The two distinctive metal ion binding domains of the wheat metallothionein Ec-1

Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn^II and Cu^I. Recently, we could show that wheat E_c-1 is coordinating its six Zn^II ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn^II ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn^II ion of the N-terminal domain is relative resistant to complete replacement with Cd^II indicating the presence of a Zn^II-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE_c-1 with an excess of Zn^II shows a certain amount of sub-stoichiometrically metal-loaded species. The possible relevance of these finding for the proposed biological functions of wheat E_c-1 will be discussed. In addition, extended X-ray absorption fine structure (EXAFS) measurements on both, the full-length and the truncated protein, provide final evidence for His participation in metal ion binding.     

Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe^II₄-MTα and Co^II₄-MTα species existing in water. Furthermore, the Fe^II₄-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe^II₄-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.     

Importance of dynamical processes in the coordination chemistry and redox conversion of copper amyloid-β complexes

Interaction of Cu ions with the amyloid-β (Aβ) peptide is linked to the development of Alzheimer’s disease; hence, determining the coordination of Cu^I and Cu^II ions to Aβ and the pathway of the Cu^I(Aβ)/Cu^II(Aβ) redox conversion is of great interest. In the present report, we use the room temperature X-ray absorption near edge structure to show that the binding sites of the Cu^I and Cu^II complexes are similar to those previously determined from frozen-solution studies. More precisely, the Cu^I is coordinated by the imidazole groups of two histidine residues in a linear fashion. However, an NMR study unravels the involvement of all three histidine residues in the Cu^I binding due to dynamical exchange between several set of ligands. The presence of an equilibrium is also responsible for the complex redox process observed by cyclic voltammetry and evidenced by a concentration-dependent electrochemical response.     

The CdII-binding abilities of recombinant Quercus suber metallothionein: bridging the gap between phytochelatins and metallothioneins

In this work, we have analyzed both at stoichiometric and at conformational level the Cd^II-binding features of a type 2 plant metallothionein (MT) (the cork oak, Quercus suber, QsMT). To this end four peptides, the wild-type QsMT and three constructs previously engineered to characterize its Zn^II- and Cu^I-binding behaviour, were heterologously produced in Escherichia coli cultures supplemented with Cd^II, and the corresponding complexes were purified up to homogeneity. The Cd^II-binding ability of these recombinant peptides was determined through the chemical, spectroscopic and spectrometric characterization of the recovered clusters. Recombinant synthesis of the four QsMT peptides in cadmium-rich media rendered complexes with a higher metal content than those obtained from zinc-supplemented cultures and, consequently, the recovered Cd^II species are nonisostructural to those of Zn^II. Also of interest is the fact that three out of the four peptides yielded recombinant preparations that included S²⁻-containing Cd^II complexes as major species. Subsequently, the in vitro Zn^II/Cd^II replacement reactions were studied, as well as the in vitro acid denaturation and S²⁻ renaturation reactions. Finally, the capacity of the four peptides for preventing cadmium deleterious effects in yeast cells was tested through complementation assays. Consideration of all the results enables us to suggest a hairpin folding model for this typical type 2 plant Cd^II-MT complex, as well as a nonnegligible role of the spacer in the detoxification function of QsMT towards cadmium. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.     

Structural features specific to plant metallothioneins

The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn^II, Cd^II, and Cu^I. The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E_c-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal–thiolate cluster structures.     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

The βE-Domain of Wheat Ec-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn^II, Cu^I, and Cd^II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β_E-domain of the early cysteine-labeled protein (E_c-1) from wheat (β_E-E_c-1), which represents the first three-dimensional structure of a plant MT. The β_E-domain comprises the 51 C-terminal residues of E_c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn^II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn^II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by ¹⁵N relaxation experiments support the results of the structure determination in that β_E-E_c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn^II with Cd^II ions and examined the effects of metal ion release on incubation with a metal ion chelator.     

Copper(II) binding properties of hepcidin

Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu^II and Ni^II through the amino terminal copper–nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu^II with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu^II than that of native hepcidin. The log K ₁ value of hepcidin for Cu^II was determined as 7.7. Cu^II binds to albumin more tightly than hepcidin (log K ₁ = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu^II present in blood will be bound to albumin. It is estimated that the concentration of Cu^II-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.     

Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.     

Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

Both Met(109) and Met(112) are utilized for Cu(II) coordination by the amyloidogenic fragment of the human prion protein at physiological pH

The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five Cu^II ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured “amyloidogenic” domain between residues 91 and 126 PrP(91-126). Herein we expand upon a previous study [J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710-719] where we demonstrated that the physiologically relevant high affinity Cu^II coordination site within PrP(91-126) is found between residues 106 and 114. It was shown that Cu^II is contained within a square planar (N/O)₃S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the Cu^II coordination environment within the PrP fragment containing residues 106-114 (PrP(106-114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106-114) with systematic Met → Ile “mutations” we show that the Cu^II coordination environment within PrP(106-114) is actually comprised of a mixture of two major species; one Cu^II(N/O)₃S center with the M(109) thioether coordinated to Cu^II and another Cu^II(N/O)₃S center with the M(112) thioether coordinated to Cu^II. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106-114) both reduces the Cu^II affinity of the peptide by two to seven fold, and renders the resulting Cu^II metallopeptides redox inactive. The biological implications of these findings are discussed.     

Spectroscopic studies on metal distribution in Co(II)/Zn(II) mixed-metal clusters in rabbit liver metallothionein 2.

Metal selectivity of metal-thiolate clusters in rabbit liver metallothionein (MT) 2 has been studied by examining the metal distribution of two similarly sized divalent metal ions, cobalt and zinc, which have different thiolate affinity. The forms of mixed-metal cluster species in (Co/Zn)7-MT generated with different ratios of both metal ions offered to the metal-free protein were investigated using EPR, ultraviolet/visible absorption and MCD spectroscopy. The results demonstrated that the distribution of these metals between the two metal-thiolate clusters is not random. Thus, the EPR absorption intensities of the bound Co(II) ions in the Zn-cluster matrix increased linearly up to a ratio of Co(II)/Zn(II) equivalents of 3:4, with the final EPR intensity of three non-interacting Co(II)-binding sites. This EPR behaviour is consistent with a binding scheme in which one Co(II) ion occupies a metal-binding site within the three-metal cluster and the remaining two Co(II) ions occupy two distinctly separate sites in the four-metal cluster. With four or more Co(II) ions in the cluster matrix, magnetic coupling between adjacent, sulphur-bridged Co(II) ions was observed. In previous studies on mixed-metal clusters in MT formed with Co(II)/Cd(II), Zn(II)/Cd(II) and Cd(II)/Fe(II), changes in the respective cluster volumes were shown to be a significant factor dictating the widely differing metal distributions in these systems. Based on the results of the current study, it is suggested that both the sizes of the two metal ions and their relative affinities towards the cysteine-thiolate ligands are important in the formation of mixed-metal clusters in MT.     

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

Three new multidimensional organic-inorganic hybrids based on polyoxometalates and copper coordination polymers with 4,4′-bipyridine ligands

Three new organic-inorganic hybrid materials with 4,4′-bipy ligands and copper cations as linkers, [Cu^II(H₂O)(4,4′-bipy)₂][Cu^II(H₂O)(4,4′-bpy)₂]₂H[Cu^IIP₈Mo₁₂O₆₂H₁₂] · 5H₂O (1), [Cu^I(4,4′-bipy)][Cu^II(4,4′-bipy)]₂ (BW₁₂O₄₀) · (4,4′-bipy) · 2H₂O (2) and [Cu^I (4,4′-bipy)]₃ (PMo₁₂O₄₀) · (pip) · 2H₂O (3) (pip = piperazine; 4,4′-bipy = 4,4′-bipyridine), have been hydrothermally synthesized. The single X-ray structural analysis reveals that the structure of 1 is constructed from [Cu(H₂O)(4,4′-bipy)₂] complexes into a novel, three-dimensional supermolecular network with 1-D channels in which Cu[P₄Mo₆]₂ dimer clusters reside. To the best of our knowledge, compound 1 is the first complex in which the [P₄Mo₆] clusters have been used as a non-coordinating anionic template for the construction of a novel, three-dimensional supermolecular network. Compound 2 is constructed from the six-supported [BW₁₂O₄₀]⁵⁻ polyoxoanions and [Cu^I(4,4′-bipy)] and [Cu^II(4,4′-bipy)] groups into a novel, 3-D network. Compound 3 exhibits unusual 3-D supramolecular frameworks, which are constructed from tetrasupporting [PMo₁₂O₄₀]³⁻ clusters and [Cu^I (4,4′-bipy)_n] coordination polymer chains. The electrochemical properties of 2 and 3 have been investigated in detail.     

Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

Spectroscopic characterization of a fruit-specific metallothionein: M. acuminata MT3

Metallothioneins (MTs) are ubiquitous low molecular mass, cysteine-rich proteins with the ability to bind d¹⁰ metal ions in the form of metal-thiolate clusters. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. The amino acid sequences of plant MTs are distinctively different to the sequences of other MT species. The protein under investigation, Musa acuminata (banana) MT3, belongs to the plant MT fruit-specific p3 subfamily. With a total of 10 cysteine residues, MT3 features a cysteine content and percentage that is more comparable to fungal and prokaryotic MTs than to the well characterized mammalian iso-forms. The gene sequence encoding MT3 was cloned into a suitable vector and the protein was recombinantly overexpressed in Escherichia coli. MT3 is able to coordinate a maximum of four divalent d¹⁰ metal ions under the formation of metal-thiolate clusters. The hitherto unknown spectroscopic behavior of MT3 in combination with the metal ions Zn²⁺, Cd²⁺, Pb²⁺, and Hg²⁺ will be presented and gives rise to the existence of a weaker metal ion coordination site. The pH stability of the investigated zinc and cadmium clusters is comparable to the values found for other plant metallothioneins though significantly lower than for the mammalian iso-forms. Possible metal-thiolate cluster structures will additionally be discussed.