S Cluster Stoichiometry in Fucus vesiculosus

Chem. Res. Toxicol. 2006, 19, 365-375

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Determination of the Cd/S Cluster Stoichiometry in Fucus Wesiculosus Metallothionein Maureen E. Merrifield,† Jennifer Chaseley,‡ Peter Kille,‡ and Martin J. Stillman*,† Department of Chemistry, UniVersity of Western Ontario, London, Ontario N6A 5B7, Canada, and Cardiff School of Biosciences, Biomedical Building, Museum AVenue, P.O. Box 911, Cardiff CF103US, United Kingdom ReceiVed July 27, 2005

The seaweed Fucus Vesiculosus is unusual when compared with other algal species, in that it can survive in toxic-metal-contaminated aquatic environments. The metallothionein gene has been identified in F. Vesiculosus by Kille and co-workers (Morris, C. A., Nicolaus, B., Sampson, V., Harwood, J. L., and Kille, P. (1999) Biochem. J. 338, 553), which suggests a possible protective mechanism against toxic metals for this species. We report the first detailed study of the metal binding properties of F. Vesiculosus metallothionein using UV absorption, circular dichroism (CD), and electrospray mass spectral techniques. The overall metal-to-sulfur ratios of this novel algal protein when bound to divalent cadmium and zinc were determined to be Cd6S16 and Zn6S16, respectively. Mixed Cd/Zn species were also formed when Cd2+ was added to the Zn-containing Fucus metallothionein. Only one conformation was identified at low pH for the native protein. Analysis of the UV absorption, CD, and ESI-MS spectral data recorded during stepwise, acid-induced demetalation supports a two-domain structure for the protein, with two 3-metal binding sites. The data suggest that one of the domains is significantly less stable than the other, and we tentatively propose from the arrangement of cysteines in the sequence that the two domains are M3S7 and M3S9 (where M ) Cd2+ or Zn2+). While the M3S9 cluster is known in the β clusters of crab, lobster, and mammalian metallothioneins, the M3S7 is a hitherto unknown cluster structure. Metallothionein in F. Vesiculosus is thought to act as a protective mechanism against incoming toxic metals. The metal binding studies reported are a putative model for metal binding in vivo. Introduction The seaweed species Fucus Vesiculosus sequesters large amounts of copper and zinc from the aquatic environment and binds these metals in sulfur-rich vesicles termed physodes (1), with levels between 103 and 104 times greater than the water in which the species inhabits (2). Metal sequestration may occur at four locations: (i) in the external medium, (ii) at the cell wall, (iii) at the plasmalemma, and (iv) within the cell (3). F. Vesiculosus has been described as an ideal bioindicator for a variety of potentially toxic group 11 and 12 metals in that it concentrates metals from the surrounding aquatic environment, is found globally and in great abundance, and can be examined year round (4, 5). It has been suggested that the uptake of metals by F. Vesiculosus is primarily from dissolved metal species rather than from the sediment (6). The reports of F. Vesiculosus functioning as a biomonitor of metal ions in the aquatic environment lead to the question of the exact mechanism for binding of group 11 and 12 metals in the organism. Metallothionein has been associated with the response to exposure of metals in groups 11 and 12 in a range of other species (7). The induction of metallothionein is considered to protect the host organism from the toxic effects of the incoming metals. This is particularly the case for Cu+ and Cd2+, where sequestration by metallothionein (MT) removes the free metal ion. The presence of metallothionein in F. * Address correspondence to Dr. Martin J. Stillman, Department of Chemistry, University of Western Ontario, London, Ontario N6A 5B7, Canada. Phone: (519) 661-3821. Fax: (519) 661-3022. E-mail: [email protected]. † University of Western Ontario. ‡ Cardiff School of Biosciences.

Vesiculosus reported by Kille and co-workers following induction by copper (8) suggests that metallothionein may provide a survival mechanism for the macroalgae that grow in the metalcontaminated environments. The metallothionein induction is a toxic response to the insult caused by the incoming metal, as is the case in many other species (1, 7, 8). However, no metalbinding studies of the F. Vesiculosus metallothionein have been reported to allow comparison with well-known metal-binding properties of metallothioneins, with the exception of recent studies of arsenic binding (9). Metallothioneins described to date bind group 11 metals in the monovalent oxidation state, particularly Cu+, and the group 12 metals in the divalent oxidation state, Zn2+, Cd2+, and Hg2+ (10-12). Characterization of metalation patterns of the metallothionein isolated from F. Vesiculosus allows a greater insight into the physiological response of species in general to metals in their environment. Metallothioneins are a family of ubiquitous proteins that are characterized by low molecular weight and high metal and cysteine content with virtually no aromatic amino acids (7, 11, 12). Cadmium and zinc binding to metallothionein takes place in metal-thiolate clusters that are well-characterized for a variety of metallothioneins (7, 12). In this paper, we report results that show that the recombinant algal F. Vesiculosus metallothionein (rfMT) binds up to six zinc and cadmium atoms in metal-thiolate clusters, using a combination of UV absorption, circular dichroism, and electrospray mass spectrometry (ESI-MS). Of these techniques, ESI-MS is a particularly powerful tool in determining the metalation status of proteins. For metallothioneins, it has been used to characterize the metalated status for a number of metals, including zinc(II), cadmium(II), mercury(II), and copper(I) (13-19). In MTs

10.1021/tx050206j CCC: $33.50 © 2006 American Chemical Society Published on Web 02/17/2006

366 Chem. Res. Toxicol., Vol. 19, No. 3, 2006

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Figure 1. The sequence for the recombinant Fucus Vesiculosus metallothionein (rfMT) produced as an S-peptide fusion protein expressed in Escherichia coli. (A) The S-peptide is 27 residues long. (B) The sequence of the rfMT with the Cys residue nomenclature outlined. There are two cysteine-rich regions, termed 7-Cys and 9-Cys, separated by a 14 residue linker region. The numbering scheme indicates residue numbers for each cysteine and the number of the cysteine in the domain (1-7 and 1-9). This sequence was reported by Morris et al. (8).

studied to date, metal chelation is achieved through the abundance of cysteine residues with conserved motifs of Cysx-Cys, Cys-x-x-Cys, and Cys-Cys, where x represents any amino acid other than cysteine (20) in one or two binding domains (12). This leads to the question concerning the structure of metalated rfMT, which has two cysteine-rich regions (Figure 1): does rfMT bind group 11 and 12 metals in a single or double domain structure? The studies carried out in this paper address this question directly. Comparison of the sequence of rfMT and rabbit liver MT2A (rlMT2A) shows a similar set of Cys-x-Cys motifs, which is typical for metal binding in metallothioneins from many organisms (11). The question of whether rfMT binds metals in clustered metal-thiolate binding sites, like those found in other metallothioneins, is key to the structural description of the F. Vesiculosus protein as a metallothionein and to an understanding of the function of this protein. The mass spectral data reported in this paper for cadmium binding directly addresses this question.

Materials and Methods Chemicals. Chemicals used were CdSO4 and Zn(NO3)2 (Fisher Scientific). All chemicals used in this study were of the highestgrade purity from commercial sources. Hi Trap SP ion exchange columns and G-25 Sephadex (Pharmacia) were used for protein purification. Protein Preparation. The recombinant rfMT protein used in this study was based on the sequence shown in Figure 1. In addition to the sequence from the rfMT, the expression system includes the amino acid residues of the stabilizing S-peptide tag (labeled “S-tag” in Figure 1) on the N-terminus of the fragment (8). The S-tag sequence is added for stability during the protein preparation. The recombinant proteins were produced from overexpression of this fusion protein in Escherichia coli grown in fresh media containing 50 µM CdSO4. Expression was induced with isopropylβ-D-thiogalactopyranoside (IPTG). The CdSO4 concentration was increased hourly in the broth over 4 h to a final concentration of 150 µM. The cells were harvested by centrifugation, resuspended with a 10 mM Tris-HCl buffer with 0.002% β-mercaptoethanol, lysed by French press, and centrifuged. The supernatant was applied to an SP ion exchange cartridge and washed with argon-saturated 10 mM Tris-HCl buffer made in deionized water to a pH of 7.40. The metallothionein was eluted with a solution of 95% 10 mM Tris-HCl buffer, pH 7.4, and 5% 10 mM Tris-HCl buffer with 1 M NaCl, pH 7.4. The cadmium concentration in the fractions was analyzed by atomic absorption spectroscopy (Varian AA-875), and the cadmium-containing fractions were pooled. The protein fractions from the SP cartridge were desalted on a superfine G-25 Sephadex column (100 cm) and eluted with an argon-saturated 5 mM ammonium-formate buffer with a pH of

7.4. The fractions eluted exhibited the Cd-S band absorption at 250 nm. Fractions containing cadmium-bound rfMT were concentrated on a 200 mL stirred cell concentrator (Millipore) with a regenerated cellulose YM3, 5000 molecular weight cutoff filter. The purity of the material used in this experiment was confirmed using three methods: (i) the concentrated and purified protein was analyzed using Western blot, (ii) the exact cysteine content of the protein was confirmed by performing an amino acid analysis on the rfMT, and (iii) the amount of cadmium was monitored by atomic absorption spectroscopy. The S-tag in rfMT has the linkage sequence of LeuValProArgV GlySer, which allows thrombin to cleave the S-tag. The fractions eluted off the SP cartridge were pooled, concentrated (stirred cell concentrator (Millipore) with a regenerated cellulose YM3, 5000 molecular weight cutoff filter), and desalted (G-25 using 10 mM Tris-HCl buffer, pH 7.6), and then pooled and concentrated again. The cadmium concentration was analyzed by atomic absorption spectroscopy (Varian AA-875). The thrombin was restriction grade purchased from Novagen. Twenty units of thrombin was added to 20 mg protein and the solution slowly stirred for 9 h at room temperature. This solution was then applied to an SP ion exchange cartridge and washed with argon-saturated 10 mM Tris-HCl buffer made in deionized water to a pH of 7.40. The metallothionein was eluted with a solution of 95% 10mM Tris-HCl buffer, pH 7.4, and 5% 10 mM Tris-HCl/1 M NaCl buffer, pH 7.4. These fractions were pooled, concentrated, and desalted as above. Analytical and Spectroscopic Measurements. The ESI-MS spectra were obtained using a PE-Sciex API 365 LC/MS/MS triple quadrupole mass spectrometer (MDS Sciex, Canada) and an LC-TOF MS instrument (Micromass, Canada). The Sciex API 365 spectrometer was calibrated using a solution of polypropylene glycol. The protein samples were dissolved in a 5 mM ammoniumformate buffer, argon-saturated at an initial pH of 6.9. The rfMT samples were analyzed in the positive ion mode using the first quadrupole only. The analyte was infused into the atmospheric pressure interface (API) at 3 µL/min. Electrospray parameters were as follows: m/z range of 500-2000 Da, step size of 0.2 Da, dwell time of 0.3 ms, ion source potential of 5000 V, orifice potential of 30 V, and ring potential of 250 V. The mass calculations were performed using the program Biomultiview 1.5.1b (P. E. Sciex (Concord, Ontario, Canada)). The LC-TOF-MS was calibrated using a solution of mixed NaI and CsI. The protein samples were dissolved in a 5 mM formate buffer, argon saturated at an initial pH of 6.9. The electrospray parameters for the spectrometer were capillary 3000.0 V, sample cone 50.0 V, RF lens 700.0 V, extraction cone 5.0 V, desolvation temperature 20 °C, source temperature 80 °C, cone gas flow 53 L/h, and desolvation gas flow 506 L/h. The m/z range was 500.0-2500.0; the scan mode was continuum, interscan delay 0.10 s. The UV-visible absorption spectroscopy was performed on a Cary 100 UV-visible spectrophotometer in the absorbance mode. The wavelength was scanned from 200 to 300 nm. A Jasco J810

Cadmium Binding in Fucus Vesiculosus Metallothionein

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Figure 2. Observed charge states and reconstructed spectra using the ESI-TOF-MS instrument. (A) The observed charge states for apo rfMT(s) at pH 1.81. (B) The reconstructed spectrum for apo rfMT(s) at pH 1.81; the calculated mass is 9512.70 Da; the measured mass is 9514.5 Da. (C) The observed charge states for Cd6-rfMT(s) at pH 6.82. (D) The reconstructed spectrum for Cd6-rfMT(s) at pH 6.82; the calculated mass is 101745.0 Da; the measured mass is 10174.3 Da. (E) The observed charge states for Zn6-rfMT(s) at pH 7.02. (F) The reconstructed spectrum for Zn6-rfMT(s) at pH 7.02; the calculated mass is 9881.6 Da; the measured mass is 9881.0 Da. (G) The observed charge states for apo rfMT(c) at pH 1.80. (H) The reconstructed spectrum for apo rfMT(c) at pH 1.80; the calculated mass is 6700.8 Da; the measured mass is 6702.0 Da. (I) The observed charge states for Cd6-rfMT(c) at pH 7.60. (J) The reconstructed spectrum for Cd6-rfMT(c) at pH 7.60; the calculated mass is 7359.2 Da; the measured mass is 7356.0 Da.

was used to perform the circular dichroism spectroscopy. The solvent was 10 mM Tris buffer at pH 7.60.

Results The studies reported in this paper were designed to determine the binding properties of Cd2+ and Zn2+ to rfMT and to determine whether rfMT adopts a single binding domain with 16 Scys or a two-domain structure, like the mammalian protein

(21, 22). Cadmium and zinc were chosen as the probe metals because these are components in the environment of the seaweed and the binding properties are very well-known for a range of other MTs (11-12). Good structural parameters are only available for Zn2+- and Cd2+-containing metallothioneins to date. In this current work, cadmium and zinc binding was characterized at neutral and acidic pH. Both these pH values hold biological significance, since intracellular vesicles in brown


Figure 3. UV absorption spectra recorded during the pH titration of Cd6-rfMT(c). Inset: the absorbance of Cd6-rfMT(c) at 250 nm as a function of pH showing the two-phase reduction in absorbance. Absorbance at 250 nm is assigned to cysteine-thiolate to cadmium charge transfer (LMCT).

algae may reach pH values below 2 (23). We have studied two forms of the protein, namely, the native protein with a metalfree mass of approximately 6700 Da and the form as expressed, which contains a stabilizing peptide tag on the N-terminus, the S-tag, to investigate the effects of additional, noninteracting amino acid chain on the metal binding properties of the cysteinerich regions. Mass Spectra of Fucus Metallothionein. We show the mass spectral data for the metal-free and the Zn2+- and Cd2+containing metallothionein in Figure 2; the measured charge states are on the left-hand side, and the calculated deconvoluted spectral data are on the right-hand side. The calculated mass of the apo or metal-free rfMT cut protein (rfMT(c)) has a mass of 6700.83 Da, and when measuring the mass of the protein with the S-tag still attached, rfMT(s), the calculated mass including the S-tag is 9512.1 Da, both masses based on the primary structure, Figure 1. Recombinant rfMT exists in the metal-free or apo form at low pH with all cysteinyl sulfurs protonated. Figure 2 shows the ESI-TOF mass spectral data for rfMT(s) (panels A-D) and rfMT(c) (panels G-J) recorded at pH 1.81

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(panels A, B, G, and H) and pH 7.6 (panels C-F, I, and J). At low pH (here, 1.8) complete demetalation takes place, and the mass spectra shown in panels A and G represent the metal-free Scys-protonated species. The spectrum comprises a series of charge states arising from peptides with different numbers of protonated basic groups. All charge states observed in Figure 2 are due to the presence of single parent species, which are shown in the panels to the right of the charge states. First, the MS data for the intact protein at pH 1.81 for rfMT(s) (Figure 2A) shows the predominant charge state as +11 (865.95 m/z), although there is a second folded conformation of the same apo rfMT(s) centered on a charge state of +6 (1586.73 m/z). Each charge state from +5 to +14 can be located in the spectrum, Figure 2B. Charge states exist in the positive ion mode due to protonation (or positive ion adduct replacement of protons) of basic residues that occur along the sequence and are solvent exposed. The +10, +9, and +8 charge states exhibit minimal counts. From the sequence shown in Figure 1, we can identify 14 basic amino acids present in this peptide chain for rfMT(s) that are available for protonation at low pH. Because of the appearance of the weak series of charge states in rfMT(s) (centered on +6) together with the predominant series centered on +11, we can conclude that there are two folded conformations of the same parent apo-protein present in solution at pH 1.81 (and pH 1.60): the +11 state indicates a more solvent-exposed open structure than the +6 charge state. The presence of the +6 charge state suggests a much more compact structure in which 5 of the 14 basic amino acids are protected from solvent access. We have noted this behavior previously for recombinant metallothioneins at very low pH (less than pH 2 down to pH 0.5) (24), but not with the metalated species at higher pH values. This is probably because in the metalated state the three-dimensional structure is much more rigidly controlled by the metal-thiolate cross-linking, which does not allow for basic amino acids that are buried to become solvent-exposed under different pH conditions (between the pH 3.5 and 8.0 needed to maintain metalation). A recent report suggests that the conformations adopted by mammalian MT3 when binding cadmium and zinc are different due to a change in peptide organization, which is manifested by the appearance of different charge states (25). For the cut protein,

Figure 4. CD spectra recorded during the pH titration of Cd6-rfMT(s). In this series of spectra, the Cd6-rfMT(s) was titrated using aliquots of 1% formic acid solution starting at pH 7.6. Inset A: CD intensities of Cd6-rfMT(s) at 255 nm as a function of pH. Inset B: the absorbance of Cd6-rfMT(s) at 250 nm as a function of pH.


apo-rfMT(c), Figure 2G, we see the predominant charge states of +7 with +8 and +6 weakly present. The deconvoluted spectra of rfMT(s) and rfMT(c) are shown in Figure 2B and 2H, with masses of 9514.5 ((0.2) and 6702.0 ((0.2) Da, respectively. Panels C and I of Figure 2 show the mass spectra for the Cd-metalated rfMT(s) and rfMT(c) proteins at neutral pH, respectively. Three charge states are equally dominant at +6, +7, and +8 for Cd-rfMT(s), and we can calculate that at neutral pH the peptide is bound to six cadmium atoms through the deprotonated sulfurs of the 16 cysteines, (illustrated in Figure 1) for a predicted total mass of 10 174.97 Da, with a measured mass of 10 174.30 ((0.5) Da (Figure 2D), which identifies a stoichiometry of 6 Cd/16 Scys. Figure 2I,J shows the measured (panel I) and deconvoluted (panel J) mass spectra for the Cd-rfMT(c) protein. The deconvoluted spectrum (Figure 2J) shows a species with a mass of 7356.00 Da for rfMT(c), close to the predicted mass of 7359.16 Da for Cd6-rfMT(c) based on the mass of 6 Cd2+ and the sequence shown in Figure 1. Because the tetrahedral coordination geometry preferred for each cadmium ion requires four attached ligands, we can determine that bridging thiolates will be involved in binding site geometry with an overall stoichiometry of M6S16. We address the evidence in Figure 3 that there are two cadmium-binding domains. Confirmation that the cadmium atoms are bound to cysteinyl sulfurs is provided by the UV absorption (Figure 3) and circular dichroism spectroscopic data (Figure 4) at 250 nm, which is the sulfur-to-cadmium charge-transfer band region that is prominent for cadmium-containing compounds with sulfur as the coordinating ligand (7). The trends in UV absorption data recorded for both Cd6-rfMT(s) and (c), Figures 3 and 4, show how the absorbance at 250 nm decreases upon lowering the pH. A decrease in absorbance at 250 nm occurs when cysteinyl sulfurs bound to cadmium are protonated, thereby releasing the metal ions (7). This is a characteristic property of cadmiummetallothioneins (26, 27) and shows that, because the absorbance is directly related to the number of S-Cd bonds, as the Scys are protonated, the bound cadmiums are displaced in a stepwise fashion. Metalation with Zn2+, another tetrahedrally coordinating metal, provides further evidence for this stoichiometry. Figure 2E,F shows mass spectral data for the zinc-containing S-tag protein. As above, the charge state distribution for the zinc-metalated rfMT(s) at pH 7.60 is quite different from that for the apo-rfMT(s). Significantly, the +7 charge state is the single most intense state, compared with the equal intensities in the +6, +7, and +8 states of the Cd6S16-rfMT(s), Figure 2C. The deconvoluted spectrum, Figure 2F, indicates a mass of 9881 Da, which is the mass of the Zn6S16-rfMT(s) species. Although the ESI-MS data for cadmium and zinc binding show that the overall stoichiometry for these metals is M6S16rfMT, there is, however, no information on the structural arrangement, with the most important question being whether metal binding takes place in either a single domain or two domains. Spectroscopic data recorded during proton-induced demetalation provides data on the relative metal binding equilibrium constants, which are related to the apparent pKa of chelating basic ligands in the presence of both the bound metals and the competing protons. The pKa is reduced dramatically by the formation of metal-ligand bonds, with the extent of the reduction in pKa depending on the stability constant of the metal binding site as a whole when that site involves bridging ligands.


Figure 5. pH titration of Cd6-rfMT(c). The pH of the solution was lowered in steps using 1% formic acid, and the ESI-TOF-MS data were recorded. pH of the solutions: (A) 7.6; (B) 6.7; (C) 4.3; (D) 3.7; (E) 2.7.

Demetalation data can indicate the presence of one or more stages in the competition for the ligands by added protons and provide evidence as to whether this protein is binding in a single Cd6S16 cluster or a two-domain structure as with mammalian metallothioneins. Both UV absorption (Figure 3) and CD spectra (Figure 4) probe the formation of cysteine sulfur-cadmium bonds (26).


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Figure 6. pH titration of Cd6-rfMT(s). In this series of ESI-TOF-MS spectra, the pH of the solution of Cd6 rfMT(s) in an ammonium formate buffer was lowered in steps through the addition of 1% formic acid, with the pH as noted between 6.6 (A) and 1.81 (F). The deconvolution was carried out, and the assignments shown are based on matching the mass of the principle species present. The NH4+ is an adduct.

The absorption data have been described above. Figure 4 illustrates the trend in the CD spectral intensity as a function of pH observed using circular dichroism spectral data of S-tag rfMT. The band centered on ca. 257 nm at pH 7.6 is characteristic of Cd3-β MT (26). Figure 4 (inset) shows the change in CD intensity at the band maximum as a function of pH. The plateau region near pH 4 in both UV absorption (Figure 3) and CD (Figure 4) clearly indicates that demetalation takes place in two distinct steps with loss of about 50% of the absorption in the first step, which we interpret as indicating loss of about 50% of the metal. Information on the species that exist at each of the pH values used in the absorption and CD spectral studies shown in Figures 3 and 4 is obtained from the ESI-MS experiment carried

out at a range of pH values. These data are shown in Figures 5 and 6. First, for rfMT(c), at pH 7.60, Figure 5A, the molecular species has a mass of 7356.0 Da, which corresponds to the Cd6-rfMT(c). At pH 6.70, Figure 5B, the predominant species is the Cd5-rfMT(c) with a mass of 7225.1 Da with a minor presence of the Cd6-rfMT(c) at a mass of 7356.0 Da. At pH 4.30, Figure 5C, the predominant species is the Cd3-rfMT(c) with a mass of 7030.80 Da, with a minor contribution from Cd4-rfMT(c) with a mass of 7134.80 Da. At pH 3.70, Figure 5D, the predominant species is the Cd2-rfMT(c) with a mass of 6900.30 Da. Only the metal-free apo rfMT(c) is present below pH 2.70 (Figure 5E). Second, for rfMT(s), Figure 6 (panels A-F) shows a sequence of ESI-TOF-MS data (reported as the



Figure 7. pH dependence of cadmium speciation in rfMT(s) and rfMT((c). (A) Cadmium stoichiometry in rfMT(s) as a function of pH. (B) Cadmium stoichiometry in rfMT(c) as a function of pH. The bars represent the range of pH values that the species can be detected in the mass spectrum. The relative abundances change according to the pH.

deconvoluted mass spectra) as a function of decreasing pH (6.60-1.81) for Cd6-rfMT(s) that mirror the UV absorption and CD results of Figure 4. The ESI-TOF-MS data in Figure 6 provide the metal composition of the protein at each pH value: from 6 Cd2+ at pH 6.6 (Cd6 + 3NH4+; mass 10220.0 Da), to 5 Cd2+ at pH 4.52 (Cd5 + 1NH4+; mass 10073.0 Da), to 3 Cd2+ at pH 3.43 (Cd3; mass 9847.0 Da), where the NH4+ ions are adducts that replace a proton and arise from the ammonium formate buffer used, a common effect in ESI-MS measurements. The plateau regions of no signal change in the absorption and CD experiments are reproduced in the ESI-MS experiment where only below pH 3.0 (panel D) does the Cd-loading change: pH 3.0 (3 Cd2+, mass 9848.0 Da), pH 2.43 to 1 Cd2+ (Cd1 + 1NH4+, mass 9641.0 Da), and at pH 1.81 metal-free (9513.0 Da). The results are summarized in Figure 7, which shows the pH range for the detection of each metalated species by ESIMS. Figure 7 shows the pH range for the speciation of the cadmium bound to rfMT(c) follows a pattern similar to that measured by UV absorption and CD, namely, that there is a plateau in the demetalation reaction as a function of pH near

pH 3. These results support a two-domain model. In more detail, we find that by about pH 4.5 the Cd5 and Cd3 species predominate and Cd4-rfMT(s) is not detected, and Cd4-rfMT(c) exists only for a very small range of pH. The stability of the Cd3 species over a very wide range of pH is evident. We interpret the loss of the first Cd2+ (from Cd6 to Cd5) as a result of the strain in the binding domain, which raises the pKa of the coordinating Scys groups, allowing concurrent protonation and demetalation at a much higher pH than expected. This is further supported by the lack of the Cd4 species, which would involve leaving just one cadmium in one of the binding sites (based on a two-domain model that we propose from the absorption and CD spectral data). In contrast, the Cd3 species is stable down to pH values normally associated with demetalation of cadmiumcontaining metallothioneins, that is, pH 2.7. Figure 8 shows ESI-TOF-MS data (as the deconvoluted spectrum) recorded when cadmium was added in mole equivalent aliquots to a solution of Zn6-rfMT(s) at pH 6.8. Addition of increasing molar ratios of Cd2+ results in greater replacement of the Zn2+ as anticipated (panels A-F). The molar sum of Zn and Cd equals 6 in almost all species, indicating the isomorphous


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Figure 8. ESI-TOF-mass spectral data recorded for a series of solutions in which mole equivalent aliquots of cadmium chloride were added to a solution of Zn6-rfMT(s) in an ammonium formate buffer. The number of mole equivalents added to each solution is noted in the captions, (A) zero to (H) 7. The assignments are based on matching the masses of possible Cd- and Zn-rfMT(s) species to the deconvoluted masses observed.

replacement of one metal by the other. However, with six Cd2+ added, a series of species with a total of seven metals are measured (panel G), with a range of metalated species from Zn2Cd5 to Zn4Cd3. We interpret these results to indicate an increased stability for the Zn2 or 3 that requires an increase in Cd2+ for displacement of the remaining bound zinc atoms. Addition of seven Cd2+ results in a single Cd6-rfMT(s) species (panel H).

Discussion In the marine environment, group 11 and 12 metal pollution poses a serious threat due to potential toxic effects and bioaccumulation in the food chain (28). The seaweed F. Vesiculosus

binds the group 11 and 12 metals copper(I), cadmium(II), and zinc(II) (8). Metal-resistant algae have adapted and thrived (30) in aquatic environments polluted with toxic group 11 and 12 metals. There have been several mechanisms proposed for these species to account for the high metal loadings present and the lack of toxic effects. Studies have been reported that show that algae that accumulate metals contain metal-binding proteins with molecular weights less than 14 kDa (30). These metalloproteins have been classified as class III metallothioneins (31, 32) which include cadystins, phytochelatins, and poly(γ-glutamyl-cysteinyl)glycine peptides (30), all based on the recurring γ-glutamylcysteinyl sequence (7). This sequence is quite unlike that of typical class I metallothioneins, which are found for vertebrates



Figure 9. Comparison between the sequence for rfMT and the 9-cysteine β domain sequence from rabbit liver MT 2A (29). (A) The 9-cysteine β domain fragment of rabbit liver MT compared with the 9-cysteine fragment of rfMT. (B) The 9-cysteine β domain fragment of rabbit liver MT 2A compared with the 7-cysteine fragment of rfMT.

(7), and unlike the sequence of rfMT studied here. No class I metallothioneins have been previously characterized for algal species. However, the evidence for metallothionein detoxification of cadmium and excess copper in cells is seen by the induction of MT upon exposure of the F. Vesiculosus to these metals (8). A cadmium inducible metal-binding protein has been isolated from the blue-green algae Synechococcus sp. (33), and although its molecular weight was determined to be only 8100 Da, no sequence was reported. The protein contained a high fraction of cysteine amino acids. In vivo algal cells exposed to cadmium produced 50 ng metallothionein/mg wet cells in contrast to cells not exposed to cadmium, which produced no metallothionein (33). In the unicellular algae Dunaliella bioculata that was grown in the presence of CdCl2, the cadmium was associated with sulfur in the vacuoles. The use of comparative X-ray microanalysis supported the hypothesis of an intravacuolar cadmium-binding protein. It was proposed that metallothionein was involved in the detoxification processes used in the algae D. bioculata (34). From the above discussion it is clear that the induction of metallothionein in algae, including F. Vesiculosus, by group 11 and 12 metals is a toxicological response (8) that is moderated by the induction of either class III or class I metallothioneins. The rfMT expressed in this work has a sequence typical of class I metallothioneins, namely, as shown in Figure 1, the presence of distinct regions within the peptide that include Cys-x-Cys and Cys-Cys motifs identified as group 11 and 12 metal binding site ligands. For a protein to be of a member of the MT family, its sequence must share a particular set of sequence-specific residues. No algal class I MT species have been reported prior to the discovery by Kille et al. (8) of the metallothionein in F. Vesiculosus. The rfMT gene isolated, and the subsequent rfMT protein expressed, could possibly represent a distinctive metallothionein family. The recombinant rfMT is composed of three regions, (i) a seven Cys residue region, (ii) a linker region comprising 14 residues, and (iii) a nine Cys residue region. The 16 Cys are located in frequencies that suggest typical metallothionein metal binding properties. This study reports data not previously observed for any metallothionein, namely, binding six group 12 metals with oxidation states

of 2+, with only 16 cysteine residues, and the probability that the metal binding takes place in a two-domain structure. Clearly, while the rfMT has little overall homology with the mammalian MT, the Cys-x-Cys motifs provide the basis for the metal binding properties observed, although no 7-Cys fragment has previously been reported. Again, the presence of the three Cys-x-Cys motifs account for the metal binding and suggest both bridging and terminal cysteinyl sulfur involvement. The data reported provide evidence for the first report of a novel metal-thiolate structure in this metallothionein. We have applied the results from mass spectrometry, UV absorption, and CD spectroscopy to determine the following three key properties for metal binding in rfMT: (i) The cadmium-to-cysteine stoichiometry is 6. In the ESI-MS data, the species observed for the cadmiumbound metallothionein corresponds to a Cd6S16 rfMT cluster. This requires that the peptide chain must be folded to form the necessary bridging and terminal Scys-Cd bonds required for the tetrahedral coordination of the Cd2+. This implies a metal binding site similar to the structures of known class I metallothioneins, for example, the crustacean or mammalian proteins (26, 35). There are no additional counterions involved in the Cd2+ binding site, so all cadmium ions are bonded to thiolate sulfurs of the cysteine residues to maintain the mass and charge balance observed in the mass spectral data. These data support Cd6S16 as the stoichiometry for rfMT as expressed recombinantly in E. coli. (ii) The characteristic number for group 12 metals with oxidation state 2+ that bind to rfMT is 6. Zn6-rfMT can be isolated; the species formed following addition of Cd2+ to the Zn6-rfMT is Cd6S16 rfMT. The presence of six bound Zn2+ ions to rfMT supports the fact that six is the natural maximum number of metals with an oxidation state of 2+ that bind to rfMT(s), so the overall stoichiometry can be written more generally as MII6S16. However, the experiment in which Cd2+ is added to the zinc-containing protein is also a very good test of the chelation properties of rfMT. In many previous studies, it has been shown that Cd2+ isomorphously replaces Zn2+ in MT (26, 36). The substitution reactions of metallothioneins, especially for cadmium added to


the zinc-proteins, have been described in a number of papers (37). NMR studies have shown that human metallothionein-2 bound to zinc has the same molecular architecture as the cadmium-bound species (36). 1H NMR data indicate that the conformations of these domains are almost identical. This is due to the presence in the folded peptide chain of loops that are interspaced between the metal-bound cysteinyl residues. In this way, the clusters formed in metallothionein accommodate a range of metals with minimal disruption to bond angles and overall conformation. The ESI-MS suggests that one of the metal binding domains in rfMT may in fact expand during substitution of the Zn2+ by the Cd2+, because seven metals are bound during the substitution reaction. (iii) Structure of the metal binding domains: a single M6S16 structure or two isolated domains, M3S7 and M4S9. Analysis of the UV absorption, CD, and ESI-MS data indicate that rfMT binds six Cd2+, Zn2+, or combinations of both metal atoms. We propose that metal binding by the Cd2+ takes place in two domains, most likely with stoichiometries of Cd3S7 and Cd3S9, separated by a linker region. One of the domains is relatively unstable, the other much more stable. Because the stable set of three cadmium atoms resembles the behavior of Cd3β hMT, we propose that the two binding domains are reasonably represented by Cd3S7 for the unstable cluster and Cd3S9 for the more stable cluster (24). The data do not provide any support for the presence of a single Cd6S16-clustered binding site. The Cd3S7 structure represents a previously unknown metalto-cysteine ratio in a metallothionein and will involve extensive bridging for the coordinating cysteine sulfurs. Further studies using NMR, EXAFS, and XANES techniques will be required to probe these proposed domain structures. In conclusion, the presence of distinct regions within the peptide that include Cys-x-Cys and Cys-Cys motifs and the determination of 6 as the number of divalent metals bound together allow the rfMT protein to be identified as the first algal class I metallothionein (using the definition of Kagi (7)) with novel 7-Cys and 9-Cys cadmium and zinc binding domains. Acknowledgment. We thank NSERC (Natural Science and Engineering Research Council of Canada), Canada (to M.J.S.) and NERC (Natural Environment Research Council), U.K. (to P.K.) for funding; NATO (North Atlantic Treaty Organization) for travel funds (to M.J.S. and P.K.) and OGS (Ontario Graduate Scholarship) for a graduate scholarship (to M.E.M). We thank Prof. Lars Konermann for use of the ESI-mass spectrometer funded through Canadian Foundation for Innovation. We thank Prof. Richard Puddephatt for use of the Micromass LC-TOF funded as part of his Canada Research Chair. We thank Mr. Doug Hairsine for valuable technical assistance with the mass spectrometric measurements.

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S Cluster Stoichiometry in Fucus vesiculosus

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