the journal of biological chemistry 262, of 5, 4538-4548 ... · 0 1987 by the the journal american...

0 1987 by The THE JOURNAL

American Society of Biological Chemists, OF BIOLOGICAL CHEMISTRY

Inc. Vol. 262, No. 10, Issue of April 5, pp. 4538-4548, 1987

Printed in U.S.A.

Cadmium Binding to Metallothioneins DOMAIN SPECIFICITY IN REACTIONS OF a AND p FRAGMENTS, APOMETALLOTHIONEIN, AND ZINC METALLOTHIONEIN WITH Cd” *

(Received for publication, July 15, 1986)

Martin J. StillmanSO, Wuhua CaiS, and Andrzej J. ZelazowskiS From the Department of Chemistp, University of Western Ontario, London, Ontario N6A 5B7,

The cadmium-binding properties of rabbit liver Zn7- metallothionein (MT) 2 and apo-MT, rat liver apo-a MT and &,-a MT, and calf liver apo-@ MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unex- pected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-a MT, and apo-#I MT), and is observed to a much lesser extent in the titration of Zn-a MT. Com- parison between the band centers of the Cd-a MT and Cd-@ MT indicates that the CD spectrum of Cd,-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the a domain, with little direct contribution from the @ domain. Analysis of the spectra recorded during titrations of ZnT-MT 2 with Cd2+ suggests: (i) that CdZ+ replaces Znz+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspe- cific, “distributed“ manner across both domains; (iii) that cluster formation in the a domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from “synthetic” Cd4,Zna-MT following a treatment cycle that allows the redistribution of cadmium into the a domain; warming the synthetic %ative,” Cd,,Zn,-MT, to 65 OC results in cadmium being preferentially bound in the CY domain; and (v) Zn,-MT will bind Cd2+ quite normally at up to 65 OC but with greater specificity for the a domain compared with titrations carried out at 25 O C . These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn- MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.

Metallothionein (MT)’ is a small, metal-binding protein that is rich in cysteine (1). The protein is commonly found in

* This work was supported by the Natural Sciences and Engineer- ing Research Council of Canada under the Operating and Strategic Grants program and by the Academic Development Fund at the University of Western Ontario. 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 Centre for Chemical Physics at the Univer- sity of Western Ontario.

§ To whom correspondence should be addressed. The abbreviations used are: MT, metallothionein; CD, circular

dichroism; MCD, magnetic circular dichroism; T, tesla; LMTC, ligand to metal charge transfer; AAS, atomic absorption spectroscopy.

the liver and kidneys of mammals, although it has also been isolated from invertebrates and microorganisms (2-4). While cadmium, zinc, and copper are usually associated with MT in uiuo, a wide range of other metals also binds to the protein, especially in vitro (5,6); among these metals are gold, silver, and mercury. Not all these metals stimulate the biosynthesis of the protein. In some cases, biosynthesis is induced, but the protein binds only zinc or copper, not the inducing agent ( 5 ) .

Extensive spectroscopic studies of the cadmium-containing protein with optical (7,8), two-dimensional ‘H NMR (P), and ‘I3Cd NMR (10-14) techniques have established the stoichiometry for cadmium and zinc binding. ‘I3Cd NMR data (10, 12) have been analyzed in terms of a two domain structure for metal binding. Results from x-ray crystallographic studies (15) support this model. Analysis of ’I3Cd NMR data (10, 12) suggested that in the mixed metal, C&,Zn3-MT protein, three zincs are located in the B domain at the amino-terminal of the protein, while four cadmiums are located in the A domain at the carboxyl-terminal. Although in native samples of Cd,Zn-MT, it appears that cadmium binds preferentially in the a domain and zinc in the domain, it is by no means established how this domain specificity actually occurs in uiuo. Indeed, Otvos and co-workers (16), in detailed ‘I3Cd NMR studies, have reported conflicting evidence concerning cadmium binding to Zn-MT in uitro. Analysis of ‘13Cd NMR spectral data recorded during a titration of Zn7-MT with Cd2+ (16) indicated that the cadmium was binding in both LY and 8 domains in a distributed manner, whereas the appearance of a “native” NMR spectrum following the mixing of stoichiometric amounts of Zn-MT and Cd-MT suggested that a domain specific mechanism must be operating (16). Domain specificity by a wide range of metals has been demonstrated by Winge and co-workers (6 , 17, 18) following in vitro chemistry using selective digestion techniques. From stoichiometric measurements of the metals bound to the fragments obtained in these experiments, Winge and co-workers (6) suggested that each of the two domains can bind up to six univalent metals, such as copper and silver. The preference for metals such as copper and silver appears to be for the /3 domain, while cadmium and zinc bind to the a domain. However, while these results have greatly advanced our understanding of the capabilities of metallothioneins as metal chelators, the experiments entail considerable chemistry, and it is not known how mobile the metals are under the conditions used in the isolation of the fragments. Spectroscopic methods applied during metal titrations of dilute solutions of the protein do offer a special view of the protein as metals bind, in the absence of any additional chemistry. Detailed spectroscopic titrations of Zn7-MT with Cu(1) (19) show that three saturation points are reached, at 6,12, and 20 mol eq of Cull). These stoichiometries coincide with the filling of the B domain, followed by the A domain, and, finally, by an unwinding of the three-dimen-

4538

Cadmium Binding to Metallothioneins 4539

sional cluster structure by 20 mol eq of Cu(I), respectively (6, 19). While the protein can be cleaved into two fragments, named a and p, after the A and B domains that they contain (17,18), spectroscopic studies have only been reported for the (Y fragment, C&-a MT (20).

In this paper, we describe a set of spectroscopic experiments in which cadmium binding to each of the major contributors to the metal-binding properties of native metallothionein is examined. Circular dichroism (CD) spectroscopy is used as a probe of geometrical change in the metal-binding site regions. The titration of Zn7-MT 2 with Cd" yielded such an unusual pattern of spectra, that we have exploredpossible mechanisms for this effect. We are able to demonstrate that the CD spectra indicate the onset of clustering in the a domain as cadmium is added to Zn-MT. We describe the formation of Cd4,Zn3- MT i n vitro that does have spectroscopic properties similar to the native, Cd,Zn-MT.

MATERIALS AND METHODS

Zn-MT was isolated from the liver of a single rabbit following a series of 8 injections of aqueous solutions of ZnSO, (20 mg/kg body weight, based on Zn) spread over a 2-week period. The protein was purified as previously reported (21). Apometallothionein (apo-MT) was prepared from Zn-MT by passage down a Sephadex (2-25 gel column equilibrated with 0.01 M HCI to remove the zinc. The (Y

fragment was prepared from rat liver Cd,Zn-MT 2 as described previously by Winge and Miklossy (17). Cu,Zn-MT was isolated (22) from fetal calf livers obtained at a local abattoir. The j3 fragment was prepared from this Cu,Zn-MT 2 after enrichment of the protein with copper. All protein samples were extensivelypurified used preparative gel electrophoresis. Aliquots of Cu(I) were added, using [Cu(CH&N),] C10, dissolved in 30% acetonitrile, up to 5 mol eq, on a per mol of protein basis. (The abbreviation mol eq indicates 1 mol of metal ions per mol of protein or per 20 mol of RSH groups). Trizma base was added to adjust the pH to 7.5. The MT solution was incubated with 5 mM EDTA at pH 7.5 for 2 h, and then subtilisin was added up to a protein/enzyme weight ratio of 301. Following incubation at room temperature for 2.5 h under anaerobic conditions, the protein com- ponents were separated on a Sephadex G-50 (2 X 160 cm) column, equilibrated with a pH 8, 10 mM Tris-HC1 buffer.

The apo-0 fragment waa obtained by incubating the Cu-j3 MT with 5 molar excess of KCN in HCI at pH 0.3 for 1 h. The solution was eluted with 0.01 M HCI through a Sephadex G-25 column already equilibrated with 0.01 M HCI. Absorption at 220 nm, and copper concentrations, were measured for the 1-ml fractions collected. Pro- tein concentrations were estimated from measurements of the sulfhy- dryl content using 5,5'-dithiobis(nitrobenzoic acid) in 6 M guanidine hydrochloride (23) and were based on the assumption that there are 11 RSH groups in the cy fragment and nine RSH groups in the @ fragment (17, 18). Total RSH plus RSSR was measured using the method of Cavallini et al. (24). Determination of both the RSH and RS-SR concentrations were made before and after many of the spectroscopic measurements; no loss of RSH was detected.

Metal concentrations were determined with a Varian 875 atomic absorption spectrometer. Absorption spectra were recorded on a Cary 219 spectrometer. Circular dichroism spectra were recorded on a Jasco 5-500 spectrometer that was controlled by an IBM S9001 computer using the program CDSCANS? Magnetic circular dichroism spectra were recorded on the Jasco 5-500 spectrometer with a field of 5.5 tesla supplied by an Oxford Instruments SM2 magnet. The MCD signal was calibrated by measuring the signal at 510 nm from aqueous CoSO,, ACM = -0.01897 liters mol" cm" tesla". The spectral data were organized and plotted on an H P 7550A plotter with the spectral database program Spectra Manager (25).

RESULTS

The CD spectrum of metallothionein has been shown to be a sensitive indicator of changes in the metal-binding site region as metals are added to, or removed from, the protein (7, 19, 26, 27). The major contributor to the CD sensitivity relates to the induced chirality imparted on all optical tran-

B. Kitchenham and M. J. Stillman, unpublished data.

sitions that involve both the protein and metal ions that are bound in the chiral-binding site. For many of the group IB and IIB metals, the most significant transitions are charge transfer in nature, thiolate sulfur to metal, i.e. LMCT. Addi- tionally, because the energies of these charge transfer transitions are dependent on the metal, the CD spectrum will often change in a systematic fashion when one metal is replaced by another. Cadmium binding to metallothionein has been in- vestigated in this work by measuring CD and MCD spectra during titrations of Cd2+ into solutions of a number of different metalIothioneins. (i) Rabbit liver Zn7-MT 2 was used as a typical example of the type of reaction that takes place when metal substitution occurs with the fully metallated protein; for Zn7-MT, this may also be the reaction that occurs in vivo following exposure of an animal to cadmlum. (ii) Apo- MT was used to provide an example of how the lack of an existing three-dimensional structure in the binding site region affects the CD spectral changes; these data contrast the Zn7- MT situation where the protein already adopts a highly organized structure (15). (iii) Titrations with the metal free cr and /3 fragments, apo-a MT and apo-0 MT, were used to provide information about the extent of specific contributions that metals bound to the individual domains (the A and B domains in the intact, native protein) make to the overall spectral intensity. (iv) Zn4-e MT was used to obtain a set of spectra that should correspond to the early part of the titration of Zn7-MT with Cd2+.

For each species, we determine the stoichiometry of the reaction from the saturation points observed in the CD spectral data. Because both zinc and cadmium are known to form tetrahedral complexes with thiolate ligands (28, 29), it is generally considered that substitution of zinc by cadmium should involve no major structural changes (10-14, 15). Re- placement reactions of this type are commonly labeled as "isomorphous." (The same cannot, of course, be said for the replacement of zinc by, say, copper(I).) While some structural change must occur as the protein backbone accommodates the slightly larger cadmium, it still is useful to use the term "isomorphous replacement" to emphasize that direct replacement has taken place, with no rearrangement of the binding site.

As Cd" replaces Znz+, the CD spectrum can show changes for three reasons: (i) the energy of the charge transfer transition, RS~-Zn2+, is greater than RS--Cd2+, and the whole spectrum will shift to the red; (ii) the charge transfer excited states may be sensitive to the binding of the same metal in adjacent sites, thus there may be a difference in the electronic states when a bridging thiolate connects zinc to cadmium, compared with connecting cadmium to cadmium, for example, as (SR)&d(SR)Cd(SR)3; and finally, (iii) some change in the induced chirality observed under these absorption bands will be observed that results from the adjustments made to the protein backbone surrounding the two metal-binding sites (the a and /3 domains), because the covalent Cd-S bond is 10% longer (29) than the Zn-S bond (28).

Titration of Z n - M T 2 with CD2+-Fig. 1 shows the absorption ( A ) and CD ( B ) spectra recorded as mol equivalent (mol eq) aliquots of Cd" were added to the rabbit liver Zn7-MT 2 protein. A quite remarkable set of CD spectra are observed. In the data shown in Fig. 1B, it is seen that the addition of Cd" profoundly changes the simple CD spectrum of the Zn- MT (line I ) that arises from an overlap of charge transfer (RS--Zn2+) and peptide bands (1, 7, 8, 30-32). The native Zn7-MT exhibits the well-known CD spectrum for Zn-containing metallothioneins (30,31), with a maximum at 242 nm and a negative inflection near 228 nm, before the signal is

4540 Cadmium Binding to Metallothioneins

1.6

0

0, . 9

3

.o

210 230 250 270 296 wavelength /ns

FIG. 1. Absorption ( A ) and CD ( B ) spectra recorded during a titration of a solution of 10 nmol/ml of rabbit liver Zn7-MT 2 with Cd*+. Each line represents a spectrum recorded with different concentrations of Cd2+ added. There are 11 lines in each figure; recorded with 0.0 (native Zn-MT), 0.82, 1.65, 2.47, 3.29, 4.12, 4.94, 5.16,6.18,6.58, and 7.0 mol eq of Cd2+. The insets in each part of the figure represent changes in intensity monitored at specific wavelengths as a function of mol eq of CdZ+ added. The upper inset shows changes in the absorption at 250 nm. The lower inset shows changes in the CD intensity at 228,240,250,255, and 260 nm.

dominated by the peptide chirality below 220 nm. Addition of Cd2+, to saturation at 7 mol eq, results in the development of a derivative-shaped spectral envelope, line 11 in Fig. 1 B, that is characteristic of cadmium-saturated metallothioneins (32).

While the cadmium-saturated trace recorded at the end of the titration, line 11, is not unusual, the lack of an isosbestic point between 240 and 270 nm in Fig. 1B is unusual, especially for a reaction that is considered to involve the isomorphous replacement of zinc by cadmium (6, 10-15). Clearly, at least two species are involved as cadmium is added to the Zn-MT. The changes in both absorption and CD intensities a t several wavelengths are shown in the insets in Fig. 1. Absorption 250 nm (upper inset) is a traditional measure of cadmium content in MT, the scan shows a linear increase from 0 up to 7 mol eq of Cdz+, where saturation just begins (8, 28). The CD intensity scans (lower inset, Fig. 1B) are much more complicated, reflecting the apparent lack of systematic change in the CD spectral envelope as cadmium is added.

Because of the complexity of the spectral changes shown in Fig. lB, we replot the CD data in Fig. 2, where (Fig. 2A) shows the spectra recorded up to 4.12 mol eq Cd2+ added, and (Fig. 2B) shows the spectra for the second part of the titration, 4.12-7.0 mol eq of Cd" added. This separation of the data emphasizes that there are at least two phases in the reaction.

210 230 250 270 290 wavelength /nm

FIG. 2. CD spectra recorded for the two phases of the reaction of rabbit liver Zn7-MT 2 with Cda+. A, the first phase, addition of up to 4.12 mol eq of Cd", lines 1-6 are for 0 (native Zn- MT), 0.82, 1.65, 2.47, 3.29, and 4.12 mol eq of Cd" added. B , the second phase, addition from 4.12 to 7.0 mol eq of Cd2+, lines 6-11 are for 4.12, 4.9, 5.76, 6.18, 6.58, and 7.0 mol eq of Cd2+ added.

During the first phase, 0.0-4.12 mol eq of Cd2+ (Fig. 2A), the positive band of the Zn-MT is replaced by a new band that grows in intensity (to a maximum for the whole reaction at 3.29 mol eq of Cd2+), while red-shifting from a maximum at 242 nm with 0.0 mol eq of Cd", line 1, to 246 nm with 0.8 mol eq, and to 252 nm with 3.3 mol eq. There is only a very slight increase in resolution of the inflection near 230 nm during this stage. Significantly, there is no indication of bands at 228 or 240 nm that are characteristic of cadmium binding in either native metallothionein (22, 32) or a fragment (20). CD intensities at wavelengths that correspond to the three major, Cdz+-related CD bands (228, 240, and 255 nm), Fig. 2 (inset), confirm that there is insignificant growth in intensity at 228 and 240 nm up to 3.29 mol eq and that the band at 250 nm reaches a maximum near 3.3 mol eq.

Fig. 2B shows the CD spectra for the second phase of the reaction, from 4.12 to 7.0 mol eq of Cd2+ added. The addition of cadmium beyond 4 mol eq results in the development of new bands, at 228 and 240 nm, but, it is important to note, the intensity of the band between 255 and 260 nm does not increase as much as expected. While we anticipated some complexity in the spectral changes associated with the filling of the different domains in the presence of zinc, these data do not appear to fit the expected pattern. Significantly, the first saturation point occurs at 3.29 mol eq of Cdz+, not at 4.0 mol eq, and the CD spectral envelope for the Cd,Zn-MT species formed at this stage, does not resemble that of either native MT or cadmium-saturated a fragment.

MCD intensity is sensitive to the symmetry of the environ- ment of the coordinating groups around the Cd2+, but not to the arrangement of the amino acids around the two metal- binding site domains (26, 33). Thus the MCD spectra provide a view from the metal centers looking out. This property


contrasts the intensity mechanism for the CD spectrum. However, despite the differences in the intensity mechanisms between the absorption, CD, and MCD techniques, in general, each uses transitions to the same LMCT states, so that the band centers and widths should coincide in spectra recorded with the three techniques.

The MCD data recorded during the titration of Zn-MT with Cd", Fig. 3A, clearly confirm that the nonisosbestic changes observed in the CD spectra, Fig. lB , arise from the appearance of new bands that begin to develop during the titration. The changes in the intensities at 236 and 257 nm saturate sharply at 7 mol eq (inset, Fig. 3A). Further addition of Cd" up to 14 mol eq, results in only a slight reduction in intensity, indicating that the symmetry around the metals does not change very much once the two domains have been filled. We conclude that the additional Cd2+ must be binding away from the cluster sites. The spectrum recorded at 7 mol eq (line 8, Fig. 3A) is characteristic of tetrahedral coordination of Cd2+ by thiolate groups (33).

It is much easier to follow the changes in the coordination of the Cd" as monitored by the MCD spectrum, when the contribution from the Zn-(RSL chromophore is subtracted out. The data in Fig. 3B were calculated by assuming stoichiometric replacement of Zn2+ by Cdz+. Clearly, the major effect of the incoming Cd" on the MCD spectrum, is the growth of

+ . . . : . . . : . . . : . . . k 10

-1

4

-12

10

-1

4 -12

-234 , , . , . . . , . , . . , , , 1 210 230 2!50 270 290

wavelength /nm

FIG. 3. MCD spectra recorded during a titration of rabbit liver Zn7-MT 2 with CdB+. A, 10 lines are shown; these represent traces recorded following the addition of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 14.0 mol eq of Cd2+. The units of ACM are L mol" cm-l T-'; the corresponding zero field CD spectrum has been subtracted from the field-on spectrum. The inset shows changes in MCD intensity a t 236 nm (-) and 257 nm (- - -). B, synthesized MCD spectra for the titration of Zn-MT with Cd", showing only the Cd-S contribution to the spectral envelope. The remaining Zn-S chromophore has been subtracted from the data shown in ( A ) . There are seven lines, representing additions of 1.0 + 7.0 mol eq of Cdz+ in steps of 1.0 mol eq of Cd*'. The inset shows changes in MCD intensity at 228 nm (-, top), 236 nm (- - -, top), 250 nm (- - -, marked with *) and 257 nm (-, marked with A).

a new, derivative-shaped band as more Cd" is added. The intensities of the 228 nm (+), 236 nm (+), 250 nm (-), and 257 nm (-) bands are shown in the inset. These effects can be understood in terms of two spectral envelopes that are overlapping (Fig. 3B). Up to 4 mol eq of Cd", a positive band at 228 nm and a negative band at 250 nm intensify (top solid line and starred line in inset). After 4 mol eq of Cd", new bands at 236 nm (+) and 257 nm (-) intensify.

Titration of Apo-MT 2 with Cd2+-The absorption and Cd" spectra recorded during a titration of apo-MT are shown as Fig. 4 (A and B). Insets identify changes in the intensities at 250 nm in the absorption spectra (upper) and at 225,239,247, and 260 nm in the CD spectra (lower). The experiment was carried out in a different manner than for the titrations of metal-containing metallothioneins. Apo-MT was held at pH 2 while the appropriate aliquot of Cd2+ was added. The pH was then raised to 7. Thus, each line in Fig. 4 corresponds to a different solution, each with a different amount of CdZ+ added at pH 2.

The set of absorption spectra, Fig. 4A, closely resembles those obtained during the titration of the Zn-MT with Cd2+ (Fig. lA), with the exception that the starting trace now exhibits no intensity above 230 nm. The absorption intensity

I

P 0 " 3

210 230 250 270 290 wavelength /nm

FIG. 4. Absorption (A) and CD ( B ) spectra of solutions of rabbit liver apo-MT 2 recorded at pH 7, following the addition of aliquots of Cd2+ at pH 2. Each line represents a spectrum of a different solution taken from the same stock solution of apo-MT. The concentration of protein was 10 nmol/ml. There are 11 lines in the figure: line 1, apo-MT at pH 2; line 2, apo-MT at pH 7; lines 3- 11 for apo-MT with different concentrations of Cd" (1.06, 2.14, 3.5, 5.0,5.95,6.05, 6.92, 7.61, and 8.82 mol eq). The insets in each part of the figure represent changes in intensity monitored at specific wavelengths as a function of mol eq of Cd" added; upper inset, at 250 nm and lower inset at 225,239,247, and 260 nm.


at 250 nm (inset, Fig. 4A) shows the same linear increase from 0 to 7 mol eq of Cd2+ that was found with the Zn-MT, Fig. 1A. Major changes in the CD spectra, Fig. 4B, take place between 0 and 4 mol eq of Cd", after which a saturation point is gradually reached by 7 mol eq (inset, Fig. 4B). Significantly, the bands near 225,240, and 260 nm develop continuously as Cd2+ is added from 1 up to 7 mol eq. Additionally, for all spectra other than up to 2.0 mol eq, there is no change in the band center of the band that grows in near 260 nm. This behavior is quite unlike that observed in Fig. 1B for Zn-MT. We expected that this set of spectral data would resemble that shown in Fig. 1B once the contribution from the Zn-S chromophore had been subtracted from the Zn-MT data (see Fig. 10 below). Quite clearly, these data are different.

Titration of Zn-a M T with Cd2+-Fig. 5 shows CD spectra recorded during a titration of zinc-saturated a fragment. Aliquots of Cd2+ were added to the Zn4-a MT at pH 7.5. Important features in these data are the growth of bands at 228, 240, and 256 nm. While there appears to be less of the complexity seen for Zn7-MT (Fig. lB) , there is still considerable intensity that initially grows in between 240 and 250 nm. The effect is not as pronounced as with Zn7-MT, but it does result in the 255-nm band red-shifting as Cd2+ is added. Thus, unlike the titration of apo-MT with Cd2+, Fig. 3B, the first aliquot of Cd", 1 mol eq, results in the development of a band near 250 nm, with no bands at 223 or 240 nm.

Titration of Apo-a M T with CdZ+-Fig. 6 shows the CD spectra recorded during a titration of apo-a MT with Cd". Cd2+ was added in mol eq aliquots to 10 nmol/ml solutions of a fragment at pH 2. The pH was then raised to 7 and the spectra recorded. Surprisingly, this set of spectral data are remarkably similar to the data shown in Fig. 4 8 , where apo- MT binds Cd" in both a and p domains. CD intensity develops at the same three wavelengths throughout the titration, at 225, 240, and 259 nm. There is no indication of any intensity near 250 nm. Saturation of the signal occurs between 5 and 6 mol eq of Cd". In view of the similarities between

2 4 6 m1 eq

210 230 250 270 2! wavelength /nn

FIG. 5. CD spectra recorded at pH 7, following the addition of aliquots of CdZ+ to a 10 nmol/ml solution of rat liver Zn-a MT. There are 13 lines in the figure: line 1, apo-a MT at pH 2; line 2, Z q - a MT at pH 7.5; then lines 3-13 are for Zn-a MT with different concentrations of Cd2+ added (0.5-5.0, every 0.5, and line 13 has 6 mol eq of CdZ+ added). The inset shows changes in intensity a t 223, 240, and 257 nm, as a function of mol eq of Cd2+ added.

20

-5

2 -30

-55

t 0 2 4 6 6

, , , , , , , I I , , , ) . -1 .p

t 210 230 250 270 290

wavelength /nm

FIG. 6. CD spectra of rat liver apo-a MT (10 nmol/ml) recorded at pH 7, following the addition of Cda* at pH 2. Each line represents a spectrum of a different solution taken from the same stock solution of apo-a MT. There are eight lines in the figure: line 1, apo-a MT at pH 7; lines 2-8 are for apo-a MT with different concentrations of Cd2+ (2.0,3.0,4.0,5.0,6.0,7.0, and8.0 mol eq Cd2+). The inset shows changes in intensity monitored at 223,240,249, and 259 nm as a function of mol eq of Cd" added.

the data for apo-a and apo-MT, it is also surprising that neither of these sets of spectral data resemble the data shown in Fig. 1 B for the titration of Zn7-MT with Cd2+. Clearly, the metal replacement reaction in which Zn7-MT-Cd7-MT involves a different mechanism, than is adopted by any of the apo-MT species, because the derivative signal that develops at low mol eq of Cd2+ in Figs. 3 and 4 is not observed in Fig. 1 B until after 4 mol eq of Cd" has been added to Zn-MT.

Titration of Apo-p M T with Cd2+-Fig. 7 shows the CD spectra recorded during a titration of metal-free apo-/3 fragment. Aliquots of Cd" were added to 10 nmol/ml solutions of apo-8 MT at pH 2. The pH was then raised to 7 and the spectrum recorded. It is striking that there are no changes in the spectrum that are recognizably characteristic of Cd2+ binding to MT (32). The spectral changes are very slight with very little intensity in any of the bands. A maximum intensity is reached at 2 mol eq of Cd", rather than the 3 mol eq expected for the 13 domain (10-14). Addition of Cd" in excess of 2 mol eq results in a decrease in the overall intensity, as indicated by the 250-nm intensity. This lack of complete filling has been noted previously for apo-MT as well, where Cd2+ binding to apo-MT results in a C&-MT rather than a Cd,-MT species (34). The titration data for apo-p MT pro- vides direct evidence that it is the p domain itself that results in the poor binding for the 7th Cd2+. Indeed, these data imply that there is little cooperativity in the binding of Cd2+ to the @ fragment because it would be expected that the last metal atom in would bind with the largest binding constant. The lack of intensity in the spectrum recorded for the ZnZ-MT species (the species for which the maximum CD intensity was recorded) appears reasonable in view of the lack of significant intensity changes during titrations of apo-MT with Cd2+ (Fig. 3) from the CQ-MT position to Cd7-MT that is, filling the 0 domain does not appear to result in any major increase in CD intensity.

Cadmium Binding to Metalhthioneim 4543

10

7

5

2

B o

-2

-?

!O 230 240 2jO 260 270 280 290 300 Wavelength / na

FIG. 7. CD spectra of calf liver apo-B MT (10 nmol/ml) recorded at pH 7, following the addition of Cdz+ at pH 2. Each line represents a spectrum of a different solution taken from the same stock solution of apo-j3 MT. The data have been smoothed with a digital filter (21). There are seven lines in the figure: line I, ap0-B MT at pH 2; line 2, apo-j3 MT at pH 7; then lines 3-7 are for apo-j3 MT with different concentrations of Cd2+ added (0.5,1.0,1.5,2.0,2.5, and 3.5 mol eq). Note, the intensity at 250 nm increases to a maximum at 2.0 mol eq, then decreases. The inset shows changes in intensity monitored at 250 nm as a function of mol eq of Cdz+ added.

Spectral Properties of CdeZn3-MT Formed in Vitro-The CD spectrum of CQ,Zn3-MT formed in oitro from Zn-MT does not resemble the spectrum recorded for native Cd,Zn- MT, Fig. 1B (line 6), yet this species has the same stoichiometric composition of metals as that found in native samples of the protein (28). There is little CD intensity at 228 nm (+), no 240 nm intensity (-), and the red band is at 252 nm. We carried out five experiments, described below as A-E, to investigate the spectral properties of the synthetic native species.

A-A solution of "synthetic" CQ,Zn3-MT was warmed to 68 "C from 20 "C while CD spectra were recorded continuously, Fig. 8A. The solution was then slowly cooled back to 20 "C, Fig. 8B. The data in Fig. 8A show that the specific spectral characteristics that are associated with both Cd,-MT and the native protein (distinct bands: at 228, 240, and 260 nm), can be obtained simply by warming the protein. The transition from a spectrum that resembles the synthetic native, to one that resembles the true native MT, occurs isos- bestically by 50 "C. The major features expected for native C&,Zn3-MT are all observed now. Two questions are raised (i) are these changes reversible; that is, are they just a temperature effect, and (ii) do these three bands (228, 240, and 260 nm) exist before warming, and just become resolved at 70 "C, or are they new bands?

Fig. 8B shows that the effect is permanent. The cooling part of the temperature cycle results in only a minor blue shift, while a second cycle from 20 to 69 "C, Fig. 8C, completely returns the "hot" spectrum. That the resolution is maintained at room temperature after the warming cycle suggests that the spectrum changed in response to changes at the metal- binding sites. Finally, Fig. 8 0 shows that the protein retains

23

B

-23

-70 55

20

B

+

210 230 250 270 2! wavelength / nm

t t

I I

1 210 230 250 270 290

wavelength / nm

FIG. 8. Temperature dependence of the CD spectrum of Cd,,Zns-MT formed by adding 4 mol eq of Cd2+ to Zn7-MT. A, the effect on the CD spectrum of C&,Zn3-MT of raising the temperature from 20 to 68 "C. Spectra are plotted for 20, 30,40, 50, 55, 58, 64, and 68 "C. The inset shows the change in intensities at 250 and 240 nm as a function of temperature. B, the effect on the CD spectrum of cooling from 68 to 20 "C, spectra are plotted for 68, 47, 37, 27, and 20 "C. C, a second warming cycle, 20-69 "C, spectra are plotted for 20,40,52,61, and 69 "C. D, titration of the sample from (C), at 20 'C, with Cd2+. The traces correspond to total additions of 5, 6, 7, 8, 11, and 13 mol eq of Cd2+. The inset shows the change in intensities at 228,240, and 260 nm.

its native property for binding Cd". A fully resolved Cd,-MT CD spectrum is observed following the addition of 3 further mol eq of Cd2+ at 20 "C, even after 2 cycles between 20 and 70 "C; in particular, the bands at 228 and 239 nm are fully resolved, just as is found for protein that has not been heat- treated (Fig. 1B). The metal-binding properties of the rabbit Zn7-MT 2 protein are apparently unaffected by the thermal cycle to 70 "C for the long periods of time required to measure the CD spectra (about 30 min/spectrum). We note, that at higher temperatures, up to 80 "C, permanent changes were observed in CD spectra recorded during the titration. This suggests that irreversible change occurs to the protein. Thus, it appears that heat treatment at above 70 "C should be used cautiously during isolation of metallothionein.

B-A pH cycle also resulted in a red shift of the 250 nm band toward 260 nm and development of bands at 228 and 239 nm. 4 mol eq of Cd2+ were added to Zn-MT at pH 7 (Fig. 9A), the pH was dropped to 2, then raised to 7 once again. A spectrum similar to that recorded after the thermal cycle, Fig. 8, was found. In this experiment all the metal dissociates from the metallothionein below pH 2, remetallation occurring as the pH is raised (32, 35). Clearly, the Cd2+-binding site changes during this cycle such that the CD spectrum resembles that observed for native C&,Zn3-MT.

C-EDTA can be used as a competitive chelator for Zn2+ in MT (17). If EDTA is added to a solution of Cd,Zn-MT, the Zn2+ will be removed first, followed at a slower rate by the


BO

A f i low pH I

70 - +EDTA I

210 230 250 270 290 wavelength / nm

FIG. 9. Effect of pH and the addition of EDTA on the CD spectrum of Cd,Zn-MT. A, the effect of acidification followed by neutralization on the CD spectrum of C&,Zn3-MT formed by the addition of 4 mol eq of Cd" to Zn7-MT. There are six lines in the figure; line I is the native Zn-MT 2 at pH 8; line 2, with 4.0 mol eq of Cd2+ added at pH 8; lines 3-6 depict spectra recorded in the sequence, at pH 3.15, 2.1, 6.7, and at pH 7.3. E , the effect on the spectrum of C&,Zns-MT of adding EDTA (1.0 mM). There are eight lines in the figure; spectra were recorded for the native Zn-MT, with no EDTA (line 1); with 4.0 mol eq of CdZ+ added, with no EDTA (line 2); then lines 3-8 at intervals of 15 up to 90 min following addition of EDTA.

Cd". A series of CD spectra were recorded over a 2-h period after addition of EDTA to the synthetic C&,Zns-MT (Fig. 9B). After 2 h, no Zn2+ remained bound to the protein. There were no changes in the band positions in the CD spectrum; intensity due to Cd" bound to the protein gradually dimin- ished. In particular, the 252 nm band did not red-shift and there was no increase in resolution at either 228 or 240 nm.

D-We examined the effect of cadmium binding at elevated temperatures in order to determine whether the temperature dependence observed in Fig. 8 required the prior presence of the Cd2+ at 20 'C. Fig. 10 shows the complicated set of CD spectra recorded during a titration of Zn7-MT with Cd2+ that was carried out at 65 "C. These data can be compared with Fig. 1B where the titration was carried out at 20 "C. A quite different pattern is found at 65 "C. Significantly, the band near 250 nm continuously red-shifts toward 260 nm with every addition of Cd". Bands at 228 and 239 nm begin to form as early as 3 mol eq of CdZ+ added. There are no isosbestic points. These spectra do not resemble those recorded for either apo-MT or apo-a MT.

E-Finally, we carried out a similar set of experiments using 80% D,O/H,O in order to examine whether hydrogen bonding was implicated in the CD spectral effects. In Fig. 11A, C&,Zn3-MT was formed by addition of 4 mol eq of Cd" at 20 "C in 80% DzO. The CD spectrum ( l i n e 2) closely resembles that observed in 100% H20 solutions (Fig. 1B). The solution was heated and spectra recorded (lines 3-11). These data show that the development of the derivative 2391 260 nm feature begins almost immediately, with saturation occurring by 40-45 "C. This compares with 55-60 "C for the

60 t

210 230 250 270 290 wavelength /nm

FIG. 10. CD spectra recorded during a titration of Zn7-MT 2 with Cda+ at 66 "C. There are 11 lines on the figure. Line 1 is the native Zn-MT at 65 "C. Spectra (lines 2-11) were recorded following additions of aliquots of Cdz+ to a total oE 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0,8.0,10.0, and 12.0 mol eq. The inset shows the changes in intensity at 228,240,250,255 and 260 nm.

same experiment carried out in 100% HzO (Fig. 8A). In Fig. 11, B-D, we show titrations of Zn-MT carried out in 80% DzO solutions at 20, 30 and 40 "C. The presence of both D20 as the solvent and the elevated temperatures clearly changes the pattern of CD spectra recorded. The last trace shown in each panel is the first spectrum that exhibited full 228-, 239-, and 260-nm character. This occurs at 4 mol eq at 20 "C and 3 mol eq at 30 and 40 "C.

DISCUSSION

In the region above 210 nm, absorption and CD intensity in the spectra of metallated metallothioneins are considered to arise from LMCT transitions between the metal and the coordinating thiolate groups (1, 7, 19, 26, 30, 31, 33). Numer- ous optical spectra of metallothioneins metallated with different metals, and of model compounds, where coordination is by thiolate analogues of the metallothionein-binding site, indicate that the wavelength and the intensity of the bands observed to the red of the 210-nm peptide transitions is determined by the energies of the excited states on the metals coordinated by the thiolate groups (26, 30,33).

Because the CD spectrum is such a good probe of changes that are taking place around the metals in the binding site, we have found that the most valuable experiments have been those in which we have recorded sets of spectral data as metals already bound to the protein are replaced by incoming metals that have a greater binding constanc thus, copper will displace both cadmium and zinc (19). We use sets of related CD spectra to indicate whether the incoming metal binds in a systematic fashion to the protein (32). If the energy of the charge transfer transitions remained constant from metal to metal, sets of spectra recorded as an incoming metal displaced an in situ metal and plotted in a three-dimensional fashion versus mol equivaient of metal added would map out the changes in the protein's overall structure that occurred in order to accommodate the incoming metal. Because the energies of the charge transfer transitions do change with different


70

30

4 - 10

-50

70

t t

20' I 30

4 -10

-50

210 230 250 270 290 wavelength /nn

C 36

.O 230 250 270 26 wavelength /nm

FIG. 11. Effect on the CD spectra recorded during titrations of Zn-MT 2 with Cda+ of using 80% DsO/HaO as a solvent. A, temperature dependence of the CD spectrum of Cd,,Zn3-MT. Line I, Zn7-MT line 2, Cd,,Zn3-MT, lines 3-11, spectra at temperatures from 20 to 70 "C. The inset shows the changes in intensity at 255 and 260 nm versus temperature. B , titration of ZnT-MT with Cd2+ in 80% D2O at 20 "C. There are five lines: line 1, Zn-MT lines 2-5 for additions of 1-4 mol eq of Cd2+ in aliquots of 1 mol eq. Line 5 represents the first point that the derivative signal appears, at 4 mol eq. C, titration of Zn7-MT with Cd2+ in 80% D20 at 30 "C. There are four lines: line I , Zn-MT; lines 2-4 for additions of 1-3 mol eq of Cd2+ in aliquots of 1 mol eq. Line 4 represents the first point that the derivative signal appears, at 3 mol eq. D, titration of ZnV-MT with Cd2+ in 80% D20 at 40 "C. There are four lines: line I , Zn-MT, lines 2-4 for additions of 1-3 mol eq of CdZ+ in aliquots of 1 mol eq. Line 4 represents the first point that the derivative signal appears, at 3 mol eq.

metals, the CD spectra are much more complicated than the simple model described above would imply, and the structural information is more difficult to extract. However, it is possible to determine both stoichiometric and structural information if enough spectra are recorded so that isosbestic points can be detected and saturation positions measured. The simplest examples are expected to be those cases where isomorphous replacement of the in situ metal occurs. We anticipated that the titration of Zn7-MT with Cd" would yield a set of spectra that would be connected by good isosbestic points and that we would see a summation of the titration data measured for the isolated a and fragments.

Analysis of the CD Data Recorded during the Titration of Zn7-MT 2 with Cd2+-The data shown in Fig. 2, A and B are very much more complicated than that recorded for apo-MT (Fig. 4). Clearly, these data are not connected by isosbestic points, and these data are not a simple summation of the spectra of the a and ,8 fragments (Figs. 6 and 7); neither do these spectra resemble data obtained for apo-MT (Fig. 4). Although there is some nonisosbestic behavior as both apo- MT and apo-a MT bind Cd2+ at low mol equivalent values (Figs. 4 and 6), systematic increases in CD intensity are found at each of the three characteristic Cd-MT wavelengths, 228, 240, and 260 nm, respectively (8, 31, 32). Initial, qualitative analysis of the Zn-MT spectral data (Fig. 2) seemed to suggest that partial saturation between 3 and 4 mol eq of added Cd2+ was followed by a second saturation point at 7 mol eq of Cd2+. Comparison between the data for apo-MT (Fig. 4) and Zn4-a

MT (Fig. 5) indicates that there is something very special about the titration of the whole protein (Z&Z@-MT) that is related both to the presence of the Zn2+ and to the presence of the two domains.

We examined the set of CD spectra recorded for Zn-MT (Fig. 1B) more closely by subtracting stoichiometrically-ad- justed spectral intensity arising from the remaining Zn-S chromophore from the overall CD spectrum of the mixed Cdn,Zn(7-n,-MT. By doing this, we obtain a set of spectra with intensity due solely to Cd(SR)4 chromophore (Fig. 12). This set of spectral traces does indeed change in a more systematic fashion than the original data does. Significantly, (i) within the limits imposed by the noise, there appears to be an isosbestic point at 234 nm for the whole titration and a second one at 255 nm that forms after 4 mol eq of Cd2+ have been added. (ii) The positive band maximum near 250 nm, red- shifts toward 260 nm, the characteristic value for Cd-MT (26, 28) but only after approximately 4 mol eq of Cd2+ have been added (inset, Fig. 10). (iii) Bands at 227 nm (+) and 240 nm (-) grow in intensity only after 4 mol eq of Cd2+ have been added. (iv) The characteristic CD spectrum of the CY domain, that we would expect to be fully developed at 4 mol eq of Cd2+, only develops after 4 mol eq Cd2+ have been added. (v) The red band between 250 and 260 nm, the "260 nm band," does not intensify as much as expected between 4 and 7 mol eq of Cd", considering its magnitude at the 4 mol eq point. Thus, while the inset shows a gradual increase at 259 nm, the band maximum is actually moving from 250 toward 259 nm (Fig. 11A).

The isosbestic changes are observed for the apo-MT species

60 t

210 230 250 270 290 wevelength /nm

FIG. 12. CD spectra recorded during a titration of rabbit liver ZnV-MT 2 with Cd*+ that arise solely from the Cd-S chromophore. Each line represents a synthetic spectrum for Cd- MT that was initially recorded with different concentrations of Cd2+ added; the original data are shown in Fig. 1B. The traces were calculated by subtracting the appropriate fraction of signal intensity due to remaining Zn-MT from the observed Cd,Zn-MT spectrum. A digital filter has been used to smooth these data (19). The inset shows changes in CD intensity at 227, 240, 252, and 259 nm as a function of mol eq Cd2+ added.


emphasizes how unusual the spectra of the Zn-MT titrations are. For apo-MT there is a shift in the band maximum of the positive CD band in the 255-nm region during the titration of the apo-MT up to the 2 mol eq of Cd2+ point (Fig. 4). In particular, intensity first appears between 240 and 260 nm, then disappears after 2 mol eq of Cd2+ have been added. Similar behavior is also observed for the apo-a MT titration (Fig. 6).

Thus, the intensity of the bands in the CD spectrum of Cd2+-containing metallothioneins increases systematically with the mol eq of Cd", as long as the titration starts with the metal-free apoprotein.

CD spectral changes are nonsystematic only for cadmium binding to Zn-MT or in the early stages of the reactions of Zn-a MT and apo-MT. Several effects could result in the nonisosbestic behavior observed in Fig. 1B. Three effects seem to be most likely, (i) nonisomorphous or even, nonsto- ichiometric replacement of Zn2+ by Cd", (ii) a wedge effect on the binding-site chelate cage, such that incoming Cd2+ expands the binding site creating strain within the clusters, and (iii) effects of clustering on the CD-active excited states. We address each of these points below.

(i) We confirmed that the replacement was stoichiometric by eluting a solution of Zn7-MT 2, containing 4 mol eq of Cd2+, through a Sephadex G-25 column. The metal content and spectral properties of the solution were compared before and after passage down the column. There was no change in the CD spectrum; the trace recorded after passage down the column resembled that shown as line 6 of Fig. 1B. AAS measurements indicated that four Zn2+ were released for four Cd2+ bound. There were no effects on the CD spectrum attributable to the excess free-Zn2+ prior to separation on the column. Because zinc and cadmium both occupy sites of tetrahedral symmetry (15), we cannot attribute the spectral changes to nonisomorphous behavior.

(ii) Cd-S bonds are 10% longer (29) than Zn-S bonds (28) in model thiolate compounds, so some expansion of the a and 0 domain cages of thiolates is required as zinc is replaced by cadmium. If the energy of the LMCT bands was dependent on the structural geometry of the chelating thiolates, then we would expect the band maxima to change as cadmium replaced the existing zinc. Fig. 13A plots the band center of the positive CD band observed between 250 and 260 nm, against mol eq of Cd". Strikingly, most of the red shift (upward drection on the plot) occurs only after 4 mol eq of Cd2+ have been added. The effect is even more pronounced when the wavelength of the crossover point between the 240-nm negative trough and the 260-nm peak is plotted against mol eq of Cd2+ added (Fig. 13B). This diagram illustrates the movement of the main CD bands in the 230-260-nm region. Clearly, the change in the CD spectrum occurs after 4 mol eq of Cd2+ has been added. Finally, Fig. 13c illustrates how the intensity of the 250-260- nm positive band changes as Cd2+ is added. All the increase takes place for the first 4 mol eq; when the band red-shifts the 260 nm, there is little increase in intensity. We would have expected that if the cage had to expand to accommodate each incoming Cd", that this would result in a smooth shift in wavelength, not a process characterized by a step at the 4 mol eq position.

Finally, (iii) we believe that the clue to the observed spectral effects lies in Fig. 13, where the cross-over point in the 250- nm region of the CD spectrum is plotted. Clearly, two new bands (at 240 and 260 nm) form at the expense of the 252- nm band. However, significantly, whatever changes the CD spectrum does not noticeably affect the intensity of absorption spectrum in the 250-nm region. If a new species is being

2 5 0 1 . . . I . , . 0 2 4 6 8

240 I . . . 0 2 4 6 8

15.0 I

0 2 4 6 8 mol eq

I . . .

FIG. 13. Changes in band positions (A) , crossover points ( B ) , and peak heights (C), in the CD spectra of the Cd-S chromophore recorded during a titration of Zn,-MT with Cda+. The complete data are plotted as Fig. 8. A, the location of the band in the 250-260 nm region versus mol eq of Cd2+ added. B, the position of the crossover point between 239 and 260 nm, plotted as a function of mol eq of Cd2+ added. C, the maximum CD intensity in the 250-260 nm region versus mol eq of Cd2+ added. The dotted line indicates the 4 mol eq of Cd" point in the titration.

formed, the spectral data described above indicate that it only does after 4 mol eq of Cd2+ have been added to Zn-MT. In the domain-specific model, cadmium would, at that point, fully occupy the a domain. Why should the spectrum change so much for only the Zn-MT, and not the apo-MT? Clearly, the cadmium must occupy sites distributed across the whole protein. Then after 4 mol eq of Cd2+ are added, clustering can begin to occur. Starting with seven zinc sites, the first cadmium binds randomly across both domains (36). As the amount of Cd2+ increases, in the distributed model, both a and f l domains are occupied on a statistical basis. However, after 4 mol eq of Cd2+ have been added, groups with adjacent cadmiums begin to form, so that (SR)3-Zn-SR-Cd-(SR)3 groups are converted into groups in which two cadmiums are linked by a bridging thiolate, (SR),-Cd-SR-Cd-(SR),. Thus, in the early stages of the titration there are few Cd-SR-Cd groups. At the 4-mol eq point, Cd-SR-Cd groups begin to dominate, and from Fig. 2 it does appear that the CD spectrum is sensitive to this change in the nature of the attached group, i.e. -SR-Zn uersus -SR-Cd.

A Model to Explain the Spectroscopic Results-While the results of many experiments involving metal binding to metallothioneins have been discussed in the literature in terms of different models, the most relevant to the data presented in this work are W d NMR studies involving Zn- MT and apo-MT (10-14), the selective blocking experiments of Bernhard et al. (36), for cadmium binding, and the selective digestion techniques of Winge and co-workers (6, 17, 18) that have identified metal specificity in the fragments for a very


wide range of other metals. Although the experimental conditions for many of these experiments are dramatically different from the CD experiments described here, and because our experiments are carried out with very dilute solutions, near pH 7 and with very low concentrations of additional metals, our data show so much fine detail that we have attempted, in the model described below, to accommodate each of the conclusions described previously. It is clear that our model has to explain two major observations: (i) the spectral properties as seen by the CD experiment following the addition of 4 mol eq of Cd2+ to Zn7-MT, and (ii) the spectral changes observed for the synthetic native, Cd,Zn-MT upon warming.

The CD data presented here support a "distributed model" for cadmium binding to Zn7-MT and a "domain-specific model" for apo-MT. This means that for Zn7-MT, once 4 mol eq of Cd2+ have been added, that statistically, two cadmiums are bound in each domain. The essential feature of our model is that the CD spectral effects can be understood in terms of exciton coupling between adjacent pairs of the Cd(SR)4 chromophore. We suggest that there is only one optical transition near 250 nm for isolated Cd(SR), chromophores. As the number of Cd(SR), groups increases, so the absorption intensity at 250 nm simply rises. But when adjacent Cd(SR), units are formed (the onset of clustering), interaction results in exciton splitting of the excited states. 2 CD bands, of equal and opposite intensity (37) are then observed. We associate these bands with the 240 (negative) and 260 nm (positive) CD bands observed at high cadmium levels. Thus, we propose that it is only after 4 mol eq of Cd2+ have been added that there exist in the a domain cluster groups of the type (SR)3-

Thus, we interpret the CD spectral changes observed for all the MT species in terms of the effects of clustering on the CD spectrum of the Cd(SR), chromophore. The derivative band at approximately 240/260 nm is a marker for cluster formation, while the 250-nm band identifies isolated Cd(SR), units. The data for Zn-a MT (Fig. 5) suggest that the CD spectrum detects the binding of one or two isolated Cd(SR), units mixed among the Zn(SR)4 units initially, followed by clustering at the 2, 3, and 4 mol eq level. The derivative CD feature does not appear to require complete C&(SR),, clusters. This implies that rather than spontaneously form S-Cd- S-Cd groups within the a domain, that even with just the 4 Zn2+ cluster in the a fragment, that RS-Cd-SR-Zn-SR is the preferred arrangement until there is no alternative, at about the 2-mol eq point, whereupon cadmium clusters do begin to form.

The CD spectra for apo-a MT, Zn-a MT, and apo-p MT suggest that the derivative features associated with the CD spectrum of both native Cd,Zn-MT and Cd7-MT arise from Cd(SR), units in the a domain; there is little CD intensity from Cd(SR), units in the ,8 domain. This lack of intensity may arise from the near-planarity of the three Cd2+ atoms in the p domain (15) which effectively introduces a plane of symmetry.

For the apo-MT and apo-a MT, we find that a band at 250 nm grows in intensity for the first 1 or 2 mol eq of Cd" added, to be followed by development of the derivative feature. These data then suggest that clusters only begin to form after 2 mol eq of Cd2+ have been added, The distinctive appearance of the isosbestic growth of both the 240- and 260-nm bands at the expense of the positive 250-nm band argues that a new spectroscopic species is formed after this stage in the titration. Clearly, Cd2+ binds preferentially into the a domain when binding to apo-MT. This conclusion is supported by the results of '13Cd NMR experiments carried out at concentra-

Cd-SR-Cd-(SR),.

tions some 500 times greater (10-14). Both in our data (Fig. 1B) and that of Nettesheim et al.

(16), we find that spectra of protein that has the same stoichiometric ratio for cadmium and zinc as the native protein (i.e. C&,Zn3-MT) do not resemble spectra of native MT. We conclude from the experiments illustrated in Figs. 8-12 that the effect of temperature or pH cycle is to redistribute Cd2+ occupying isolated sites (Le. Zn-SR-Cd-SR-Zn) into clustered sites in the a domain. The cadmium must be quite mobile under these experimental conditions. Thus, the protein species with cadmium in both domains (following metal binding in a distributed fashion) is thermodynamically unstable with respect to domain-specific binding. Clearly, conclusions reached concerning the domain specificity of a particular metal, where the data are obtained by chemical means, might find a dependence in the data on the type of chemistry involved. We tested this by repeating the Winge type of selective digestion experiment (6) for Zn-MT and apo-MT. The results3 do indicate that there is significant mobility of the metals bound to MT, because, even when only 2 mol eq of Cd2+ have been added to Zn-MT, a significant yield of a fragment is obtained.

Our model does then explain many of the spectroscopic features observed as cadmium binds to metallothionein. Cad- mium binding to Zn7-MT involves a distributed mechanism; the appearance of a derivative signal only after 4 mol eq of Cd2+ have been added, suggests a complete lack of cooperativity, and clusters form only when there is no other isolated zinc that can be replaced. Because of the well-known examples of clustered cadmium and zinc complexes in inorganic chemistry (28, 29), as well as the example of the native protein itself, it is curious that the cadmium does not form clusters of the type Cd-SR-Cd, but rather Cd-SR-Zn clusters at lower mol ratios.

The temperature cycle, and the binding studies carried out at elevated temperatures, suggests that the distributed species is a kinetically controlled product, so that the thermodynamically more stable product does involve domain-specific binding. Why should the lower energy, domain-specific mechanism not operate for Zn-MT? Perhaps, when zinc already occupies the same type of binding site, the thermodynamic gain is not realized because the binding site is "frozen" in place by hydrogen bonding that holds the two clusters to- gether. The Cd-S bond length in [c&(sC6H5),,]" is 246 pm (29) compared with 228 pm for Zn-S in the analogous zinc complex (28). It is possible that the incoming Cd2+ initially occupies sites that are not connected by a bridging thiolate because the strain in expanding the cage is less when adjacent positions are occupied by zinc. From X-ray data of Stout and co-workers (15), it is known that the two domains lie against each other, with possible connections by amino acids at positions KZ5, CZ6, TZ7, and SZ8 in the p domain, and A4', K43, and C" in the a domain. We envisage that hydrogen bonds formed initially during metallation of apo-MT with zinc hold the amino acid backbone, and thus the two metal binding domains, in a configuration specific to the Zn-S bond lengths (228 pm (28)). The addition of the Cd2+ into either domain causes the cage size to change; however, the relative geome- tries of the domains cannot change unless the hydrogen bonds break. With the apo-MT it would be expected that there would be a significant template effect as the protein bound metal ions in sequence. This would tend to favor the location of metals in adjacent sites, hence clustering would predomi- nate, as observed (Figs. 4 and 6). The factors that contribute to the enthalpy change for the reaction (Cd2+ + apo-MT) are

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

4548 Cadmium Binding

quite different than for (Cd2+ + Zn7-MT). Titrations at elevated temperatures allow the rearrangement of cadmium from distributed sites into domain specific sites to take place on a CdZ+ by Cd2+ basis. Both the pH and thermal cycles allow the cadmium to bind in a domain-specific manner, which results in the CD bands characteristic of the native protein being observed before saturation with Cd2+. Binding experiments carried out in DzO solvents exhibited a greater extent of domain specificity than observed for pure H20 solvents, pre- sumably because the hydrogen bonds are weaker than a DzO solvent so rearrangement takes less thermally supplied acti- vation energy. This would suggest that for NMR experiments, more clustering should be observed because there would be a greater preponderance of Cd-SR-Cd units in the (Y domain.

CONCLUSIONS

1) Cadmium binds in a distributed manner to Zn7-MT at room temperatures. Whereas cadmium binds to apo-MT in a domain specific manner, the (Y domain filling before the ,B domain.

2) Protein with the native stoichiometric ratio of Cd2+ and Zn2+ present, C&,Zn,-MT, exhibits a CD spectrum quite unlike that observed for the native protein. Warming the protein to 68 "C results in the appearance of spectral features associated with native Cd,Zn-MT.

3) The onset of cluster formation in the (Y domain can be associated with the appearance of a derivative feature in the CD spectrum. It is suggested that this feature is characteristic of S-Cd-SR-Cd-S units in the (Y domain.

4) The CD spectrum from Cd(SR), units in the ,B domain is much weaker than the spectral intensity from the (Y domain.

Acknowledgments-We wish to thank Dr. William Browett and Robert Kitchenham for expert programming assistance. We wish to thank Professor P. A. W. Dean at the University of Western Ontario for stimulating discussions.

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