR-detectable copper under anaerobic conditions (Fig. 3c). Following admission of oxygen to the sample, Cu(II) could once again be detected by EPR (Fig. 3d). We attribute these effects to an oxidation b y oxygen of Cu(I) released b y HC1 or p-hydroxymercuribenzoate. In the absence of oxidizing agents or thiol reagents, the copper-thiolate clusters in the protein were exceptionally stable.

Effect of EDTA Under anaerobic conditions EDTA (10 mM) did not release copper in an EPR-detectable form at 0°C (Fig. 4d) whereas at 100°C (5 min), 42% of the g -value 22 i

2.1 i

20 i

(f]

(d)

(a)

i

Q30

~3~ Magnetic

0'.32

~33

0 .'3 4

field (T)

Fig. 4 . E P R s p e c t r a o f C u - t h i o n e i n i n 5 0 m M Tris-HCI, p H 8 . 7 . I n t h e a b s e n c e o f E D T A : (a) a t 0 ° C , u n d e r a r g o n ; (b) k e p t at I O 0 ° C f o r 5 r a i n , u n d e r a r g o n ; (c) a t 1 0 0 ° C , f o r 5 r a i n , u n d e r air. In t h e p r e s e n c e o f 1 0 m M E D T A : (d) 0 ° C , u n d e r a x g o n ; (e) 1 0 0 ° C , 5 r a i n , u n d e r a r g o n ; (f) 1 0 0 ° C , 5 mix*, u n d e r air. C o n d i t i o n s of m e a s u r e m e n t w e r e a s for Fig. 2.

469 total copper was detectable (Fig. 4e). Introduction of oxygen increased the a m o u n t of detectable copper to 56% (10 mM EDTA, 100°C, 5 min) (Fig. 4f). By contrast, in the absence of EDTA the signal of Cu-thionein heated at 100°C for 5 min was only very small (2%) and was increased in the presence of oxygen to 7% (Fig. 4b and c). The presence of EDTA, therefore, alters the reactivity of Cu-thionein with oxygen and the release of copper during heat treatment. It is, therefore, n o t justified to add EDTA as a routine procedure to Cu-thionein for quantifying copper with EPR b y referring to a copper-EDTA standard.

Effect of H202 Cu-thionein showed a sluggish reaction with H202. Thus, H202 at a ten-fold excess over copper in Cu-thionein had no effect on the EPR properties after 1 min, whereas after 30 min 52% of total copper appeared as EPR-detectable Cu(II). However, at a lower H202 to copper ratio (1 : 1 and 1 : 2) no significant EPR signals were observed. Furthermore, Cu-thionein in the presence of a high concentration of H202 (10%, b y vol.) did not show any EPR signal. Oxygen had no detectable effect on the reactivity of H202.

Oxidation reactions with hexachloroiridic(IV) acid In Cu-thionein b o t h the metal ions and the ligands are potential electron donors/acceptors. For following oxidation-reduction reactions of Cu-thionein, we used a potentiometric titration m e t h o d [25]. The oxidizing and reducing agents used should ideally carry o u t their electron accepting or donating function w i t h o u t binding either to the oxidized or reduced site, as this would result in a change of the oxidation-reduction properties. As oxidizing agent we employed hexachloroiridate(IV) which was successfully used for the titration of laccase [28], and as reducing agent we used ascorbate. The oxidation of the copper b y hexachloroiridate(IV) occurred in t w o stages, characterized b y different EPR spectra. The first oxidized species is represented by the spectrum at +217 mV (Fig. 5a). On further oxidation it changed to that illustrated at +339 mV (Fig. 5d). The spectra at +257 mV and +278 mV consist of mixtures of these t w o forms. The sizes of the t w o overlapped signals were measured from the amplitudes of the characteristic features given in Fig. 5. The low signal to noise ratio of the spectrum did n o t allow a quantitative evaluation of the respective signals in the g~ region. The spectral features outlined in'Fig. 5 were plotted against the oxidation-reduction potential (Fig. 6). The size of the signal due to the first species was maximal around +250 mV while the second was half-maximal at +290 mV and showed no further increase above +350 mV. Since the spectral features were overlapped, the a m o u n t of the first oxidized species did apparently not decrease to zero at high potentials. The respective points were, therefore, n o t given in Fig. 6. These oxidation processes, required the presence of mediators which presumably assisted the transfer of electrons from the copper sites. On reversal of the titration, reducing the sample with ascorbate, the signal did n o t disappear until lower potentials were used. The same effect was observed during titrations regardless, whether equilibration times of 1 min or 3 min were used. This result indicates that the characteristic potentials for oxidation might n o t represent

470

24

i

i

g- value 22

i

i

20

i

i

(d)

0.126

0.128 01.30 Magnetic field (T)

QI32

0134

Fig. 5. EPR s p e c t r a of C u - t h i o n e i n in 50 m M Tris-HCl, p H 8.7, o x i d i z e d w i t h h e x a c h l o r o i r i d a t e ( I V ) ; (a) at a p o t e n t i a l of + 2 1 7 m V ; (b) + 2 5 7 m V ; (c) + 2 7 8 m V , a n d (d) + 3 3 9 i n V . C o n d i t i o n s o f m e a s u x e m e n t w e r e as f o r Fig. 2.

[]

~3 b

i

i

÷200

a

i

i

÷ 300 Redox potential (mV)

i

* 400

Fig. 6. O x i d a t i o n - r e d u c t i o n p o t e n t i a l t i t r a t i o n c u r v e s f o r C u - t h i o n e i n in 50 m M Tris-HC1, p H 8.7 a t 25°C; t h e o x i d a t i o n - r e d u c t i o n p o t e n t i a l s w e r e a d j u s t e d using h e x a c h l o r o i r i d a t e ( I V ) as o x i d i z i n g a n d a s c o r b i c acid as r e d u c i n g a g e n t ; f o r t h e h e x a c h l o r o i r i d a t e t i t r a t i o n d a t a t h e p o i n t s ( s , o) w e r e d e r i v e d f r o m t h e h e i g h t s o f t h e t w o signal f e a t u r e s m e a s u r e d as s h o w n in Fig. 4. T h e c u r v e s f i t t e d t o t h e p o i n t s w e r e c a l c u l a t e d a s s u m i n g c h a r a c t e r i s t i c p o t e n t i a l s of + 2 4 2 m V f o r t h e a p p e a r a n c e , a n d + 2 4 7 m V f o r t h e disa p p e a r a n c e , o f t h e first c o m p o n e n t ( a ) a n d + 2 9 0 m V f o r t h e a p p e a r a n c e of t h e s e c o n d c o m p o n e n t (o).

471 true reversible midpoint potentials, b u t may be accompanied b y other irreversible changes. The results were reproducible in three titrations with hexachloroiridate(IV). It is n o t e w o r t h y in this c o n t e x t that also for the oxidation of tyrosinase no quantitative relationship was observed, although the reduction process showed an ideal behaviour [27]. The spectra of oxidized Cu-thionein (Fig. 5) are markedly different from those of the blue copper proteins. The nuclear hyperfine splitting constant [A~ I = 0.020 cm -1 of Cu-thionein, poised at +257 mV, is large compared to [Aul of 'blue' copper which is in the order of 0.003--0.009 cm -1 [21]. Based on the EPR criteria proposed by Peisach and Blumberg [29], we have to conclude that a co-ordination in the form 2S and 2N or 4S does not occur in Cu-thionein oxidized with hexachloroiridate(IV).

Oxidation reactions with hexacyanoferrate(III) Hexacyanoferrate(III) as an oxidizing agent showed a more complex reactivity than hexachloroiridate(IV) acid. In the first place, the oxidation of Cu-thionein occurred at higher oxidation-reduction potentials using hexacyanoferrate(III). In the potential range from +140 mV up to +295 mV no significant EPR signal, due to copper, was detected. The molar ratio of hexacyanoferrate to copper in Cu-thionein was 0.9 at potential +295 mV. Above +410 mV the EPR spectra gradually changed, the oxidation involving at least three different spectral species (Fig. 7). An isotropic line (g -- 2.15) was observed as the final specg-volue 26

2.4

o.' 6

22

20

..•

(e)

- ~

(c)

0.% Mognetic field (T)

Fig. 7. E P R s p e c t r a o f C u - t h i o n e i n in 5 0 m M T r i s - H C l , p H 8 . 7 , o x i d i z e d w i t h h e x a c y a n o f e r r a t e ( I I I ) ; (a) at a p o t e n t i a l o f + 3 9 9 m V ; (h) + 4 1 1 m V ; (c) + 4 2 7 m V ; (d) + 4 4 1 m V ; (e) + 4 5 4 m V . C o n d i t i o n s o f m e a s t t r e m e n t w e r e as f o r Fig. 2.

472 trum for a sample poised at +454 mV (Fig. 7e). The integrated areas of the spectra (Fig. 7b--e) corresponding to +411 mV, +427 mV, +441 mV and +454 mV were all the same within experimental error. Thus, the overall oxidation occurred within a maximum of 120 mV. The back titration with dithionite resulted only in a decrease of the size of the isotropic line. At +229 mV the .g = 2.15 signal was reduced b y a factor of 0.5. A similar behaviour was found in experiments where the Cu-thionein was oxidized with stoichiometric amounts of hexacyanoferrate(III). A hexacyanoferrate(III) to copper ratio of 2 : 1 resulted in EPK spectra which were similar to the spectrum of Cu-thionein poised at potential +441 mV (Fig. 7d). The integrated intensity accounted for 77% of total copper present. Isotropic signals (g = 2.15) were observed for samples containing an excess of hexacyanoferrate(III) (4 mol and 10 tool hexacyanoferrate/per mol copper). In order to clarify further the interaction of Cu-thionein with hexacyanoferrate, the oxidation-reduction potential was adjusted with hexacyanoferrate(III) to +295 mV, then the titration was continued using hexachloroiridate(IV). An increase in the oxidation-reduction potential resulted in composite EPR spectra which clearly showed a contribution of an isotropic line typical for Cu2.4

22

g-value

2.0

~

,~'

(e)

(¢)

Ca)

~26

o'28

d~o

~

434

Ivlognetic field ( T )

Fig. 8, EPR s p e c t r a o f C u - t h i o n e i n in 50 mM Tris-HCl, pH 8.7; t h e P o t e n t i a l w a s a d j u s t e d w i t h h e x a c y a noferrate(IIT) t o (a) +295 mV, and t h e n w i t h h e x a c h l o r o l r i d a t e ( l V ) t o (b) +312 rnV, (c) +339 rnV, (d) +367 mV, (e) +385 inV. C o n d i t i o n s o f m e a s u r e m e n t w e r e as for Fig. 2.

473 thionein interacting with hexacyanoferrate (Fig. 8d). At a b o u t +385 mV an abrupt change in the EPR properties was observed. The potential dropped suddenly to a b o u t +320 mV. On readjustment to +385 mV, the spectrum (Fig. 8e) was changed to one characteristic of Cu-thionein oxidized with hexachloroiridic(IV) acid. In addition, the intensity of the signal was considerably increased. Above +385 mV, the intensity remained constant. A possible explanation is that hexacyanoferrate b o u n d to Cu-thionein was released at a b o u t +385 mV, followed by a fast oxidation of free Cu-thionein. Discussion

From the evidence presented in this report and elsewhere [6,15,17], it is concluded that the EPR non
474 sulphurs. Our data indicate that the potentials required to oxidize copper in Cu-thionein are about +150 to +350 mV. These are not true midpoint potentials, however. The reversible potential for Cu(II)/Cu(I), if it could be measured, would almost certainly be higher. The oxidation of Cu-thionein is a relatively complex process; two intermediates were involved with hexachloroiridate(IV) as oxidizing agent, and three with hexacyanoferrate(III). The species observed during the first stage of oxidation by hexachloroiridate(IV) (Fig. 5a) stongly resembles that produced by reacting the thiol ligands with p-hydroxymercuribenzoate (Fig. 3d). This is an indication that oxidation of the protein b y this agent causes release of the copper from its binding site; subsequently it presumably attaches to other ligands on the protein. Apparently, the oxidizing agents employed in this study do not carry out their electronaccepting function without perturbing the molecular structure of the metal binding site. It is indeed well d o c u m e n t e d that hexacyanoferrate(III) has secondary effects on copper proteins. The oxidation-reduction potentials of type 1 copper and the two-electron acceptor in R h u s laccase were found to be dependent on the concentration of hexacyanoferrate [33]. The oxidationreduction potential for type 1 copper was increased by 40 mV and for the twoelectron acceptor by 49 mV, both in the presence of a b o u t three times excess of hexacyanoferrate. Strong binding of hexacyanoferrate was also observed for haemoglobin [34] and plastocyanin [35]. Furthermore, an isotropic signal (g = 2.16) was found for superoxide dismutase treated with hexacyanoferrate(II) at pH 3.0 which was attributed to a binding of hexacyanoferrate(II) to the copper site [36]. It may be noted that a similar t y p e of isotropic line witho u t resolved hyperfine splitting was observed for Cu2(glutathionedisulphide) [37]. In this complex an interaction between t w o copper ions via the disulphide group was assumed. However, we find for this complex an isotropic line with an apparent g value of 2.07. In addition, the peak to peak width is 180 gauss which is markedly greater than the signal of Cu-thionein (100-110 gauss). Therefore, we prefer to interpret the isotropic signal of Cu-thionein, poised at potentials above +450 mV (Fig. 7e) as a binding of hexacyanoferrate to the copper site, with the cyanide anions as bridging ligands. In this respect, it is interesting that the complex (CN)sFe-CN-Cu-(SR)n was found to be involved in the rate
Acknowledgements This study was supported b y a grant from the NATO Research Programme (No. 1256), the U.K. Science Research Council and the Deutsche Forschungs-

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