the of vol. 264, no. 15, pp. q by the and molecular …the journal of biological chemistry vol. 264,...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264, No. 29, Issue of October 15, pp. 17091-17099,1989 Q 1989 by The American Society for 13ioehemistry and Molecular Biology, Inc. Printed in U. S. A.

Silver Binding to Rabbit Liver Metallothionein CIRCULAR DICHROISM AND EMISSION STUDY OF SILVER-THIOLATE CLUSTER FORMATION WITH APOMETALLOTHIONEIN AND THE a AND 0 FRAGMENTS*

(Received for publication, February 21, 1989)

Andrzej J. IZelazowskiS, Zbigniew GasynaS, and Martin J. StillmanSQ From the Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada

We report new spectroscopic properties for a range of silver-metallothiomein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apoa and -B fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 “C and 55 OC reveal for the first time the formation of a sequence of clusters (Age-MT, Aglz-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently ireported for the HglS-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chern. SOC. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leadls to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random “cross-linked” regions in place of the cluster structure. C.D spectra were recorded as Ag+ was added to the a and B fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The a and /3 fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ags-a MT 1 and Age- a MT 1 is suggested by the spectral data obtained at 20 and 55 “C. Formation of Ag3-B MT 1 is suggested by the spectral data recorded at 20 “C for the B fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag,,-MT 2. A band at 575 nm reaches a maximum for Agle-MT 2. Analysis of the emission

* This work was supported by the Natural Sciences and Engineer- ing Research Council of‘ Canada under the Strategic grants (Open) program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Associated with the Center for Chemical Physics a t the Univer- sity of Western Ontario and the Photochemistry Unit in the Depart- ment of Chemistry (publication No. 419).

§ To whom correspondence should be addressed.

data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.

Metallothionein (MT)’ is low molecular metal binding protein, which is rich in cysteine (1). The protein was first isolated from equine kidneys (1, Z), but liver is also a good source of metallothionein (2,3). MT has been found in almost all mammalian tissue (4, as well as in microorganisms and invertebrae (5). Concentrations of metallothionein can be elevated in the liver and kidneys by a broad range of metals. Typically, metals such as cadmium, zinc, copper, mercury, and bismuth induce MT synthesis in either the liver or kidneys. Metals like silver, gold, and platinum (6) also either induce biosynthesis of the MT (7, 8) or bind to the MT already present (9). The overall structure of the metallated protein and the precise arrangement of the metals within the binding sites represent one of more difficult challenges of current investigations, especially for metals other than cadmium and zinc. Mammalian class 1 metallothioneins can be divided into two broad sub-forms, labeled as MT 1 and MT 2, that differ according to charge. Rabbit metallothionein are polymorphic in nature, with several subforms existing, each with one or more substitutions in the polypeptide chain not involving the essential structural amino acids (42).

The MT peptide forms two independently reacting, metal binding site domains, named A and B (11-15). Cadmium and copper exhibit a strong domain preference when binding to apoMT, binding first in domains A and B, respectively (10- 13, 15). Separate peptides containing either one of the two domains can be prepared from the holoprotein by selective enzymatic digestion. Fragments that contain one or other of the domains, named a and 8, have been characterized (12, 13). It has been suggested from detailed selective digestion studies (15) that metals like copper and silver adopt a stoichiometry of six in both domains. Similar digestion experiments that monitored the formation of the C&-a fragment indicated that the mobility of metals bound in one domain could be enhanced by competitive chelation agents (16). Spec- troscopic measurements made during titrations of Zn7-MT with copper demonstrated that it is possible to follow with high precision changes that take place at the metal binding site and to draw conclusions about the behavior of the whole polypeptide chain from these spectral data (17). Although a stoichiometry for copper:MT of 6 in both domains is now reasonably well established (13, 17), the stoichiometry for other metals is much less well known.

The early reports on the metal binding properties of metal-

’ The abbreviations used are: MT, metallothionein; apoMT, metal- free metallothionein; Zn-MT, zinc metallothionein; Cd-MT, cadmium metallothionein: a MT, a fragment; (3 MT, (3 fragment.

1709 1

17092 Silver Binding to Metallothionein

lothionein arose from studies of the physiological chemistry of zinc and cadmium (1, 2). However, the metabolism of platinum from cisplatin and gold from anti-arthritis drugs (e.g. Auranofin) may be closely linked to the presence of thiol- containing molecules, and in particular, metallothionein, in the liver or kidneys (18-21). The monvalent group 11 triad metals (copper, silver, and gold) bind strongly to metallothionein both in vitro and in vivo. Because each of these is a dlo ion, we expect similar but not identical chemistry. Because the CD spectral properties of Cu-MT are difficult to characterize (17) (and titration data can be complicated by redox reactions of the Cu(1) and thiolate groups) and the in vitro aqueous chemistry of gold(1) complexes in the presence of metallothionein is difficult to follow, we chose Ag+ as a model for the reactions of Cu‘ and Au+ with MT. Ag+ exhibits several coordination preferences in its inorganic chemistry, including linear and trigonal structures (22). Like Cu-MT (17, 23,24), Au-MT, and Pt-MT (25), Ag-MT (26) is luminescent.

In this paper, we report a series of experiments performed with apometallothionein and its a and j3 fragments, in which detailed sets of CD spectra have been recorded at different temperatures, as silver has been added to the protein. As with our previous work, we find that the examination of the pattern of the spectra recorded as the mole equivalents of silver increases provides remarkable insight into how silver binds to the MT peptide. We also report that these Ag-MT complexes are strongly luminescent with intensity that is dependent on the stoichiometric ratio of Ag:MT.

MATERIALS AND METHODS

Zn-MT 2 was isolated from the livers of rabbits injected eight times with a solution of %SO4 (20 mg of zinc/kg body weight) over a 2-week period and purified as described previously (10,27). ApoMT 2 was prepared from this Zn-MT 2 by passing the protein down a Sephadex (2-25 column, which had previously been equilibrated with 0.01 M HCl. a fragment was prepared from rabbit liver apoMT 1 as described

previously by Winge and Miklossy (12). 4 mol eq of cadmium were added to an apoMT 1 solution, and the pH of the solution was brought up to pH 8. Partial digestion of the protein was achieved using subtilisin. The p fragment was prepared from apoMT 1 after substitution with 6 mol eq of Cu+ and digestion as described previously (13). Apoa MT was produced from C&-a MT by stripping off cadmium on a Sephadex G-25 column, previously equilibrated with 0.01 M HCI. Apop MT 1 was obtained by incubation of the C%-@ fragment with 5 M excess of KCN in HCI at pH 0.3 for 1 h. The solution was then separated on a Sephadex G-25 column which had been equilibrated with 0.01 M HCI (10). Absorption at 220 nm and the cadmium and copper concentrations were used to monitor the concentration of the fragments.

Protein concentrations were estimated from measurements of -SH groups using 5,5’-dithiobis(nitrobenzoic acid) in 6 M guanidine hy- drochloride (28). Calculations were based on the assumption that there are 20 -SH groups in the whole protein and 11 or 9 in the a or p fragments, respectively. The total -SH, plus RSSR, concentration was estimated using the method of Cavallini et al. (29). Metal concentrations were determined with a Varian 875 atomic absorption spectrophotometer. Circular dichroism were recorded on a Jasco J- 500 spectrometer, controlled by an IBM 9001 computer using the program CDSCAN5 (30). Ag+ was added to solutions of the apoprotein under argon. Emission spectra were recorded from frozen solutions at 77 K. The emission intensities were digitized from a Perkin-Elmer MPF-4 spectrometer. The spectra were processed using the programs Spectra Manager (31) and Plot3D2 and replotted on an HP 7550A plotter.

The essence of the research described in this paper lies in clearly identifying those stoichiometric ratios between silver and the protein which result in formation of discrete complexes. We report series of CD spectra recorded as the molar ratio of si1ver:protein increases, using the induced chirality in silver-dependent bands to follow

Z. Gasyna and M. J. Stillman, unpublished program.

changes in the binding site. Unlike the reaction of Cd2+ with apoMT (101, we find that the CD intensity for AgMT is weak, and the bandwidths are broad. This means that the spectral data are more difficult to analyze when plotted in the usual two-dimensional manner. However, even with this weak amplitude (and, therefore, increased noise level), we have found that the three-dimensional sur- faces (plotted as amplitude versus wavelength versus mole equivalent Ag+ added) clearly identify the speciation that occurs. In particular, the contour plots extracted from the three-dimensional data provide unambiguous evidence of species formation. We include a selection of traditional two-dimensional plots, because these are the spectral data that were used to calculate the three-dimensional representation. Throughout this paper, wherever we refer to the mole equivalents of silver added, we use the form, for example, of 6 Ag+, in place of “6 mol eq Ag+,” or “a molar ratio for Ag+:MT of 6.”

RESULTS

Design of the Spectroscopic Experiments-Circular dichroism spectroscopy has proven to be very effective and precise in identifying structural changes in the metallothionein metal binding sites as metals have been introduced (10, 32,33). We used the CD spectrum to monitor changes in the chirality of the peptide chain as Ag+ was added. Carrying out experiments at different temperatures is helpful, because the ability of the thiolate groups within the protein to wrap round the incoming metal in the preferred cluster arrangement appears to be favored at elevated temperatures (perhaps as high as 65 ‘C) (33). Titrations carried out at lower temperatures (less than 20 “C) emphasize kinetic rather than thermodynamic prod- ucts (33).

We interpret changes in the intensities and spectral shapes of the absorption, emission, and CD spectra recorded as Ag+ is added to apoMT 2 and the a and /3 MT 1 fragments in terms of the formation of specific Ag,-MT species. There are four steps in our interpretation of these data. First, we assume that the initial conformation of the polypeptide chain is that of a random coil (each MT peptide used in the titration is initially metal-free). The very weak CD intensity in the 200- 230 nm region of the spectrum of apoMT supports this assumption: the lack of intensity suggests that there are almost no regions of a helix or /3 pleated sheet structure (10). Second, we assume that when metal ions bind to apoMT and the two fragments they can do so in a manner that results in cross-linked, SR-M-SR units, where M represents the coordinated metal cation, which form regions of distinct structure. In the well known three-dimensional structure adopted by native Cd+Zna-MT (11, 14), the metal-sulfur bonds form a network of cross-linkages that hold the protein’s polypeptide chain in a rigid three-dimensional arrangement. This structure bears no relationship to the interactions expected between amino acids in the apopeptide chain in the Chou- Fassman manner, rather the MT peptide chain exists as a multidentate chelating ligand. Third, we can follow changes that occur in the Ag,-MT binding site by monitoring the metal-related spectral intensity in the absorption and CD spectra to the red of 220 nm and the emission intensity to the red of 400 nm. Finally, fourth, we make the assumption that the development of a spectral signal that reaches a maximum intensity at a certain stoichiometry of si1ver:protein is related to the formation of a single species that is characterized by a specific stoichiometric ratio. We suggest that the MT species which adopt these well defined three-dimensional structures is characterized by specific CD spectral intensity under the metal-thiolate transitions, because this CD signal arises di- rectly from the chirality of the two binding site cages. It is the clusters Miss and MkSl1, where j and k represent stoichiometric values of metals, in the j3 and CY domains, respectively, that exhibit the chirality rather than the sum of the CD

Silver Binding to Metallothionein 17093

spectral envelopes from single cysteine-metal bonds. The three-dimensional representations also convey better our no- tion that these CD spectra are a property of the whole protein, not of isolated metal-thiolate units, so that the CD intensity surface that is seen in the figures relates structural changes that occur to the peptide chain as it folds around the incoming metal ions.

In most cases for the apoMT 2 samples used in these titrations, we have had to use separate solutions for each spectrum, unlike the procedures used for metallated MT, where aliquots of the incoming metal solution were simply added, repetitively, to the same solution to displace the previously bound metal. Use of different solutions gives rise to much greater "wobble" between adjacent spectra because a completely different solution is being used, this is especially apparent in the region below 300 nm and for titrations of the fragments.

Titration of ApoMT i? with Ag+ at 20 "C-Figs. 1 and 2, show changes in the absorption and CD spectra, respectively, recorded for a large number of identical solutions of apoMT 2. Silver was added in 1-mol eq aliquots to 10 nmol/ml solutions of apoMT 2 at pH 2. The pH was then raised to 7.5 and the spectrum recorded.

The series of absorption spectra depicted in Fig. 1 shows the growth and subsequent saturation of the Ag-MT 2 band near 260 nm. The intensity reaches a maximum at Ag,,-MT, although the absorbance continues to increase because of absorption by the silver salt (the last line plotted is for 20 mol eq of Ag+ added and is clearly not part of the set that saturates at AgIs-MT). The spectral pattern for the CD spectral intensity (Fig. 2), which is shown in the three-dimensional plot, exhibits three systematic effects. (i) A derivative-like envelope (( +) 240 nm =and (-) 260 nm) develops isodichroically up to 6 Ag+, with the 245-nm positive band reaching a maximum between 6 and 8 Ag+. (ii) The band intensification changes character between 6 and 12 Ag+ as both isodichroic points blue-shift. (iii) A broad positive band centered on 295

1.0

. e

.6 LD 0 4 \ u)

. 4

. 2

. o 210 240 270 300 330 3

w a v e l e n g t h / nm FIG. 1. Absorption spectra measured at 20 "C for a series of

separate apoMT 2 solutions with increasing molar ratio of Ag+. The Ag+ was added at low pH. The pH was raised to 7.5 and the spectra recorded. Spectra are shown for each 1-mol eq addition, from native apoMT (0 Ag+) to 20 Ag+. The inset shows changes in absorbance at 245,260, and 295 nm.

220 250 200 310 340 220 250 200 310 340 220 250 200 310 340

wavelength I' nm /I APO-MT 2 t Ao 200

15

9

3

At -3 20

-9 Q P '

-15

220 250 280 310 340 " wavelength / nm

FIG. 2. CD spectra recorded at 20 O C from different solutions of rabbit liver apoMT 2, each containing an increasing concentration of Ag+. An aliquot of Ag+ was added to each solution of apoMT 2 at a constant temperature (here 20 "C) and at low pH, the pH was then raised to 7.5 before the spectrum was measured. A, spectra recorded for the native protein (apo), and with 2, 4, and 6 Age added. B, spectra with 6, 8, 10, and 12 Ag'. C, the final set of spectra, with 12, 14, 16, 18, and 20 Ag+. The inset (in A ) shows how the CD band intensities change as a function of molar ratio Ag+:MT at three different wavelengths (245, 260, and 295 nm). The three- dimensional representation was constructed from a larger set of data than is displayed as A-C. The z axis is plotted in units of the Ag+ added to each of the solutions of apoMT. The grid lines added to the contour diagram are drawn for Agf molar ratios of 6, 12, and 18.

nm grows in intensity up to 17 Ag+. We consider first the derivative signal in the CD spectrum.

For the first few Ag+ added there is little signal intensity in the 240-260-nm region. The spectrum is broad and centered on 248-250 nm. With 4 Ag+ added, this new signal develops strongly and isodichroically (at 252 nm in Figure 2A) as a function of Ag+, so that by 6 mol eq a plateau forms at 245 nm. The isodichroic point changes to 250 and 268 nm (Fig. ZB), as a new species forms with 12 Ag+. The contour diagram picks up the steep change in signal a t 245 nm as a plateau forms in the CD spectrum. The contour lines overlap to lower wavelengths, because the peptide CD signal is strongly negative between 212 and 230 nm. The peak of the 240-nm band occurs at 12 Ag+, a gradual reduction in intensity of both the positive (240 nm) and negative (260 nm) components follows, until the intensity collapses with greater than 19 Ag+ (Fig. 2C).

The 295-nm band, on the other hand, intensifies linearly up to between 17 and 18 Ag+. This band is very much broader than the bands recorded at 240 and 260 nm. The continuous growth of this broad band as the As+ is added is clearly seen in the contour diagram. The breadth of the band, which is


about 50 nm full width at half-height, suggests that the negative intensity from the 260 nm band due to Ag12-MT at 260 nm loses some of its intensity to this band. Eventually, past 14 Ag+, the derivative band rapidly diminishes in intensity as the 295-nm band reaches a maximum. Addition of more than 18 Ag' quenches the CD intensity at 295 nm.

Titration of ApoMT 2 with Ag+ at 55 "C-Fig. 3 shows the change in the CD spectral data as a function of increasing molar ratio of Ag+ added to separate samples of apoMT 2 at 55 "C. A major difference between Figs. 2 and 3 is the appear- ance of a spectral feature at 12 Ag+, seen clearly in the three- dimensional plot (Fig. 3). The change in spectral intensity also clearly pauses at the 6 Ag+ point, with the 2501270-nm derivative band reaching a maximum at 6. In the next phase of the reaction, as 12 Ag+ are added, the derivative band blue- shifts slightly, with saturation at 245 and 260 nm being reached at 12 Ag+. Similarly, the 295-nm band reaches a maximum at 12 Ag+. Addition of further Ag+ quenches the

230 260 290 320 350

10

0

A E

-10

-20

230 260 290 320 350 wavelength / nm

20

230 260 290 320 350 wave leng th / nm

. -

FIG. 3. CD spectra recorded at 55 "C from different solutions of rabbit liver apoMT 2, each containing an increasing concentration of Ag+. The procedure was the same as for Fig. 2. A, spectra recorded for the native protein (apo), and with 4 and 6 Ag+ added. E , spectra with 6,8,10, and 12 Ag+. C, the final set of spectra, with 12, 14, 16, 18, and 20 Ag+. The inset (in E ) shows how the CD band intensities change as a function of molar ratio Ag+:MT at three different wavelengths (245, 260, and 295 nm). The three-dimensional representation was constructed from a larger set of data than is displayed as A-C. The z axis is plotted in units of the mole equivalents Ag+ added to each of the solutions of apoMT. The grid lines added to the contour diagram are drawn for Ag+ molar ratios of 6, 12, and 18. Note how the raised temperature allows the formation of Ag6- MT, Ag,,-MT, and Ag,,-MT to be visualized compared with the spectra recorded at 20 "C.

intensities of each of these bands, until a new band also at 295 nm intensifies towards 18Ag'. The contours show how this band intensifies rapidly from 16 to 18 Ag+, then diminishes between 18 and 20 Ag+. Each of these species is characterized by quite specific CD spectral intensity patterns in the contour level diagram (we have marked the 6, 12, and 18 Ag+ positions in Fig. 3). These data suggest that the kinetic product is normally formed at room temperatures, with the thermodynamic product requiring thermal energy or a cata- lyst. The data presented in Fig. 3 indicate that Agn-MT is stable above room temperature and that spectroscopic measurements of specific species should be carried out on complexes formed above room temperature.

Titration of Apoa MT 1 with Ag+ at 5, 20, and 55 "C- Spectral data for three experiments carried out at three different temperatures are reported for apoa MT 1. For the 5 and 20 "C experiments, separate solutions were used as for apoMT described above; for the 55 "C experiment, a single solution was used and the Ag+ titrated into the solution as in our previous studies with Zn-MT (10). The solutions were mixed, equilibrated, and the spectra recorded using a constant temperature water bath. The instrument amplification is so high that we observe some CD band structure for the apoa MT in this region (the lines labeled 0). The signal magnitudes in the CD spectra for the apoa experiments are about half those of the apoMT experiments described above.

Fig. 4 shows the spectral data recorded at 5 "C for different solutions that contained apoa and increasing amounts of Ag+. Addition of 5-6 Ag+ results in a new spectrum developing with a minimum at 262 nm and a maximum at 296 nm (Fig. 4B). The three-dimensional representation is difficult to follow, because the spectral data is so noisy under the conditions of this experiment. However, the contour diagram indicates that a Agb-6-MT species does form. With excess Ag+ only a negative band near 230 nm remains.

At 20 "C, Fig. 5, it is clear that new bands develop at 264 nm (negative) and 296 nm (positive) once 6 Ag+ have been added (the inset in Fig. 5A shows the changes at 245,260, and 295 nm). When further Ag+ is added, we observe a number of bands that are hard to disentangle from the noise. Finally, for the titration of Ag+ with the single solution of apoa MT 1 at 55 "C, Fig. 6, we find a similar spectral pattern to that of apoMT, Fig. 3. A derivative-shaped band forms initially, with a strong dependence of the stoichiometry of si1ver:protein. The 2401270 nm derivative-shaped band grows in isodichroically, reaching a very well-defined maximum at 3 Ag. Addi- tional Ag+ results in the collapse of this signal intensity. Although the band intensities change isodichroically from 3 to 5 Ag+, it is probable that at this elevated temperature complexes formed are so unstable that the Ag6-a does not survive.

Titration of Apop MT 1 with Ag+ at 20 "C-Few spectral data have been reported for the p fragment. This is especially the case for metal binding reactions involving apop MT, in part, probably because the fragment is so unstable. Fig. 7 shows the CD spectra obtained from separate samples each with increasing amounts of Ag+ added. A new CD spectral envelope forms which reaches a maximum intensity with 3 Ag+. The band maxima are at 250 nm (+), 270 nm (-), and between 300 and 310 nm (+). The wavelength positions of the maxima of this derivative envelope matches quite closely the bands that form when Ag+ is added to apoMT, especially in the spectrum recorded at 55 "C (Fig. 3A). Additional Ag+ results in the development of a second, but much weaker, spectral envelope centered on 295 nm which is maximal at 5 Ag. Further Ag+ causes a loss of the CD signal intensity,


3

2

-1 w

-4

-7

-10 210240 270 300330 210240 270 300330 210240 270 300330

w a v e l e n g t h / nm

A &

210 240 270 300 330 ' V

wavelength / nm

FIG. 4. CD spectra recorded from individual solutions of apoa MT 1 fragment containing increasing concentrations of Ag+ at 5 "C. The procedure was the same as for Fig. 2. A, spectra recorded for the native 01 fragment (0) and with 1,2 and 3 Ag+ added. B, spectra with 3, 4, 5, and 6 Ag+. C, the final set of spectra, with 6, 7, 8, 9, 10, and 11 Ag+. The inset in A shows the change in CD intensity at 245, 260, and 295 nm. Grid lines for 3 and 6 Ag+ are marked. The noise is observed because these CD are very weak.

which suggests that the clusters fell apart. The contour diagram shows clearly the development of the Ag3-MT species, followed, in a less defined manner, by an Ag6-MT species.

Emission Spectra-Fig. 8 shows the dependence of the emission intensity, in the 350-650-nm region, on the molar ratio of Ag+: protein when excited at 300 nm. Ag+ was added in increasing molar ratios to separate solutions of apoMT 2 under argon, and the solutions were then frozen. Measure- ments were made at 77 K. The Agn-MT species (where n = 1-20) are all luminescent, with both the emission spectrum (Fig. 8) and the yield of light intensity (Fig. 9) being clearly dependent on the silvecprotein stoichiometry. Fig. 9 shows how the intensities at three wavelengths vary with the molar ratio of Ag+. At low loadings of Ag+, we find a peak near 465 nm, as more Ag+ is added, the band maximum red shifts. The contours in Fig. 8B show how the blue edge of the band is maintained up to 12 Ag+. Addition of Ag+ up to 20 mol eq results in a new band forming near 575 nm.

DISCUSSION

The silver-protein species described for metallothionein in this paper are identified from saturation in the chiral intensity as increasing amounts of metal are added to the protein. Isodichroic points are also essential in providing evidence that a single transformation from one species to another occurs. Decomposition, which might be caused by oxidation or simply an instability of the metal cluster itself within the protein, will lead to loss of the isodichroic point. Because the apoMT adopts a random coil structure (IO), there is almost no con-

-8

210 240 270 300 330 210 240 270 300 330 210 240 270 300 330 wavelength / nm

10

5

A E o

-5

-10

11

" 210 240 270 300 330

wave leng th / nm

FIG. 5. CD spectra recorded from individual solutions of apoa MT 1 fragment containing increasing concentrations of Ag+ at 20 "C. The procedure was the same as for Fig. 2. A, spectra recorded for the native 01 fragment (0 ) and with 1,2, and 3 Ag+ added. B, spectra with 3, 4, 5, and 6 Ag+. C, the final set of spectra, with 6, 7, 8, 9, 10, and 11 Ag+. The inset in A shows the change in CD intensity at 245, 260, and 295 nm. Grid lines for 3 and 6 Ag+ are marked. The noise is observed because these CD spectra are very weak.

tribution to the CD spectrum above 220 nm from ordered peptide chain that is not bound to the silver with a well defined structure. This means that the intrinsic chirality of the component amino acids does not contribute to the signals that are recorded between 215 and 400 nm. The CD spectrum arises solely from a chiral, silver-binding site. If CD spectra are measured during zinc or cadmium displacement reactions, then the chirality of the zinc- or cadmium-binding site will contribute to the measured spectrum of the Ag-MT.3 The CD spectra measured during the experiments discussed here for apoMT are weak, and the spectral envelope is located in a region of the spectrum that is prone to low signal to noise values. Presentation of the CD data in terms of three-dimensional plots improves the clarity of the spectral effects that depend on the molar ratio of metal added to the protein. CD spectral intensity observed for molecular electronic transitions that are specifically centered on the metal arises when the transitions take place within a chiral electric field from the metal binding site (34). The simplest examples of this type of molecule are the trisethylenediammine cobalt(II1) complexes which involve "propeller"-like bidentate chelating ligands that occupy all six vertices of the octahedron. While the d and 1 enantiomers of the complex will result in CD

A. J. Zelazowski and M. J. Stillman, unpublished data.

17096 Silver Binding to Metallothionein 1"

6

2 0 Q

-2

-6

-10

3

2.0

-3

-6

3

2.0

-6 -3u wavelength / nm

APO-U + Ag 55O

210 240 270 300 - u 330

wavelength / nm

FIG. 6. CD spectra recorded from the same solution of apoa MT 1 fragment containing increasing concentrations of Ag+ at 55 OC. The procedure was the same as for Fig. 2. A , spectra recorded for the native a fragment (0 ) and with 1,2, and 3 Ag+ added. B, spectra with 3, 4, 5, and 6 Ag+. C, the final set of spectra, with 6, 7, 8, 9, 10, and 11 Ag+. The inset in A shows the change in CD intensity at 245, 260, and 295 nm. Grid lines for 3 and 6 Ag+ are marked.

spectra of opposite sign under the Co(II1)-based transitions, the ethylenediammine is not itself optically active (34).

We suggest that a similar molecular effect results in the CD spectral intensity that is observed for metal-containing metallothioneins. In the most studied case, Cd,Zn-MT, the transitions are not solely metal-based, rather they are ligand (RS-) to metal (Cd2+, Zn2+, etc.) charge transfer in nature. The wavelength of such transitions are dependent on the combination of the redox potentials of the donor and acceptor atoms. Vasak et al. (35), using rules devised by Jorgensen (36), assigned the absorption transitions observed for Cd,Zn- MT in the 250-nm region as ligand to metal charge transfer, RS- + Cd". Our previous absorption, CD, and magnetic CD studies have shown that the CD intensity reaches a maximum when the a cluster is full (10). The key component in the assignment of the Cd7-MT CD spectrum was the recognition that exciton splitting of the main ligand to metal charge transfer band took place once the full CQ-a cluster was formed, so that a derivative-like spectrum is measured, which has its cross-over point at the band center of the absorption band (10). We associate large changes in the CD spectrum to the red of the peptide band (X > 215 nm) with bond formation between the thiolate groups on the MT and the Ag+.

Recent observations of luminescence from several metallothioneins (17,24-26) suggests that emission properties may

230 260 290 320 350 . . . .

230 260 290 320 350 230 260 290 320 350 wavelength / nm

A /I

/ I APo-8 + Ag 2oo / I

230 260 290 320 35OU wavelength / nm

FIG. 7. CD spectra recorded from individual solutions of apop MT 1 fragment containing increasing concentrations of Ag+ at 20 "C. The procedure was the same as for Fig. 2. A , spectra recorded for the native a fragment (0) and with 0.5,1,1.5,2,2.5, and 3 Ag+ added. B, spectra with 3,3.5,4,4.5,5,5.5, and 6 Ag+. C, spectra with 6, 6.5, 7, 8, and 9 Ag+. The inset in A shows the change in CD intensity at 245, 260, and 295 nm. Grid lines for 3 and 6 Ag+ are marked. Note that the CD sensitivity is very high for these measurements, which contributes significant noise to the spectral data.

be a sensitive probe of metal-protein complex formation. The data plotted in Figs. 8 and 9 show that the band maximum and emission intensity are dependent on the mole ratio of Ag:MT. Normalizing the emission intensity to the effect of each silver added shows that the emission spectrum is an indicator of metal-thiolate cluster formation. The data in Fig. 9B demonstrate that each additional Ag+ between 1 and 10 increases the yield of emission intensity in a cooperative manner. In Fig. 9A, we see how maximum intensity for the 575-nm band at 16 Ag' is accompanied by a general red shift of the spectrum at these high molar ratios (Fig. 8). We associate the retention of the emission intensity at 575 nm with the formation of the tightly wound AgI6-,,MT species. The emission intensity for Cu,-MT from rabbit livers (where n = 1-20) is quenched once more than 12 Cu+ have been added (17,24), which we associate with the opening up of the Cu-MT peptide chain at high Cu:MT ratios. This is in complete contrast to the increased coiling proposed for Ag-MT with molar ratios of Ag:MT greater than 12. We interpret this effect as follows. Silver binds to thiolates on 2 or more cysteines, which cross-links the peptide chain in the region of the binding site. As the peptide chain becomes more tightly wound round the metals, so the solvent is extruded from the metal binding site cage, which reduces the efficiency of the nonradiative deactivation pathways of the metal-dependent


wavelength / nm

350 450 550 650

Wave leng th / nm

FIG. 8. Emission spectra from Ag,-MT 2. Upper panel, uncorrected emission spectra recorded for the three distinct species, AgG- MT ( 6 ) , Ag,,-MT (IZ), and Ag,,-MT (18), following excitation of frozen aqueous samples at 77 K at 300 nm. Lower panel, three- dimensional representation of the uncorrected emission intensity observed from frozen (77 K) samples of Ag,,-MT, with increasing molar ratios of Ag:MT, between 350 and 650 nm, following excitation at 300 nm. The z axis shows the molar ratio of Ag:MT, n, which increases from 0, for apo-MT 2, to 20, for Ag,,-MT. The contour lines show how the intensity varies as the molar ratio of Ag:MT increases. Note especially how the intensity at 530 nm reaches a maximum at the 10 Ag+ point. New bands intensify between molar ratios of 12 and 20 Ag:MT.

excited states that are responsible for the emission. We characterize the emission bands observed in the 400-

650 nm region as silver-centered, 3[5s’3d”] * 1[3d10] phospho- rescence. In this case, the formation of Ag-S-Ag links in the clusters, leads to delocdization of the metal electrons, which modifies the observed luminescence. Three bands, located at 465,520, and 575 nm, ,appear to make up the intensity of the spectral envelope as Ag+ is added to apoMT. The intensities of the 465- and 520-nm bands are a maximum for Ag:MT = 10, which we associate, tentatively, with formation of a AgI2- MT species. It is not known why the maximum is at 10 mol eq rather than 12. The significant temperature dependence of the CD reactions displayed as Figs. 2 and 3 suggests that perhaps mixtures of species forms at the 20 “C used for the metal binding reaction.. (The spectra were recorded at 77 K.) The 575 nm band appears to be a marker band for high molar ratio species.

At low Ag:MT ratios, it is probable that the coordination will be trigonal for isolated units of Ag(RS)3 because of the excess thiolate ligand that is present. As the molar ratio of Ag:MT increases, we expect “cluster” formation begins. In this context, clusters involve shared thiolate groups (11, 13, 14) of the type determined for [AgSC(Si(CH3)3]4 (22). The S-

1.6 575 nm

m 0

, 1.2 CI

> .rl c, m 0) c, C H

c . 0

. 4

.o

0 4 0 12 16 20 Ag m o l eq

FIG. 9. Emission spectra from Ag,-MT 2. A, relationship between emission intensity of Ag,-MT 2 at three wavelengths (465,520, and 575 nm) and the molar ratio of Ag:MT. The intensity values were extracted from the spectral data shown in Fig. 8. Note how the intensity of the 575-nm band is retained between 12 and 18 Ag+. 13, relationship between emission intensity per silver in Ag,-MT 2 at three wavelengths (465, 520, and 575 nm) and the molar ratio of Ag:MT. The intensity values were extracted from the spectral data shown in Fig. 8. This graph shows the effect of additional silver atoms on the emission intensity. The growth in intensity is associated with a reduction in the emissive state quenching by solvent as cluster formation makes the metal binding sites region more hydrophobic.

Ag-S-Ag units in this structure contrast the metal-metal bond formation reported by Chiari et al. (37) for Au(I), (dithiocarboxylate)4, where the internuclear distance of 301.3 pm suggested direct bonding between the four gold atoms. The complicated changes in coordination number that can accompany metal binding to MT have been discussed by Laib et al. (20) for Au-MT, and by Nielson and Winge (13) for Cu- MT. The inorganic chemistry of Ag+ might suggest that 2-, 3- and 4-fold coordination might be possible, whereas, for Au+, only 2-fold coordination is to be expected. At very high molar ratios, we expect that linear coordination will exist, possibly with chains of Ag-RS-Ag forming, as with the syn- thetic silver-thiolate complexes of Dance et al. (38).

We summarize the data described above in Fig. 10. We justify the entries in this diagram as follows.

Ag3-a, Ag3-p, AgG-a, and Ags-p MT 1-Figs. 2-5 show the spectra obtained for the isolated fragments. These silver binding experiments were difficult to perform as the concentrations used were very low. The spectral data shown for the cy and p fragment titrations are weak, and we believe that the


apo-0 apo-MT

FIG. 10. Pathways for silver binding to apoMT. A diagram showing a series of proposed binding mechanisms that are followed when Ag+ is added to apoMT 2 and to the two fragments, apoa MT 1 and apop MT 1. The formation of each species, with the exception of Ag6-8 MT, can be observed within at least one of the sets of CD spectra shown in Figs. 2-9.

complexes formed are relatively unstable. The CD spectra of both fragments are at a maximum for the Ag3 complex, rather than Ag6, and the spectral envelopes exhibited for both Ag, species initially appear to resemble that recorded for Ags-MT in the complete protein. However, there are differences in these spectral data. The p data, whereas weaker, exhibit the asymmetric envelope observed for the holoprotein, whereas the envelope for the a fragment is quite symmetrical, as is observed when between 6 and 12 Ag+ are added to apoMT. As each line shown in the figures is a spectrum of a separate solution, equilibrated with a single amount of Ag', we suggest that the bands that are a maximum at molar ratios of 3 or 6, point to the formation of the species Ag3-a, Ag3-/3, and Ag6-a. We find no evidence for the formation of Ag6-@. Whereas oxidation of the thiolate groups in the fragments during the course of these reactions is a very real possibility, we feel that the precision of the isodichroic points for other samples, even at 55 "C under identical experimental conditions, Fig. 3, indicate that the environment was sufficiently anaerobic to reduce the rate of oxidation.

Ag6-MT and AgIz-MT 2-Titrations carried out at 55 "C reveal how the series of complexes, Ag6-MT, Aglz-MT, and Ag18-MT, form in sequence as Ag+ is added to rabbit apoMT 2. We suggest that at elevated temperatures the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product formed at room temperature and below, in which metals are bound across the peptide chain forming more random cross-linked regions in place of the cluster. Ag6-MT is characterized by a spectral envelope which exhibits a derivative band with a maximum at 240 nm and minimum at 265 nm. This band intensifies as further Ag+ is added, until the band maximum is located near 243 nm and the minimum near 260 nm, with 12 Ag+ present. At the same time the 304-nm peak at 6 Ag+, shifts to 292 nm at 12 Ag. These results are indicative of cluster formation. Initially at 6 Ag+, the derivative band suggests that exciton coupling occurs much like we have described for C&-a or C&-MT (prepared from apoMT) (10). From a domain specificity stand point, we had hoped that we could identify which domain contributed which band, and, therefore, we would be able to assess a domain preference as we have been able to do for cadmium (10). However, although the data in Fig. 6 for a-MT

and Fig. 7 for 8-MT resemble the sequences observed in Fig. 3 for the apoMT, the differences are too small to allow identifications as to which domain is involved.

Agla-MT 2-Chelation of more than 12 metal atoms by metallothioneins has only been observed previously for Hg,,- MT 2 (39). The CD spectral intensity displayed in Fig. 2C and 3C for Ag,-MT arises from the same transitions that give rise to the spectral intensity associated with Ag6-MT and Aglz-MT. Clearly, and unambiguously, the spectral data observed for Agle-MT also arise from transitions based on the silver. The emission spectra in Fig. 8 also offer evidence for the formation of this Ag16-I~-MT species, because unlike the emission data for Cu(1)-MT (17), the intensity at 550 nm does not collapse once 12 Ag+ have been added to the apoMT. Although we find the strong spectroscopic evidence presented in Fig. 2 compelling, we are surprised that a species with such a well defined three-dimensional structure exists. However, there are several reports in the literature that saturation of MT with Ag' occurs at the 18 level. The spectroscopic data show why this is the case. We can only speculate on the structural form of the Agls-MT protein at the present. We have used the following assumptions in making suggestions for the structure. (i) Because the CD spectral intensity arises from chirality around the Ag-S bonds and at most only two thiolate groups are unsaturated, the CD intensity must arise from chirality of the whole binding domain. (ii) If the binding site for all 18 Ag+ is highly chiral, then the Ag-S bonds must form a complex with a specific three-dimensional, rather than a random coil, structure. (iii) The emission intensity requires a tight structure that can isolate the emissive exited state from deactivation by the solvent. (iv) The Ag+ must bind with at least two-coordination. Based on these assumptions, we suggest that the structure involves a tight stack of linear RS- Ag-RS goups, using bridged cysteine thiolates. Elucidation of the structural form of Agla-MT will require considerable further analysis.

Among metals that bind to MT, some preferentially bind to one or other of the two domains. Cadmium is strongly associated with the a domain, whereas zinc does not have a strong preference for either a or p (12). Once the binding site is filled with metals, the tertiary structure of the protein is defined by the clustered metals. By using apoMT, we ensure that the binding site structure depends only on the incoming metal, here, the Ag+. The optical spectra recorded both as CD and emission arise solely from transitions related to the silver bound to the protein (the incoming metal, Ag+, exhibits no CD or emission intensity when not bound to the protein). The a and /3 fragments were used in this study to help identify the location within the protein of transitions observed for reactions of apoMT with Ag+. In studies of cadmium binding to apoMT 2 and to Zn-MT 2, we found that the CD spectrum measured during the titration was dominated by transitions located on the a domain, with surprisingly little contribution from the /3 domain (10). The emission spectra, Fig. 8, provide direct information on the binding mechanism. Comparision between these data for apoMT and previous data obtained for a titration of Zn7-MT with Ag+ (26) shows that with the apoMT there is a distinct separation of the band maxima when plotted against molar ratio of Ag:MT. Band maxima can be associated with Ag:MT of 6,12, and 18. The analogous set of data for displacement of Zn by Ag+ in the Zn-MT (26) do not show such a clear distinction between band maxima and molar ratio. We interpret this difference in the resolution of band maxima and molar ratio, with domain specificity for silver binding to apoMT and distributed binding to the Zn7- MT. In the case of the distributed mechanism we expect that


filled domains, for example, Ag6-8, only form when 12 Ag' have been added. The domain-specific mechanism allows for filled domains to form before 12 Ag' have been added. Winge and co-workers (15) have reported preliminary results in which they proposed that Ag(1) binds cooperatively, initially in domain 8. By analogy with copper, it is quite probable that silver does bind in the p domain first.

Thiolate complexes of Ag' have been reported by a number of authors (22,38,40). These reports include complexes where silver is coordinated with different stoichiometries. The preferred coordination number for silver(1) with thiolate ligands is two, with a linear geometry strongly favored (40). Other types of geometry are possible, with trigonal and T-shaped structures being known. Such complexes are yellow due to charge transfer from thiolate to the silver, which occurs at a lower energy than that in linear complexes (40). Shaw and co-workers (20) have discussed the possible structures adopted by gold-containing metallothionein. We expect that complexes between Ag+ andl apoMT will exhibit a similar range of structures. Neither CD nor emission spectra provide coordination numbers. These data can only provide the stoichiometric ratios between the metal and the protein. In these complexes, both terminal and bridging ligands are used to maintain a molecular structure, because of this property a range of Ag:MT molar ratios are possible. There seems no reason at present not to suggest that the silver-thiolate clusters follow the trigonal coordination currently proposed for copper-containing metallothioneins (41).

From the spectral titrations of apoMT shown as Figs. 2 and 3, we find that Ag7-MT does not form, but that Aglz-MT does form, a species most probably an analog of Cu12-MT. In which case, following the suggestions of Winge (12), we suggest that Aglz-MT will adopt trigonal coordination. We expect that coordination in Ag1,,,-MT will be linear, possibly like Scheme 4 from Shaw's work (20), which is similar to the double-stranded chain arrangement reported for (-Ag-SR-)n by Dance et al. (38). It is also possible that a more complex structure, one involving -RS-Ag-RS- units stacking as suggested for Hgl6-MT (3'3), is adopted. The one constraint on any structure proposed is that it has to generate significant chirality in the metal binding site region in order to induce the strong CD spectrum observed, especially at higher temperatures.

In summary, (i) apoMT 2 binds Ag' in several stages, with distinct species forming at Ag6 and Ag12. We have observed the formation of a third novel structure, namely Ag,,-MT. (ii) Titrations carried out ;at elevated temperatures exhibit much greater cooperativity, so that each of the possible clusters (that is Age-MT, Agl2-MT, and Agle-MT) form before the development of the next structure, whereas a t room temperatures a mixture of colmplexes forms. (iii) Ag-MT complexes exhibit luminescence intensity that is dependent on the Ag:MT molar ratio.

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