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The Bioinorganic Chemistry of Mammalian Metallothioneins
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The Bioinorganic Chemistry of Mammalian Metallothioneins
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Cite this: Chem. Rev. 2021, 121, 23, 14594–14648
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https://doi.org/10.1021/acs.chemrev.1c00371
Published October 15, 2021

Copyright © 2021 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time.

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1. Introduction

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Three human generations have witnessed the development of the field of metallothioneins. It is crucial to start the narration with the early history to understand the facts and why and how contentions developed. In a discussion of discoveries seriatim, the etymology of “metallothionein” and the epistemology of the field will foster an understanding of how our present ideas about metallothionein (MT) were shaped. Trials and tribulations emerged from some assumptions in lieu of facts and from not interpreting chemistry in the context of biology. Resolutions of conjectures had to await additional discoveries, especially insights into the molecular basis of how cellular metal metabolism is controlled. MT is emblematic of the difficulties faced when trying to define functions of proteins without known activities. Seen with this perspective, the search for a function of an entire family of proteins turns out to be a quite educational lesson in the history of science. The article will start with the discovery of metallothioneins and definitions of the term with an account of the first 40 years of MT research culminating in the determination of three-dimensional (3D) structures of mammalian MTs. It will then address two significant developments that challenged the general perception about their structure, chemical reactivity, and metal complexation, proceed to discuss how the chemical properties of MTs are limited by the biological environment and how the highly dynamic properties of MTs relate to our current knowledge in mammalian metal biochemistry, and finally attempt a synthesis of our knowledge with the goal of advancing the understanding of their functions. By necessity, a scientific literature with over 20 000 original articles, numerous reviews, books, and conference proceedings will have to be reduced to ∼2% with representative articles. We hope that we do due diligence in citing key articles from a wide community of scientists whose disciplines have been and will be impacted by the fundamental roles of MTs.

1.1. Search for a Role of Cadmium in Biology: Discovery of Metallothionein

In the middle of the last century, an ongoing objective was to find biomolecules associated with the metal ions present in biological tissues (“biometals”). On the basis of reports in the late 1940s that cadmium is present in various living species, Margoshes and Vallee began to address the question whether cadmium is naturally present for an essential function or as a result of environmental contamination. Incidentally, the same question was posed for zinc 30 years earlier. (1) Among different animals screened, they chose equine kidney cortex because of its relatively high cadmium content. In 1957, they reported the isolation of a Cd(II)-binding protein from horse kidney. (2) They noted that the protein also contains zinc and that it does not absorb light at 280 nm, signifying the absence of aromatic amino acids. Further purification and characterization of the protein showed the presence of 2.9% cadmium, 0.6% zinc, and 4.7% sulfur (per gram of dry weight), the latter in the form of cysteine bound to the metal ions. (3) Since the protein contained different metal ions and is rich in sulfur, Kägi and Vallee named the holoprotein metallothionein and the apoprotein thionein. After clarifying onomastics, there is an issue of semantics from the beginning, namely, focusing the field on metallothionein (MT) with different metal ions and not on the protein thionein (T). On the ribosome, T is made, and whether or not MT is formed depends on the tightly controlled availability of metal ions. Further purification of MT using anion-exchange chromatography yielded two fractions and refined the composition of the major fraction to 5.9% Cd, 2.2% Zn, 0.2% Fe, 0.1% Cu, and 9.5% sulfur. (4) The electronic absorption of the protein at 250 nm was assigned to Cd-mercaptide bonds; 95% of the sulfur was found in the form of cysteine thiols. A comparison between MT from the liver and the kidneys of horses showed that the former contains mainly zinc. (5) Likewise, MT from adult human liver contains mostly zinc and small amounts of copper and cadmium. (6) In human and bovine fetal liver, however, a major Cu(I)-binding protein was identified as MT. (7,8) Thus, the metal composition depends on the tissue from which MT is isolated, containing mainly Zn(II), Cu(I), or Cd(II) or a combination of these metal ions. It also depends on the specific physiological and pathological condition of the tissue and its developmental stage. Ensuing work confirmed the presence of MT in basically all tissues from different mammalian species, albeit at different amounts. The conspicuous properties of binding rather high amounts of different metal ions, the large number of cysteines, and the absence of aromatic amino acids are “unusual and unconventional” for most proteins and guided the search for a function for years to come.

1.2. MT1 and MT2

Following initial reports of isolated horse kidney MT being heterogeneous with two or even three components detected by electrophoretic and chromatographic methods, two forms of liver metallothionein, MT-I and MT-II, were prepared from rabbits that received cadmium injections, which Magnus Piscator demonstrated to induce the protein. (9,10) The forms had pI values of 3.9 and 4.5 and showed slightly different amino acid and metal (Zn vs Cd) composition. Two fractions were obtained from human liver after anion-exchange chromatography at pH 8.6 and called MT1 (eluting first) and MT2 (eluting second) with the former having 6.05 total metal and the latter 7.24 total metal per protein. (6) These isolations, however, did not reveal the molecular identity of the forms. The difference between the two charge-separable forms MT1 and MT2 was later found to be due to an aspartate at position 11 in MT2.

1.3. MT1 Proteins

An additional purification step, that is, reversed phase high-performance liquid chromatography (RP-HPLC) at pH 7.5, demonstrated MT2 to be homogeneous but resolved five different forms of MT1 from human liver. (11) This observation was preceded by a report that the human genome contains at least a dozen MT genes. (12) The multiplicity and molecular identity of MT1 proteins will be addressed later in this article.
Two additional mammalian MTs were discovered later in 1991 and 1994 in specific tissues, one via a biological assay and the other through molecular cloning. They define a different branch in the evolutionary tree, and their discovery brought the number of expressed human MTs to at least 11. We will briefly describe these additional forms and then resume the chronology of discoveries.

1.4. MT3 (GIF)

A growth inhibitory factor (GIF) that is deficient in Alzheimer’s disease brain turned out to be a new metallothionein. (13) It has 68 amino acids, that is, seven additional amino acids compared to the MT1/MT2 proteins, with an additional amino acid (Thr) at position 5 near the N-terminus and a six amino acid, Glu-rich insert near the C-terminus. Otherwise the cysteines are conserved. The GIF activity, which refers to inhibiting the survival and neurite outgrowth of cortical neurons in vitro, is not shared by other MTs and abolished by specific mutations, in particular, replacing the prolines in the unique CPCP sequence starting at position 6. (14) Though GIF is the first MT found on the basis of a biological activity, the molecular basis for the GIF activity remains unknown. Isolated GIF contains Cu(I) and Zn(II), but the zinc protein is active as well. As a new member of the MT family, the protein was subsequently named MT3 and found not to be induced by metal ions or other common inducers of MTs. (15) It is, however, inducible, for example, by hypoxia, and this aspect of regulation is discussed below in section 5.1.

1.5. MT4

MT4 was discovered by molecular cloning. (16) Like MT3, it has an insertion at position 5 but, in this case, a glutamate instead of a threonine; it contains Cu(I) in addition to Zn(II) when isolated, and the positions of the cysteines are conserved. Its expression was found to be rather restricted to stratified squamous epithelia. It is believed to be important for the differentiation of these epithelia.

2. The Structures of Mammalian Zinc and Copper Metallothioneins

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2.1. Primary Structures

The first protein sequence determined was that of horse kidney MT1b. (17) It showed a special clustering of the cysteines in CC (3), CXC (7), and CXXC (2) motifs, and an absence of histidine and aromatic amino acids as noted earlier (Figure 1). The protein has 61 amino acids; thus, it is a relatively small protein, in agreement with earlier determinations of its molecular mass of 6000 Da. Noteworthy are the presence of seven Ser-Cys pairs and the association of cysteines with basic amino acids in four Cys-Lys, three Lys-Cys, and one Cys-Arg pair (Figure 1).

Figure 1

Figure 1. Amino acid sequence of horse MT1b with the Cys-containing motifs indicated, including CC (blue), CXC (red), CXXXC (green), SC (gray), and CK, KC, CR (underlined).

All sequences are referred to as metallothionein, though they represent the protein thionein only. The sequence per se does not indicate the types and numbers of bound metal ions, an issue to keep in mind as critical for the discussion to follow. Until today, investigators variably refer to the holoprotein in two ways, for example, copper metallothionein (employing the given name for the protein and indicating the type of metal ion bound) or copper thionein (employing the name of the protein and indicating the metal bound). Although the first term is a pleonasm, it is used more widely, similar to, for example, a zinc protein being called a zinc metalloprotein.
When sequences of small sulfur- and metal-rich proteins from invertebrates and fungi became available, it became obvious that the sequence similarity among so-called MTs is limited. A division into three classes was proposed: (18) class I─polypeptides with cysteine motifs closely related to horse kidney MT, class II─polypeptides with cysteine motifs distantly related to horse kidney MT, and class III─nontranslationally synthesized metal–thiolate polypeptides, such as phytochelatins. This division exacerbated the problem of nomenclature: What constitutes a metallothionein? This article focuses on mammalian class I MTs as the quintessential MTs for which sufficient information is available to discuss the structure–function relationships. Mammalian MTs are the prototypes on which most of the work has been performed, and they define the field.
The issue lingers why gene duplications for MT1 proteins occurred in some but apparently not all mammalian species. (19) The term MT1 has historic reasons to distinguish the protein(s) from MT2 on the basis of a charge difference due to Asp(D) in MT2 (at position 13 in the alignment) (Figure 2). MT3 and MT4 do not have an Asp at this position. All 20 cysteines are conserved in the human MT family. The total number of strictly conserved amino acids is 29.
From the standpoint of evolution, MT1 proteins and MT2 are in one phylogenetic branch, while MT3 and MT4 are in a different branch (Figure 3). Gene duplication occurred before speciation occurred, indicating functional differentiations. (22)
If one extends the analysis from mammalian MTs to examples of vertebrate MTs, at least three of the original tenets that define a prototype MT already break down (Figure 4). Only 16 out of 20 cysteines are strictly conserved, a His is found at the C-terminus of chicken MT, and an aromatic amino acid (Tyr) is found in African chameleon MT. This variability is important to note, as some human MTs were thought not to form functional proteins simply on the basis that they contain “unusual” amino acids.

2.2. Metal Composition: Zn(II), Cd(II), Cu(I)

It is essential to understand how MT is prepared for a chemical characterization in the ensuing work to determine its structure that serves as a pivotal point of reference. Several methods are employed, and they include harsh treatments. Since the metal-containing protein is rather heat-stable, a common assay, the so-called hemoglobin-binding assay, includes a step of boiling the tissue homogenate at 100 °C for 1 min. (25) A standard procedure for the isolation of the protein includes the precipitation of a tissue homogenate with a solution of 96% ethanol/chloroform (1.05:0.08). (26) The purification of MT is followed by an assay of its metal content. Hence any apoprotein (thionein) that may exist in tissues will not be detected unless it copurifies. Another matter─and a limitation─is that MT is prepared from cytosolic fractions. Therefore, MT present in cellular organelles is also not investigated routinely. Because of the harsh treatment, one cannot exclude that the heterogeneity of the isolated MT in terms of different bound metal ions, at least in part, is due to metal binding or metal swap during isolation. To prepare homometallic forms after isolation, T is prepared by removing Zn(II) or Cd(II) by an acidification of MT to pH 2 in the presence of dithiothreitol (DTT) to reduce any oxidized sulfhydryls either initially present or formed during isolation. In order to remove Cu(I), a short exposure to pH 1 or a treatment with diethyldithiocarbamate at pH 5 is necessary. (27) The protein is rather resistant to acid treatment, except for an Asp-Pro bond following the N-terminal Met. T thus obtained is then subjected to gel filtration and reconstituted with seven molar equivalents of divalent metal ions. (28) In this way of isolation and preparation, information about the natural metal composition and redox state of MT is lost, and one chooses intentionally a particular stoichiometry with a single metal ion based on an average content of approximately seven metal ions (sum of Zn(II), Cd(II), Cu(I)) generally found in isolated MTs and based on spectroscopic data that demonstrate a titration breakpoint when seven divalent metal ions are added. (28) Working with this stoichiometry, one assumes that metal ions are available in vivo to saturate the protein and to reach such a stoichiometry. This assumption is certainly not correct for Cd(II). To increase the yield of isolated MT, it is often induced with Cd(II) in laboratory animals, thus biasing the metal content toward the inducing metal ion. Even when rather high doses of Cd(II) cadmium are injected into an animal, the maximum incorporation of Cd(II) yields Cd5Zn2MT. (29) Cd7MT can be made in vitro but does not form in vivo. (30) A stoichiometry of seven metal ions has been referred to as the “magic number”. (31) Its significance is at the center of our discussion of structure and function later in this article. Forms with additional metal ions (up to 20 monovalent or at least one extra divalent metal ion, i.e., Cd(II) in the formation of MT dimers) (32) and forms containing less than seven metal ions can be prepared. Thus, with this modus operandi of preparing specific homometallic forms with seven divalent metal ions─or other stoichiometries with yet additional monovalent or trivalent metal ions─in vitro, investigators moved away from investigating the metal composition of the native protein.With the advent of molecular cloning starting in the late 1970s, a heterologous expression of the recombinant proteins became possible. In this case, the addition of metal ions to the growth media and the availability of metal ions in the host determine the metal composition of MTs. MTs expressed in Escherichia coli do not have an acetylated N-terminus. Currently it is unknown whether the N-terminal acetylation of MT affects its reactivity or metal affinity.

Figure 2

Figure 2. Sequences of human MT1–4 with conservation of Cys residues. There is one exception, though: human MT1b has an extra cysteine, yielding a total of 21 Cys. Only some of the Lys (K) residues are conserved, one CKC motif in the N-terminal part and three (KCA, CKG, and KCS) in the C-terminal part─KCS is not conserved in MT1m and MT1b, though. The KK motif in the middle of the protein is conserved with the exception of MT4, where it is RK. Blue codes on the right denote UniProt entries. Alignment was performed using MAFFT software (20) and visualized using Jalview. (21) Yellow and black bars stand for conservation and consensus, respectively.

Figure 3

Figure 3. Phylogram (left) and cladogram (right) of human metallothioneins (based on protein structures, translation of pseudogenes is not included). MT2 is part of the MT1 branch, while MT3 and MT4 have a separate root. The tree was generated in Clustal Omega using the neighbor-joining clustering method and visualized by iTOL. (23,24) The bar indicates the number of changes per residue. The number 0.01 corresponds in length to a 1% difference in sequences.

Figure 4

Figure 4. Alignment of MT sequences from representative species of vertebrates. In this selection, 23 amino acids are strictly conserved, but among them only 16 Cys are strictly conserved. Besides Coelacanth and African chameleon MTs, all other MTs contain 20 Cys residues. Yellow and black bars below the alignment show the patterns of conservation and consensus. Blue codes on the right denote UniProt entries. Alignment was performed using MAFFT software (20) and visualized using Jalview. (21) Yellow and black bars stand for conservation and consensus, respectively.

2.3. 3D Structures

2.3.1. Structures of the Metal Sites

With 20 cysteines and seven divalent metal ions resulting in a metal–sulfur stoichiometry close to 1:3 and every metal ion bound in a tetrahedral coordination environment with sulfur donor ligands, (33) there are not enough cysteines to bind the metal ions individually, as 28 would be needed to do so. Therefore, a solution to the riddle of the coordination structure was not a straightforward matter. It turned out to be an exceptional, homoleptic coordination in bioinorganic chemistry in the form of “clusters” with bridging and terminal sulfurs of cysteines. (34) The cluster organization was known before the 3D structure of the protein was solved. On the basis of 113Cd NMR and the homonuclear coupling of the cadmium nuclei, it was proposed that the metal ions are organized in two clusters in rabbit liver MT, a three-metal cluster with three sulfur bridges and a total of nine cysteine sulfurs bound and a four-metal cluster with five sulfur bridges and a total of 11 cysteine sulfurs bound. (35) These stoichiometries result in an overall net charge of −3 for each cluster. MT1 and MT2 are indistinguishable in terms of the metal environments. The clusters can be described as cyclohexane-like (chair) and adamantane-like (tricyclic structure not fully formed) (Figure 5). (36) A limited proteolysis of the apoprotein led to the preparation of a C-terminal peptide in which four metal ions reside and the suggestion of a structure of MT with domains. (37) The reconstitution of this peptide with Cd(II) and a demonstration by 113Cd NMR that it contains the four-metal cluster established that the protein has two domains, with each one harboring one of the clusters. (38)
Remarkably, seven Cd(II) resonances are resolved in the 113Cd NMR spectra, demonstrating that each metal ion resides in a slightly different environment despite each Cd(II) ion being surrounded by four sulfurs in a tetrahedral geometry. The group of Kurt Wüthrich then went on to determine which cysteines are bound to each Cd(II) ion from the heteronuclear 113Cd–1H connectivities (Figure 5). (40) It revealed a criss-cross pattern of binding, in which the ligands are not employed linearly in the sequence and are therefore not predictable from (a) signature(s). MxCysy clusters are not restricted to MTs. A Zn3Cys9 cluster─characteristic for the β-domain of mammalian MT─is present in domains of histone lysine methyltransferase (41) and the E3 ubiquitin-protein ligase of male-specific lethal 2 (MSL2). (42) Further examples of Zn(II)/thiolate clusters in proteins will be discussed in section 5.1 with reference to Figure 26. The primary sequences of these proteins harboring the clusters, however, differ significantly from those of MTs in the pattern of cysteine residues and the presence of histidine and hydrophobic/aromatic residues that promote the formation of more stable secondary structures. In the crystal structure of the corresponding domain of the human histone methyltransferase ASH1L, only two Zn(II) were found to be bound to seven out of the nine Cys residues, indicating differences in the Zn(II) occupancy with functional implications yet to be determined. (43)

2.3.2. Structures of the Proteins

The protein is virtually devoid of a secondary structure in the absence of metal ions. In their presence, several half-turns and two 310 helix segments are formed, especially in the α-domain. With disorder-producing amino acids (G,S,P), no aromatic amino acids, and few aliphatic amino acids (I,V,L), both of which support the formation of a secondary structure, T classifies as an intrinsically disordered protein. Solution structures of rabbit, rat, and human Cd7MT2 were determined by NMR spectroscopy before a revised crystal structure of rat MT became available. (44,45) The peptide chain wraps around the clusters in either a left-handed (α-domain) or right-handed (β-domain) manner (Figure 6). With NMR spectroscopy, it was possible to obtain structures of the individual domains, but how the domains are positioned relative to each other remained unknown and had to await X-ray diffraction investigations in crystals of MT.

Figure 5

Figure 5. NMR assignments of Cd(II)/thiolate coordination environments in the MT clusters. Cluster A (or α) is in the C-terminal α-domain, and cluster B (or β) is in the N-terminal β-domain. Black and red numbers indicate Cys residues in the rabbit MT2 sequence and the Cd(II) ions assigned from 113Cd NMR (inset). (35,39)

Crystallography was performed on Cd5Zn2MT2 from rat liver, and the structure was refined to 2.0 Å (Figure 7). (47) The two Zn(II) ions reside in the β-domain cluster (N-terminal domain). The domains are aligned linearly and are connected by H bonds between the side chain of K31 and C21 and the sulfur atom of C19. This interaction includes a phosphate ion that interacts with the carbonyl of C19 and the side chain of K31. Besides H bonds between the domains, there are H bonds in each domain, and those between sulfur atoms and peptide amides (N–H···S) deserve special mention due to a charge compensation. Likewise, Ser residues in both domains are responsible for a stabilization of the structure by enhancing the H-bonding network, which is discussed further in section 3.2 with reference to Figure 15. Lys residues are an additional factor responsible for the charge compensation. They are conserved in the α-domain but not in the β-domain. The protein forms a dimer in the crystals, and there are several intermolecular contacts. A sodium ion binds to the carbonyl of C29 from one subunit, the carbonyl of A42 and the side chain oxygen of S45 of the other subunit, and three water molecules. Thus, in addition to describing the tertiary structure of the protein, crystallography also demonstrated that MTs can have a quaternary structure. The quaternary structure may have biological implications for the polymerization of the protein and its intermolecular metal exchange as discussed in sections 2.3 and 3.1.
X-ray crystal and NMR solution structures of rat MT2 are identical. (48) The 3D structures with Cd(II) and Zn(II) differ in cluster size only. The 3D structure with Cu(I) is different, however, demonstrating that not only the metal itself but also the type of metal determines the conformation of the protein.
An NMR solution structure of the α-domain of MT3 shows that the metal cluster is identical with that in MT2, but the additional amino acid residues in the α-domain of MT3 have a significant impact on the structure of an extended loop as observed in the individual domains and in the entire protein (Figure 8). (49,50)

Figure 6

Figure 6. NMR structures of mammalian α-domains (PDB: 1MRB, 1MRT, 1MHU) and β-domains (PDB: 2MRB, 2MRT, 2MHU) of MT2. (A) Comparison of the structures of β (left) and α (right) domains of rabbit (blue ribbon), rat (beige ribbon), and human (pink ribbon) Cd7MT2. (B) Sequences of both domains with criss-cross Cd(II) (M) binding sites. (44−46)

The solution structures of the individual domain peptides of murine MT1 were also determined by NMR spectroscopy (Figure 9). Cu(I) titration into the 3-Zn(II) or 4-Zn(II) containing domains yielded defined structures when three Cu(I) ions reside in the α-domain and four in the β-domain. (51) Principally, with Cd(II) isotopes, a heteronuclear coupling allowed a determination of how the sulfur donor ligands connect with the metal ions and hence provided structures of the clusters. Since Zn(II) and Cu(I) do not have this NMR property, neither the cluster structures nor the number of Zn(II) ions remaining bound could be determined in this copper MT. Importantly, the protein structure is significantly different from those with divalent metal ions bound, emphasizing the role of the metal ions in organizing structure. When the two domain peptides were mixed they retained their Cu(I) stoichiometry and did not exchange Cu(I). When more than the seven Cu(I) ions were titrated into the two domains, no defined structures were observed. However, species with a higher number of Cu(I) ions have been observed by spectroscopic methods, for instance, Cu12MT with six Cu(I) ions in the α- and β-domain (51,52) or Cu15MT as confirmed by mass spectrometry. (52−54) It indicates the variability in Cu(I) coordination in forming diagonal and trigonal geometries as shown in the crystal structure of yeast copper MT (Cup1). (55) A trigonal geometry is expected to predominate at a low-copper load, while the chances for a diagonal geometry increase with more Cu(I) ions bound to MT.

Figure 7

Figure 7. Structure of the entire rat MT2 molecule solved by X-ray crystallography. (A) Structure of Cd5Zn2MT2 with the indication of Cd(II) (beige color) and Zn(II) (gray color) ions in both domains. (B) Connectivities of the metal ions with the Cys residues in the β-cluster. (47)

A third method for visualizing a 3D structure is scanning tunnelling microscopy (STM). It provides a direct image of the molecule in a buffered solution and shows the compact structure of MT with its elongated shape and the bound metal ions (Figure 10). (56) Moreover, MT becomes bent when it interacts with adenosine triphosphate (ATP). (57)

Figure 8

Figure 8. NMR structure of the α-domain of human MT3 in the complex with Cd(II). (A) Comparison of the MT2 (blue) and MT3 (beige) structures of the individual domains (PDB: 1MHU vs 2FJ4). (B) Comparison of the MT3 structures of the individual domain (beige) and the one in the entire protein (pink) (PDB: 2F5H). (50)

Figure 9

Figure 9. NMR structures of Cu(I)-containing mouse MT1 domains. (A) 20-structure family of ZnyCu4βMT1; (B) 20 best structures of ZnxCu3αMT1; (C) superposition of the mean Cd3βMT1 structure (red) and the structure family of ZnyCu4βMT1 (blue); (D) superposition of the mean Cd4αMT1 structure (red) and the structure family of ZnxCu3αMT1 (blue). (A, B) Gray, blue, and yellow colors represent the polypeptide backbone, cysteinyl side chain, and sulfur atoms, respectively. Figures were adopted with permission from ref (51), copyright 2007 by John Wiley and Sons.

Figure 10

Figure 10. STM image of rabbit liver Zn7MT2 showing the shape of the molecule and the contrast due to the bound metal ions (A). The metal ions bound in two clusters are also seen in the contour plot (B). (57) Figures were adopted from ref (57) with permission of the American Chemical Society.

The clusters are not static. They are thermodynamically stable and kinetically labile. A fluxional behavior allows a facile exchange of Cd(II) or Zn(II) intramolecularly─within the clusters and between the clusters, with metal ions in solution, and intermolecularly between MT molecules. Cd(II) exchange within the three-metal cluster occurs with rate constants of 0.2–2.7 s–1 (35 °C), while an exchange within the four-metal cluster was too slow to be detectable. (58) On the basis of first principles, Zn(II) exchange rates should be an order of magnitude lower. Zn(II) exchange between rabbit liver MT1 and MT2 is biphasic with rate constants of 5000 and 200 min–1 M–1 (pH 8.6, 25 °C) and was suggested to reflect the intermolecular exchange between three-metal clusters and four-metal clusters, respectively. (59) However, it could also reflect the exchange of four tightly bound metal ions and three less-tightly bound metal ions in the protein in a nondomain-specific way (see below). While the kinetic lability has been discussed primarily in terms of the lability of the divalent metal ions, it also signifies lability in the coordination of the sulfur ligands. Metal exchange requires the thiolate ligands to dissociate, which enhances the reactivity of the thiolates.

2.4. From Structure to Reactivity and Regulation

Starting from approximately the early 1980s, molecular biology and genetics began to contribute to the field of MTs and widened its scope significantly. (60) New MT genes were cloned, defining mammalian MT gene clusters, and the mapping of genes identified their coding and noncoding regions and resulted in a much improved understanding of the regulation of gene expression and silencing of expression by DNA methylation. While the induction of MTs by many endogenous and exogenous substances and conditions of stress was known, (36) the DNA recognition elements of MTs, the transcription factors that bind to MTs, and signaling pathways that control induction became known. Incidentally, the concept of zinc fingers in transcription factors developed at approximately that time. It significantly changed the understanding and scope of the then-known Zn(II) coordination environments in proteins by revealing the prevalence of Zn(II) coordination to the thiolate sulfur of cysteine. Many of the transcription factors that control the MT expression turned out to be zinc proteins with a Zn(II)/thiolate coordination. The pathway of metal induction of MTs was established with the discovery of the metal response element (MRE)-binding transcription factor-1, MTF-1. (61) MTF-1 has six zinc fingers that are thought to be involved in sensing an excess of cellular Zn(II). Perhaps the most important outcome of all the new insights from molecular biology and genetics was that it showed that MTs are integrated into many pathways of the cellular signal transduction network, underscoring the fundmental importance of MTs in many aspects of cell biology: proliferation, differentiation, and programmed cell death (apoptosis). (62,63) How changes in MT and zinc availability are associated in the proliferation, differentiation, and apoptosis of cells is discussed at the end of section 3.1. Until now in this article, we discussed the structure without a reference to function─the holy grail of MT research. The initial characterization of MT as a cadmium protein in horse kidney cortex did not establish a biological function but led to speculation about its roles in catalysis, storage, immune phenomena, or detoxification. (3) The idea that T rather than MT might be the functionally important species was advanced. (5) Subsequent findings of MT1/2 being mainly zinc proteins in many tissues and containing copper under some conditions, MT3/4 in particular, then focused research on the metabolism of these essential elements. Because of the association with multiple metals and the dynamic changes under a variety of conditions, the protein was considered multifunctional. After ∼30 years of research, a consensus developed in numerous review articles that the protein primarily has a role in zinc metabolism overlapping with copper metabolism under specific conditions and with cadmium metabolism under conditions of exposure. However, the molecular mechanism eluded all attempts to define it. Bert L. Vallee compared MTs to sphinxes that guard their riddles (and the career paths of many!). There was some expectation that the emerging gene deletion technology of molecular biology would answer the question. (60) The hope was dashed however when mice with genetic knockouts of MT1 and MT2 were found to be viable under laboratory conditions; that is, the mice reproduced and grew. (64,65) The interpretation of these genetic experiments is discussed in section 6.2. Molecular biology and genetics added a lot of important information about the number of MT genes and their induction, but naturally such investigations did not focus on the protein and therefore did not further our understanding of its mechanism of action. On the basis of the exquisite regulation of gene expression, it was concluded that MTs have a pivotal role in cellular metabolism under normal conditions and conditions of stress. (66) After these initial 30 years, knowledge about the structural and biophysical properties of MT left the scientific community with the specious impression that each MT is essentially one form as determined by the structure of Zn7MT, the elucidation of which undoubtedly was a feat and a culmination in the work on MTs. The work indicated that MT sequesters metal ions and stores them with high affinity. We will now re-examine these postulates after describing subsequent discoveries that were game-changers. Contemporaneous advances in the field of zinc and copper biology make it clear, retrospectively, why the conundrum of function could not have been solved at this earlier point in time. It had to linger for the next 30 years despite a myriad of investigations in both biology, describing conditions of changed gene expression of MTs in health and disease, and chemistry, characterizing the binding of many different metal ions to MT. We believe that the time has finally come to interpret this vast literature in the context of what is now known about zinc and copper metabolism, thus leading to a full exploration of the functional potential and potential functions of MT.
At least three discoveries changed the view about a static, passive role of MT in storing metal ions to a dynamic, active one, in which the protein serves as a metal donor and acceptor: first, the reactivity of its sulfurs, in particular, their redox activity; second, the realization that not all the metal ions are bound with the same high affinity; and third, the complex and dynamic regulation of multiple MTs. In addition to the cysteine sulfurs as ligand donors to metal ions, a series of investigations emphasized their reactivity with other agents as part of the mechanism of action of MTs. The work challenged the dogma of MT as a protein with seven divalent metal ions bound with higher affinity than in most other metalloproteins. At that time, it demonstrated that the prevailing view about the structure of MT and its reactivity was too simplistic and suggested how MTs can have a much more dynamic role in metal metabolism as metamorphic or multimorphic proteins, the structures of which depend on the redox state (section 3.1) and the availability of metal ions (section 3.3), both of which change in biology. MTs turned out to be remarkably reactive, engendering many more structures than implied by their 3D structures. The significance of these dynamic aspects was amplified through discoveries not concerning MT itself but how Zn(II) and Cu(I) ions are controlled and traffic in the cell. The work transiting from structure to reactivity was a turning point and provided different and new perspectives on structures and functions.

3. Reactivities and Metal Affinities of Metallothioneins

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The two properties of the cysteine sulfhydryls, metal coordination and reactions with electrophiles, are linked, because metals will dissociate when the sulfhydryls are chemically modified. The linkage is important because the reactivity of the sulfhydryls depends on the metal load of MTs. Free thiols are generally more reactive, but Zn(II)/thiolates can react, too.

3.1. Redox Chemistry and Biology of MT

The observation that MT reacts with hydroxyl radicals with a 340-fold higher rate constant than glutathione shifted interest from the metals to the reactivity of the sulfurs in MT. (67) It resulted in a significant number of investigations of MT as a scavenger of free radicals. (68) With a possible biological significance for leukocyte biology, it was shown that the three principal neutrophil oxidants, namely, hydrochlorous acid, superoxide, and peroxide, mobilize Zn(II) from MT. (69) The dissociation of Zn(II) was thought to cause cellular injury, but a beneficial role of this process was not ruled out.
Reactions of the thiolates in MT with organic and inorganic electrophiles were known. (70) They include a thiol/disulfide interchange with Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)), which occurs with the metal-bound thiolates in the clusters, albeit with a slower rate than with free thiols (71) as supported by quenching the reactivity when four Zn(II) ions are added to thionein. (72,73) This quenching supports the hypothesis that four Zn(II) ions are tigthly bound across the protein to make it more resistant to electrophilic attack. While the reaction with MT is biphasic, the reaction with the individual α-domain peptide containing Cd(II) is monophasic, suggesting that each cluster reacts separately. (74) However, the reaction of both individual domain peptides containing Zn(II) with an excess of DTNB is biphasic. (75) At sub-stoichiometric amounts, DTNB generates inter- and intramolecular disulfides in MT. Mixed disulfides form only at stoichiometric and higher amounts of DTNB. (76) A biological disulfide, glutathione disulfide (GSSG), triggers the dissociation of Zn(II) from MT. (76,77) While the first report concluded that GSSG is unlikely to react with MT in vivo, the second suggested that such a reaction would link the metal content of MT to the cellular glutathione redox state. Indeed, other biological disulfides such as coenzyme A disulfide and cystamine react more rapidly than GSSG, suggesting that (i) such chemistry is feasible with biological oxidants in the cell, (ii) MT is a Zn(II) donor when the cellular redox state changes to more oxidizing conditions, and (iii) conditions of oxidative stress perturb metal metabolism with important consequences for the progression of many diseases associated with oxidative stress such as neurodegeneration. (78) A comparative investigation of chemical and biological oxidants with different redox potentials was then employed to estimate the redox potential of the thiols in MT. (79) The redox potential of MT was found to be quite low, demonstrating that MT is a redox protein that reacts readily with a host of biological oxidants in the cellular milieu. This novel redox biology of MT suggested a way of how Zn(II) is made available when its availability is low and linked it to the known induction of T to sequester Zn(II) when its availability is high, (80) essentially establishing how the cellular redox state controls the cellular Zn(II) availability via the MT/T couple (Figure 11). (81)

Figure 11

Figure 11. Function of the MT/T couple as a homeostatic Zn(II) system. An increase in the amount of available Zn(II) induces the synthesis of T through the action of Zn(II) on Zn(II)-dependent transcription factors and leads to the formation of MT and a sequestration of Zn(II) (left). When available Zn(II) is low and needed for the synthesis of zinc proteins, Zn(II) dissociates from MT and T is formed (right). For clarity, the effects of oxidants and reductants are omitted. The redox effects are illustrated in Figure 13.

Such redox activity is remarkable because Zn(II) is redox-inert in biology, and zinc sites in zinc proteins therefore had not been considered to participate in redox metabolism. Thus, while transition-metal ions such as Fe(II)/Fe(III) and Cu(I)/Cu(II) are involved directly in redox reactions, in the case of Zn(II), another d-block metal but not strictly a transition metal (Zn(II) has a filled d-shell ion and therefore is not considered a transition-metal ion per IUPAC recommendation), (82) the sulfur donor ligand in the coordination environment and not the central atom confers redox activity on the complex. The redox chemistry and reactivity of the sulfur donor ligands in MT add an important principle to redox biology, which hitherto was based on either redox-active transition metals or sulfur chemistry in the absence of metals. (83) The new principle is that, in seemingly redox-inert zinc proteins, the thiolate ligand has a direct redox function and the dissociated metal ion an indirect function on redox metabolism. It is a typical example of a context-specific biological chemistry that turned out to be different from the focus of purely chemical in vitro investigations of MT until that point in time. Since the Zn(II)/thiolate coordination is quite frequent in cellular proteins, in particular, in the large number of zinc finger type of proteins, the redox biology of MT also inspired investigations into the redox functions of other zinc proteins with Zn(II)/thiolate coordination environments, in which catalytic or structural sites in proteins were shown to be redox-modulated. In this way, these proteins can serve as redox sensors and transducers when Zn(II) dissociation and disulfide formation change their conformation. Thus, unlike in MT, where Zn(II) dissociation serves as a trigger of biological events, the changed protein conformation can become the trigger. The rather extensive subject matter of the redox activity of Zn(II)/thiolate coordination in proteins other than MT has been reviewed and is beyond the scope of this article. (84−86)
The redox chemistry and redox biology of MT suggested a set of new functions of the protein. In particular, it initiated the search for the metal-free protein (TR, thionein) and the oxidized protein (TO, thionin). It was known that the MT fraction in homogenates of neoplastic cells was able to bind additional Zn(II), suggesting that T exists in cells together with MT. (87) Further evidence for the existence of forms of MT that are not fully saturated with metal came from the investigations on rat tissues, in which commensurate amounts of MT and T were found by employing a differential modification assay with ABD-F (7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide) (Figure 12A). (88,89) Because of various Zn(II)-to-thionein affinities (see below), we now understand that T (the species entirely devoid of metal ions) cannot coexist with MT (the species that is saturated with metal ions), and that instead Zn7–xMT forms are present in cells. (73)

Figure 12

Figure 12. Differential modification of MT with ABD-F uncovers the presence of significant amounts of metal-depleted protein. (A) Schematic reaction of ABD-F with the sulfhydryl group resulting in a fluorescent adduct. (B) Amounts of MT and T (TR+TO) found in various rat tissues. (88) (C) Changes of MT and T (TR+TO) content in HT-29 cells pretreated with ZnSO4. (88) (D) Percentage of MT, T, TR, and TO in rat liver stored on ice for up to 30 h as determined with a modified differential ABD-F assay. (89) Gray, red, blue, and green bars correspond to MT, T (TR+TO), TR, and TO species. MT, T, TR, and TO correspond to fully metal-loaded, fully depleted, reduced and depleted, and oxidized MT, respectively.

3.1.1. TR (Thionein)

ABD-F is a fluorescent sulfhydryl labeling reagent that reacts rapidly with free sulfhydryls but only very slowly with the Zn(II)/thiolates in MT. It was employed for a differential labeling assay in which tissue extracts were analyzed by a modification with the reagent in the presence or absence of the chelating agent ethylenediaminetetraacetic acid (EDTA) and a subsequent chromatographic separation. (88) The assay is based on the observation that Zn(II) quenches the reactivity of cysteine residues with thiol-modifying reagents, whereas the removal of Zn(II) from MT with chelating agents restores the reactivity of the sulfhydryls. The incubation of MT-containing samples with ABD-F in the presence of a strong metal chelator such as EDTA and the reducing agent tris(2-carboxyethyl)phosphine (TCEP) results in a total metallothionein modification (MT+T) with ABD-F as manifested by a strong fluorescence at 512 nm when excited at 384 nm. A lower fluorescence observed when ABD-F reacts with MT in the absence of EDTA showed that a certain amount of protein must not be saturated with metal ions. A high-performance liquid chromatography (HPLC)-based quantification with fluorescence detection allowed a calculation of the MT amount as the difference between two reactions in the presence and absence of EDTA; that is, MT = (MT+T) – T. (88) Importantly, the terms “T” and “MT” in the assay refer to metal-free and metal-saturated protein because the only existing model at that time was one with highly cooperative Zn(II) binding, which would allow T and MT to coexist without any intermediates. When we consider later findings that this model is inadequate, the assay in fact examines an average of multiple species, namely, a ratio of modified and unmodified Cys residues in the assembly of MT proteins that includes partially metalated species (see below). Employing the assay, significant amounts of T were detected in biological tissues. The amounts found in rat liver, kidney, brain, and testis were 27, 54, 53, and 9% of total metallothionein (MT+T), respectively (Figure 12B). Similar analyses performed on HT-29 colorectal cancer cells showed the presence of T over total MT+T as 30%, an amount that decreased to 25 and 21% when cells where incubated with 100 and 200 μM ZnSO4, respectively, for 6 h prior to cell homogenization (Figure 12C). (89) Concurrently “free” Zn(II) concentrations increased from 0.8 pM in nontreated cells to 5.2 nM in cells incubated with 200 μM zinc sulfate. It demonstrated that T/MT molar ratios correlate with freely available Zn(II) and indicated a fundamental role of the ratios in the buffering of cellular zinc. (89,90)

3.1.2. TO (Thionin)

A second differential modification assay was developed to interrogate the redox state of the cysteine thiols in MT and to distinguish TO from TR, that is, oxidized from reduced species not fully saturated with metal ions. It uses a derivatization of the thiols with the fluorescent probe 6-iodoacetamide-fluorescein and a colorimetric determination of the product. (91) In the presence and absence of the nonsulfhydryl-based reducing agent tris(2-carboxyethyl)phosphine (TCEP), the assay gives the amounts of TO and TR, respectively. Similar to the reaction with ABD-F, the assay in the presence and absence of EDTA then gives the total amount of the protein (MT + TR) and TR, respectively, and thus allows the determination of all three species MT, TO, and TR. In this work, chemical (DTNB) and enzymatic (sulfhydryl oxidase (SOX)) oxidations were used. Likewise, a modified ABD-F method employing quenching of the sulfhydryl groups with N-ethylmaleimide allowed the determination of the three species in homogenized rat tissues and demonstrated an increase of TO over time (Figure 12D). (90) Thus, oxidized forms of the protein indeed exist in vivo. Disulfides form in both domains when MT is overexpressed in the heart of mice and increase under conditions of oxidative stress. (92) Cysteines 44 and 48, which are ligands to metal number 5, are involved (Figure 5). Also, the human ovarian surface epithelial (HOSE) cell line was used together with the same cell line transformed with an H-rasv12 oncogene, which produces reactive oxygen species, to determine the oxidation state of MT. In the parent cell line, less than 10% of the thiols were found to be oxidized, an amount that corresponds to the formation of one disulfide bridge. But in the transformed cell line, a threefold increase in oxidation was observed. (90)
Chemical oxidation in vitro generates intra- and intermolecular disulfides in MT. (76,93) MT polymers form in the reaction with DTNB. (76) Multimers are found when the protein is oxidized in vitro and analyzed electrophoretically after fluorescence labeling with 5-iodoacetamide-eosine. (94) In this oxidative polymerization, MT polymers from dimers all the way to decamers were detected. Arrangements of MT molecules as dimers in the crystal structure and a possible trimer interface suggest how this polymerization proceeds structurally. (47,95)
With the finding of TR and TO forms, it became clear that MT undergoes a redox cycle (Figure 13) and that there are minimally two species in addition to MT, an oxidized form, thionin, TO, and a reduced form, thionein, TR. The redox cycle is extended to a dual cycle to account for the later finding of partially metalated species (see below).

Figure 13

Figure 13. MT and T redox cycles. In this expanded dual cycle, TR and TO represent the fully reduced (thionein) and oxidized (thionin) metal-free protein. Zn7MT, Zn7–xMTR, and Zn7–xMTO refer to fully loaded, partially Zn(II)-depleted reduced, and partially Zn(II)-depleted oxidized protein, respectively. It is presumed that thionein/thionin can serve its own role as a redox couple in vivo under conditions of restricted metal ion availability.

From prior investigations, it had been known that MT is a rather reactive protein, prone to oxidation during protein isolation, purification, and storage, but this information was not related to its biology. One may ask why additional forms, TR and TO, had not been discovered earlier when MT was isolated from biological tissue. There are several answers. Because the protein lacks aromatic amino acids and there are no assays based on a biological activity, MT was largely isolated by following its metal content, thus precluding the detection of other forms that do not contain metals. The discovery of an efficient induction by heavy metal ions changed significantly the procedure of MT preparation and purification. To increase the yield of the protein prepared from livers, rats or rabbits are injected with sublethal doses of Zn(II) or Zn(II) and Cd(II) salts for several days. (5,96) With this procedure, the protein is forced into the metal-bound form. Yet, isolated MT remained heterogeneous. The metal-to-protein ratios did not always indicate a full saturation of the protein with seven divalent ions, demonstrating that partially saturated MT may be present in some tissues even when the protein is metal-induced. Also, MT was mostly isolated from liver, the hepatocytes of which normally do not proliferate and are rich in metals, but rarely from other sources. Nowadays, overproduction of a specific MT in a bacterial expression system is the most straightforward method of MT preparation. It requires a cloning of the cDNA encoding MT into an appropriate vector, for example, the plasmids pETYB or pET, and the subsequent induction of thionein production with isopropylthio-β-galactoside (IPTG). (97) Although metals are not inducers in these constructs, they may increase the yield of protein during purification. Heterologous expression increases protein purity due to the production of only one MT isoform, but conditions are different in terms of redox state and metal ion availability from native tissues as metal ions are added to bacterial growth media. Also in this case, the isolated MT can be partially oxidized and not fully saturated by metal ions, and the expressed MT is not a chemically pure species regardless of the method of isolation or induction, though using Cd(II) instead of Zn(II) for induction increases the metalation of induced MT due to its higher affinity for the protein. (28) Thus, MT isolated from either natural sources or bacterial cells is heterogeneous. In order to obtain homogeneous MT a reconstitution procedure is required. It includes the reduction of any oxidized thiols in the protein, removing the bound metal ions and, subsequently, a controlled reconstitution of the fully reduced T with the desired metal ion. (98)
In the search for the biological oxidant(s), many proposals were made for the redox biology in vivo. Glutathione (GSH)/glutathione disulfide (GSSG) received particular attention as a biological redox pair interacting with MT and metal ions. Changing the GSH/GSSG ratio affects the Zn(II) dissociation from MT and Zn(II) transfer to the apoforms of zinc proteins. (99) In addition to GSSG releasing Zn(II) from MT, GSH modulates the transfer. GSH binds to invertebrate copper MT and rabbit liver Zn(II),Cd(II)-MT. (100) For the latter species, the binding constant is 14 ± 6 μM. Molecular modeling and energy minimization suggests a stabilization of MT and a prevention of the dissociation of Zn(II) when the GSH sulfur replaces the sulfur ligand of Cys26 in the N-terminal domain of MT. (101) The biological significance of the interaction is not entirely clear, but it could indicate that the metal affinities of MT are further modulated by a ternary complex formation. GSH inhibits the Zn(II) transfer, and a Cu(I)/GSH complex has been postulated to be in equilibrium with copper MT. (99,102) The role of Zn(II)/GSH complexes in cellular zinc trafficking is even less clear than in the case of Cu(I), though the binding equilibria have been well-characterized. (103,104) The putative role of GSH in copper metabolism is addressed in section 4.4.
Another type of reaction is the S-glutathionylation and S-homocysteinylation of MT. Glutathionylation was investigated with commercial rabbit liver MT in vitro with either S-nitrosoglutathione or GSH and hydrogen peroxide or diamide and detection of the glutathionylated protein with an anti-GSH antibody. (105) Glutathionylated, highly aggregated MT was also found in peripheral blood mononuclear cells (PBMCs) in vivo. Solvent-accessible Cys7, Cys13, and Cys59 were implicated in the reaction. (105) Cys7 and Cys13 have been shown to be involved in the nitrosylation of MT. (106) The homocysteinylation of MT was detected in human aortic endothelial cells (HAECs) when using 35S-homocysteine. (107) The intermolecular disulfide bond is stable in the presence of 10 mM GSH. This stability is based on the higher sulfhydryl pKa value of l-homocysteine (10.0) compared to l-cysteine (8.3). The proposed biological significance of this reaction is that it provides a pathway of how increased blood levels of homocysteine, which are a risk factor for cardiovascular disease, effect metal redistribution.
Numerous investigations showed that a subjection of cells to various types of oxidative stress increases the availability of Zn(II) ions in the cell. It includes disulfide stress. 2,2′-Dithiodipyridine, a cell-permeable disulfide, leads to Zn(II) dissociation. (108)
Enzymes have been shown to be involved in the redox chemistry of MT in vitro, but the significance of these reactions in vivo remains to be shown. One example is avian sulfhydryl oxidase, a flavoenzyme of the quiescin sulfhydryl oxidase family of proteins that introduce disulfide bonds into cellular and extracellular proteins. (109) A kinetic characterization of the reaction demonstrated that T, but not MT, is one of the best substrates of this enzyme with a kcat/Km value of 2.1 × 105 M–1 s–1. (91,94) Another protein involved in the disulfide formation in proteins is protein disulfide isomerase (DsbA). It reacts with MT under stoichiometric conditions. (80) Further kinetic characterization of the reaction also demonstrated a rather high (103 higher than glutathione) rate constant of 4.4 × 105 M–1 s–1. (110) Last but not least, MT thiols are a substrate for the selenium enzyme glutathione peroxidase. (111)
Biology uses selenium, the congener of sulfur in the periodic system of the elements, in redox biology. The redox chemistry of selenium compounds with the thiolates in MT has been investigated. (111) It is remarkable, because, in addition to reacting stoichiometrically in higher oxidation states than the selenol─the equivalent of a thiol─selenium compounds catalyze reactions of MT with reactive disulfides and the subsequent reduction of the oxidized proteins. (112,113) It suggests a relationship between the metals carried by MT and the biologically essential micronutrient selenium and its anti-inflammatory and anticarcinogenic actions.
The redox chemistry of MT and the presence of MT species that are not saturated with metal ions changed the field from a perception of MT as a protein storing metal ions to one being involved dynamically in metal and redox metabolism. A characterization of isolated MT and investigations of its state in cells and tissues demonstrate that MT is unlike any other zinc or copper protein with catalytic or structural sites that are generally fully occupied with the metal ion to maintain function. MT’s metal content is variable, and MT has a dynamic structure that depends on the metal availability, type of metal ions, and redox conditions in its biological environment at extracellular and intracellular locations. A correlation between the cellular redox state, the redox state of “MT”, and the concentrations of free Zn(II) has been observed in four defined states of the same human cell line (growth-arrested, proliferating, differentiated, and apoptotic). (89) Thus, observations in both isolated systems and cells suggest that the redox state of MT is one factor determining the availability of cellular Zn(II) ions.
A limited number of investigations have suggested alternative or additional functions of the redox chemistry of MT and T, namely, that Zn(II) controls their redox function rather than their redox function controlling Zn(II). For example, in the presence of EDTA, MT assists the protein disulfide isomerase-catalyzed formation of native pancreatic ribonuclease A in vitro. (114) In another interaction with enzymes, it was shown that TR/TO could be part of a redox chain between thioredoxin and methionine sulfoxide reductase, essentially linking nicotinamide adenine dinucleotide phosphate (NADPH) to the reduction of methionine sulfoxide. (115) In a search for a reducing factor for human methionine sulfoxide reductases (Msr) B2 and B3, a heat-stable factor from bovine liver was isolated, shown to serve as such as a reductant in the presence of EDTA, and identified as MT. It was then shown that TR can reduce human MrsB3 and TO can be reduced by the thioredoxin system. Selenocystamine or selenocystine increases the potency of thioredoxin or TR as reducing agents. (116)
In terms of redox chemistry, MT is often discussed as an antioxidant. Any such antioxidant function must consider the metal load of MT and the role of the dissociated Zn(II) ions, which are potent effectors of protein function and have their own indirect effects on redox biology. (117) Depending on concentrations of dissociated Zn(II) ions, they can have either pro-antioxidant or pro-oxidant functions in the cell.
For the reasons discussed here, we link both MT and T primarily to zinc homeostasis rather than redox homeostasis, namely, that global redox changes or changes of particular redox pairs determine the availability of cellular Zn(II) (and Cu(I)). This link has gained further support when experiments in cells examined how signaling with with reactive species targets MT and affects Zn(II) dissociation to generate “zinc signals” as potent effectors of biological processes. These observations were critically important for the emerging field of zinc signaling (see below), as they demonstrated that the Zn(II)/thiolate clusters in MTs are transducers of redox signals into zinc signals. The observation that MT reacts with nitric oxide (NO) followed by a Zn(II) dissociation continued the focus on the sulfhydryl reactivity of MT. (118) The possibility that MT is a target of redox signaling became apparent when it was shown that cellular NO formation triggered by hormonal stimulation of nitric oxide synthase reacts with MT and increases concentrations of cytosolic and nuclear Zn(II) ions. (119,120) To bring about specificity in terms of which sulfhydryls are targeted, transnitrosation is a mechanism. For example, MT3 is the only MT that has an acid–base sequence motif indicative for S-nitrosylation by transnitrosation at Cys22 and Cys42, and it reacts with S-nitroso-l-cysteine faster than other MTs. (121) On the basis of observations that extracellular S-nitroso-albumin and a cell surface protein disulfide isomerase are involved in transnitrosation of intracellular proteins, MT was identified as a protein of such transnitrosation in endothelical cells. (122) Thioredoxin and lipoic acid then could be involved in the denitrosation. (123) It was suggested that oxygen is needed for the S-nitrosothiol formation because an MT-SNOH radical intermediate forms under anaerobic conditions. (124) Reactions of the sulfhydryls of MT are not restricted to species with bound Zn(II), as redox signals can also lead to the dissociation of bound Cd(II) or Cu(I). (125,126) These reactions were mostly studied in vitro. Analyses in which Cys thiol(ates) are nitrosylated in vivo have yet to be performed. Uncertainties remain about these reactions under normal conditions versus conditions of redox stress.
Another type of modification of the sulfhydryls in MT is alkylation. Reactive carbonyls react with the sulfhydryls in MT with a concomitant Zn(II) dissociation. (127) Reactants can include endogenous aldehydes, aldehydes formed under carbonyl stress such as observed in lipid peroxidation, and xenobiotic aldehydes in the diet or the environment. For example, oxidized dopamine products react with MT. (128,129) Alkylation has also been observed with pharmaceuticals, in particular, with highly reactive compounds used in cancer chemotherapy. The important message is that all these reactions affect the metal redistribution or perturbation of the control of metal homeostasis with implications for many mechanisms of cytoprotection or cytotoxicity. (130)
A determination of the redox state of MTs, their polymerization and modifications, the presence of Cu(I)/Cu(II) and Zn(II) under some conditions, ligand binding, and the existence of multiple MTs makes speciation a hugely challenging analytical problem that has not been resolved to this date. What the species MT, TR, and TO are molecularly is a different matter, as all of them are not exactly one species but rather an assembly of species (see below), making the name “metallothionein” very unspecific in terms of what it actually refers to─a key consideration when associating functions with molecules that have a generic name only. And the species undergo dynamic changes, which introduce biological time and space as yet other variables. With the assays described above the metal load of MTs is measured, the MT/T ratio, which is the averaged sum of different forms. Finding a given percentage of T indicates that the protein is not fully saturated with metal ions. Metal binding depends on metal affinity and metal availability in addition to biological factors such as the rates of synthesis and degradation of the proteins. Assays of liver homogenates in the absence and presence of added Zn(II) ions demonstrate that the proteins indeed do not carry a full complement of seven Zn(II) ions because the metal load increases when metal ions are added. (89) The interpretation of highly cooperative binding needed to be revised when affinities of the seven Zn(II) ions for human MT2 were found to differ, (73) and this is the next development to be discussed, which further increases the number of species that the epithet “MT” refers to.
Much as we made the point before that redox biology added to the chemistry of MT in the biology milieu, the opposite is equally important, namely, how chemistry adds to biology as now discussed by chemical investigations on the affinity of MT for Zn(II) ions. To emphasize this aspect, a quote from Professor Colin Thorpe’s webpage (University of Delaware) is provided: “Biology cannot ignore chemistry, much as I wish it could” (John Maynard Smith).

3.2. General Notes on Metal Affinities and the Essence of Metal/Thiolate Coordination

Metal binding is at the center of the properties of MTs. The determination of the affinities of metals for proteins is challenging. (131) In the case of MT, the challenge is even greater due to the presence of multiple and alternative coordination sites. The affinity of metal ions for MTs depends on numerous factors. Definitely the most important one is the tendency of a particular metal ion to form a metal–thiolate complex. According to the hard and soft acids and bases (HSAB) concept, soft metal ions prefer binding to soft bases such as sulfur donors. (132) Therefore, the softest metal ions such as Hg(II) and Cu(I) have the highest affinities toward MTs, although their exact stability constants are still under scrutiny. Cd(II) has a much softer character than Zn(II) and therefore forms significantly more stable complexes. Zn(II) has a moderately soft character, resulting in binding to nitrogen and oxygen donors in addition to cysteinyl sulfur in proteins. Neither nitrogen nor oxygen donors are involved in metal ion coordination in mammalian MTs. Only sulfur donors are responsible for efficient but not necessarily equal binding in terms of affinity. The presence or absence of sulfur donor bridges in MT may or may not impact affinity depending on the particular metal ion. Metal ions such as Cu(I) and Cd(II) have a higher tendency for thiolate cluster formation, whereas the tendency of others such as Zn(II) or Co(II) is lower. (98,133,134) One question in the history of MT research always has been what the significance is for the formation of thiolate clusters.
The interaction of MTs with various metal ions has been investigated with multiple techniques, mostly different types of spectroscopies and mass spectrometric aproaches, among which only a few have provided limited information about metal-to-protein affinities. More sensitive methods have added to our knowledge and define the order of metal ion affinity in MT (Figure 14). This order is not absolute, as other chemical and biological factors may affect the range of affinities.

Figure 14

Figure 14. Affinity series of metal ions binding to metallothioneins. The order of affinities is based on experimental data and estimations from exchange experiments and model studies. (28,135−142) The series also defines the tendency of free metal ion concentrations buffered by MTs. Its implication for the metalation of proteins is discussed in section 4.1.

Affinity data have been collected with multiple methods at various conditions. Considering only the essential metal ions Zn(II) and Cu(I), the Kd values in Figure 14 correlate well with published cellular free metal ion concentrations, (89,143−145) suggesting that MTs function as a buffering system for these metal ions. Moreover, the range of affinities also should be commensurate with fluctuations of free metal ions, as Zn(II) ions are signaling ions, and more recently Cu(I) ions also have been suggested to have signaling functions. At this time, our knowledge of the stability of metal complexes is limited to a few MTs with a single metal ion, and investigations on affinity constants of mixed metal MTs are lacking. Given the different affinities and stereochemical preferences of Zn(II) and Cu(I), it is likely that a partial occupation with one metal ion will affect the affinity of the other one. Since mixed MT complexes (Zn(II)/Cu(I), Zn(II)/Cd(II), or Cd(II)/Cu(I)) form and are found in tissues, it is an important issue awaiting scientific inquisitiveness to be addressed. In addition to serving as a dual metal buffer for the two most competitive metal ions, MTs could have a role in keeping strict ratios between these metal ions or, perhaps more importantly, in changing these ratios depending on metabolic or energetic demands of cells. Therefore, much more emphasis needs to be placed on the properties of mixed MT complexes, especially with essential metal ions such as Zn(II) and Cu(I). It is insufficient to base our knowledge about MTs on the binding properties of only one metal ion because pools of many different metal ions exist in the biological environment. How the metal ions cooperate in the cell will determine how they interact with MTs. Since MTs bind various metal ions with affinities covering a wide range, it is important to understand how the formation of mixed-metal complexes affects buffering and hence the availability of metal ions. How this control occurs from a molecular and cellular point of view is a critical question to be addressed experimentally. Do the mixed metal complexes still serve as metal donors and acceptors in the same way or does one metal ion inhibit or enhance the availability of the other? How does the cellular redox status affect the buffering properties of such mixed-metal complexes? Do MTs retain one metal ion under certain conditions and tune the availability of another when needed? How would such subtle control work? Do MTs function as Zn(II)-modulated Cu(I) chaperones?
Because MTs do not form stable secondary or tertiary structures in the absence of metal ions, the most important factor that drives particular metal ion affinities is the enthalpy of the metal–sulfur bond formation. While the thiophilicity is an overriding factor in explaining why Hg(II) forms more stable complexes than Cd(II) and the Cd(II) complex is more stable than that of Zn(II), several other factors are critically important for the metal-to-protein affinity, for example, differences of metal affinities provided by the different primary structure of the domains and particular coordination environments of each site in the two domains.
In principle, the difference in stabilities between metal ions with filled d-shells, that is, Zn(II), Cd(II), Hg(II), and Cu(I), and those with partially filled d-orbitals derives from the ligand field stabilization energy (LFSE) in the latter. For instance, Zn(II) and Co(II) binding to Cys-rich sites is often accompanied by a transition from an octahedral geometry in water to a tetrahedral geometry in protein sites. With Co(II) (d7), a change of geometry leads to a thermodynamic penalty due to the LFSE. The penalty arises from different splittings of the d-orbital energy levels in an octahedral versus a tetrahedral ligand field. In the case of Zn(II), the d10 shell leads to the same average electronic energy in either ligand field. The different splitting of d-orbitals into eg and t2g levels in both geometries gives Co(II) a preference for an octahedral geometry compared to Zn(II), which, on the basis of model complexes, amounts to a difference of −4.5 kcal/mol in free energy. Indeed, experimental data, mostly for zinc finger proteins, show that formation constants of the Zn(II) complexes are 2–4 orders of magnitude higher than the ones for Co(II). (135,146,147) Another important factor that affects the coordination number of metal ions is the overall net charge of the complex formed. The formation of complexes with higher coordination numbers becomes increasingly more unfavorable energetically due to a Coulomb repulsion. The higher stability of Zn(II) over Co(II) complexes is also due to the greater charge/size ratio, as the Zn(II) cation is smaller than Co(II). Data on metal affinities of Co(II) or Ni(II) toward MTs are fragmentary, for example, using only the α-domain of MT2 by spectroscopically monitored titrations. (148) Therefore, the order Zn(II) > Co(II) > Ni(II) is based not only on these limited data sets but on LFSE rules and metal binding affinities of other Cys-rich sites (Figure 14).
For metal complexation in MT, several specific factors need to be considered. One effect that is mostly neglected in discussions of metal-to-protein affinity is proton dissociation from binding amino acid residues driven by metal ion coordination. From a biological perspective, it is a factor to contend with, as pH values vary in different cellular compartments. Metal binding to MT causes several energetically important events at the same time. Besides water molecules dissociating from the metal ion and the formation of the coordination bonds between the metal ion and the sulfur donors, the ionization of R-SH groups impacts the energetic cost of the whole process. The proton disscociation is endergonic, while the proton association to solution components is exergetic. The pKa values of thiols therefore have an impact on the process. Deprotonated thiol groups clearly do not contribute to the energetics of proton dissociation, and in this case the enthalpy of the metal–ligand bonds is the most important component of metal-binding thermodynamics. Overall, the more acidic the cysteine thiol the more likely is the formation of a more stable binding site. There is limited information about the acid–base properties of T. The average reported pKa value of the cysteine thiols in MT is 8.6–8.9, depending on the investigation. (141) However, the acid–base properties of individual thiol groups in T remain unknown. Various Cys motifs in the MT sequences and variable environments of particular cysteines are expected to fine-tune the acidity of the thiol groups. Thiols and thiolates can interact with amide protons. (149,150) Fluorescence resonance energy transfer (FRET) probes, molecular modeling, and mass spectrometric investigations have shown that T does not adopt an absolute random coil conformation and that electrostatic interactions including hydrogen bonding determine its conformation. (151−153) All of the above-mentioned energetic effects are important for the metal complexation in individual or clustered sites in MTs. In individual sites, four Cys residues are involved in complexation, while the formation of an M2Cys6 cluster requires the deprotonation of three Cys thiols per one metal ion. The coordination of a second metal ion to a preformed MCys4 core requires only two deprotonations. One cannot neglect entropy, which changes during metal complexation to the protein depending on the metalation mechanism. The significance of entropy is even more obvious, as the enthalpy of binding of each metal ion is very similar in the same coordination spheres. Thus, entropy is a determining effect in the thermodynamics of the binding process. (154) In T, there are no preorganized coordination environments, and protein folding, that is, the spatial organization of the protein’s main chain, is coupled to the metal binding with a potentially very large conformational space in the process. There will be significant differences between the binding of the first couple of metal ions, which serve a role in nucleation of protein folding, and the binding of the last metal ions, which bind to an already mostly folded protein. Protein folding therefore is a very important factor in the overall affinity of the metal ions. Since the only secondary structure elements in MT are two 310 helical segments and a few half-turns, the formation of a secondary structure does not seem to be critical. Numerous hydrogen bonds have been observed in both domains of the crystal structure of rat Cd5Zn2MT2 (Figure 15). They provide rigidity to the entire structure and impact the protein’s affinity and physicochemical properties. Without a crystal structure of zinc MT2 or crystal structures of other MTs, there is no comparison how this hydrogen-bonding network affects the metal affinity in other forms. Structures of partially metal-depleted species are not available. Our knowledge about them is based only on computational methods and a characterization of structure at low resolution by mass spectrometry and pertain almost exclusively to Cd(II) species. (154−158) Importantly, all discussed energetic effects that control metal-to-protein affinity can be different in states with a different metal load and, therefore, should be considered separately when discussing specific metal association or dissociation processes. Overall, despite formally being in the same tetrahedral coordination sphere, the metal ions in MT have various affinities. In Zn(II)-MT2, they vary by almost 4 orders of magnitude, corresponding to ∼5 kcal/mol in free energy difference of complexation (see below). The thermodynamic effects responsible for such significant variations in affinity are not obvious in this complicated system. More insights into the mechanism of protein folding/unfolding are needed to understand the coordination dynamics of MT and thus to ultimately define its molecular functions.

Figure 15

Figure 15. Intramolecular hydrogen-bonding network (red dashed lines) in the structure of rat liver Cd5Zn2MT2 (PDB: 4MT2). Gray and beige spheres denote Zn(II) and Cd(II), respectively. (47)

Another important issue concerns the conditions of the experimental procedures employed to determine the metal-to-protein affinities. Notably, MT is not a simple ML complex, in which a metal ion binds to a strictly defined binding site with a stoichiometry of 1:1. In a simple metal complexation such as in a zinc finger (ZF) domain, the binding is represented by one event, for example, at neutral pH, and depends on the relative ratios of the reactants, the M-ZF complex, and the apo-ZF present. The pZn (−log[Zn(II)]) dependence of complex formation is characterized by only one binding isotherm with an inflection point corresponding to the −log Kd value of the M-ZF complex. If the point is shifted to a slightly lower or higher pH, only one event will be observable in the pZn graph (Figure 16); however, conditional dissociation constants are shifted due to their dependence on pKa values of the ligand donors of amino acid residues. How significant the shift of affinity as a function of pH is will depend on the composition of the coordination sphere. For example, if the metal binding occurs in a mixed coordination environment of His (H) and Cys (C) ligands, the pH-dependent shift of affinity will be different from that of a site with only Cys ligands because His is mainly deprotonated close to neutral pH.

Figure 16

Figure 16. Illustration of how the metal-to-protein affinity shifts when the pH increases or decreases. The case corresponds to a model metal site composed of Cys residues with a −log Kd value = 11.5. Inflection points of the binding isotherms numerically correspond to −log Kd values under the conditions used.

In three different metal binding sites (CCCC, CCHH, and HHHH) one obtains three quite different relationships between the pH value and the stability of the complex formed. Assuming that the Zn(II) coordination system has the same Kd value at pH 7.4 in all cases, the site with His residues will only form the most stable complexes at lower pH and the weakest above pH 7.4 in comparion to Cys residues. Morever, the Kd value remains constant in the alkaline pH range, because one N–H in the imidazole ring is completely deprotonated at pH ≈ 7 or above, and metal binding above this pH does not cause a deprotonation of the second N–H. The scenario is different if only Cys ligands are involved in the Zn(II) binding site. In this case, a pH-dependent increase of the complex stability occurs in the alkaline pH range until a pH value above 10 is reached, where all thiol groups are ionized (Figure 17). A mixed coordination sphere of Cys and His ligands behaves in between the two discussed examples. (135,159−161) A determination of the stability at different pH values, therefore, undoubtedly will result in major differences of metal affinities. A shift of only one pH unit will result in ∼4 orders of magnitude difference in Kd values. Clearly, this pH dependence makes a comparison of Kd values determined at different pH values problematic, a fact to consider in experimental procedures and discussions of MT thermodynamics. In conclusion, these considerations illustrated in Figure 17 resolve a long-standing issue about the exclusive coordination to Cys ligands in mammalian MTs. There are at least three purposes for the use of sulfur. In addition to their redox activity, the sulfur donors impart a higher affinity and hence selectivity for the more thiophilic metal ions, in particular, the competitive essential metal ions Zn(II) and Cu(I) and a pH sensitivity of the affinity in the range of physiological pH values with yet to be fully explored biological consequences for the pH control of metal coordination.

Figure 17

Figure 17. Influence of the composition of the coordination environment in zinc sites on the metal affinity and the pH dependence of conditional dissociation constants. With HySS software, the relationships were simulated based on the assumed protonation and stability constants in such a way that all formed complexes reach −log Kd = 12 at pH 7.4. (162) The pKa values of Cys thiols and His imidazoles have been limited to the 8.6–8.9 and 6.1–6.4 ranges, respectively, based on previously determined data. (135,160,161)

With the clusters, MTs are a much more complicated coordination system than zinc fingers. (135) The next chapter will discuss the metal affinities and metal binding mechanism of MTs, which was the subject of debate for several decades. New evidence was provided that Zn(II) binding occurs differently from that of Cd(II) and Cu(I) and with much less cooperativity than assumed hitherto. Retrospectively, these findings are understandable because the clusters in MT are not as symmetrical as clusters in inorganic models, and metal binding occurs in a specific order determined by energy minima influenced by the folding of the peptide chain. The main reason why results suggested that Zn(II) binding to MTs occurs with an indistinguishable affinity is that investigators examined the binding process as an average of affinities. The observation of metal binding events in metal-controlled media is much more robust and avoids easy-to-miss differences in affinities or an overestimation of stability constants. To illustrate step-by-step the origin of errors in relying on data that are averaged, one needs to dissect the binding process molecularly and eschew the assumption that Zn(II) binding is highly cooperative. UV absorption is followed in a pH titration of MT to determine affinities because Zn(II)/thiolate and Cd(II)/thiolate coordination results in several ligand-to-metal charge transfer (LMCT) bands in the UV (followed at 218 nm for Zn(II) and at 254 nm for Cd(II)). In the case of the Zn7MT molecule, there are 28 Zn–S bonds; 12 are present in the β-domain, and the remaining 16 are in the α-domain. Clearly, it is impossible to distinguish the contribution of the terminal Zn–S bonds and the bridging Zn–S–Zn bonds to the UV absorption. All Zn–S bonds are expected to differ from each other, but it would require quantum-chemical calculations to resolve the contributions from the protein’s environment. Considering that the first four bound Zn(II) ions are distributed across the protein (in the α- and β-domains), they generate up to 16 Zn–S bonds. This maximal number assumes total independent events with the same high affinity, but a more likely scenario includes some bridges between metals as well. The binding therefore is observable as one event and causes a huge increase in absorption amounting to ∼60% of the total absorbance in the UV range (Figure 18A). The remaining three Zn(II) ions bind to the protein by docking to already coordinated metal ions, mostly by generating bridged connectivities that result in the formation of the maximally remaining 12 Zn–S bonds. Because those three Zn(II) ions bind to the protein with lower affinities, pH-dependent events in the pH titration are shifted to higher pH values. This shift is readily noticeable. The fifth and the sixth Zn(II) ions bind with only slightly lower (moderate) affinity compared to the first four, resulting in a small shift, and all the effects from 0 to 6 Zn(II) ions overlap each over. A careful analysis of titration curves shows that they are not symmetrical at both ends. The curve has a sharp turn at low pH when metal binding begins and a much broader turn above the inflection point. Such asymmetry is further increased when the weakest, seventh Zn(II) ion binds. The binding causes a deprotonation of two SH groups (Cys21 and possibly Cys5) and the formation of four Zn–S bonds, including two bridged ones. (15) It results in a significant pH shift of the titration curve with a relatively small effect on absorbance (4 bonds over 28 giving a maximal 14% change of total absorbance). As a result, the last Zn(II) binding is almost indistinguishable in the pH titration curve. To observe the binding of the last Zn(II) ion, one needs to titrate the protein to at least pH 7 and analyze the shape of the curve. It requires enough signal/noise to observe the increase in absorption. Figure 18A illustrates an experimental Zn7MT2 titration curve over a wide pH range with an indication of the curve’s asymmetry. As a confirmation of the discussed effects, Figure 18B shows simulations of the absorbance increase and includes the experimental binding model (solid line) with gradually lowered affinities of Zn(II) toward MT (73) and a theoretical binding model (dotted line) that assumes all Zn(II) ions to be bound with the same high affinity. The comparison indicates that the simulated curve (solid line) is similar to the experimental one (Figure 18A), despite the assumption in the simulation that the thiols have the same pKa values; that is, affinities are differentiated, but thiol pKa values are averaged. The situation is different when the pH-dependent formation of Cd7MT2 is considered. 113Cd-NMR spectroscopic investigations have shown that Cd(II) binding occurs in such a way that the α-cluster in the C-terminal domain is formed first at a lower pH and then the β-cluster in the N-terminal domain is formed with the remaining three metal ions at a higher pH. (141,157,163) This binding can also be observed when UV absorbance is followed in pH titrations. The experimental curve (Figure 18C) demonstrates two independent regions of metal binding events, the first with an inflection point at pH ≈ 3 and the second with one at ∼3.8. Doubtlessly, the two steps confirm the conclusions from the NMR experiments and clearly prove that the four Cd(II) ions in the C-terminal domain bind with significantly higher affinity (∼2 orders of magnitude) than the three Cd(II) ions in the N-terminal domain. The steepness of the observed curve at the first stage of metalation shows that the affinities of all metal ions in the α-cluster are identical or rather similar. Cd(II) ions in the β-cluster also bind with very similar affinities; however, the shape indicates some differences as concluded previously. (164)

Figure 18

Figure 18. Experimental and simulated UV-monitored pH titrations of Zn7MT2 and Cd7MT2. (A) Experimental pH titration of 1 μM Zn7MT2 from pH 3 to 7. (B) The simulations of pH-dependent saturation of the protein with Zn(II) (see main text for details). The dashed line corresponds to a case where all seven Zn(II) are bound with the same affinity (−log Kd = 11.7). The solid line indicates a case where four sites are bound with high affinity and the remaining three with gradually lower affinities. (73) (C) Experimental pH titration of 1 μM Cd7MT2 from pH 3 to 7.

To summarize, it is very important to understand the pH-dependent equilibria in MTs as a structural component of their functions, especially to realize the significance of homoleptic coordination with only sulfur donors endowing such a complex system with a strong pH dependence around physiological pH and multiple pH-dependent equilibria, which we are unable to describe ab initio with the ionization constants of each of the 20 thiol groups. These considerations illustrate why pH control is critical for the purification and investigation of MTs and a discussion of their affinities for metal ions. For instance, size-exclusion chromatography at neutral or slightly acidic conditions of Zn(II)-reconstituted proteins results in the preparation of proteins that are not fully saturated with metal ions and leads to drawing the wrong conclusions about reactivity or function. Only purification steps at higher pH, for example, pH 8.6, warrant the preparation of fully saturated proteins.
Another source of overestimation of metal-to-protein affinities in MTs is the assumption that all metal ions bind with the same affinity. Inasmuch as pH titration of T provides average pKa values of the thiols, a pH titration of MT provides average pKa′ values of the thiols that compete with metal ions. In the latter case, the inflection point in Figure 18A is shifted to lower pH values, and the magnitude of the shift is an indication of the affinity of the metal to the protein. The assumptions that all metal ions bind with the same affinity and that one metal ion causes a deprotonation of three Cys residues allows obtaining an average −log Kd value for all metal ions. In such a procedure, weaker metal binding sites are averaged and dominated by high-affinity sites, and, due to the assumption of indistinguishable −log Kd values and pKa values of thiol groups, the average −log Kd value is overestimated and does not provide information about variation of stabilities of individual sites. The only robust way to address the actual metal-to-protein affinities of such a complex system is an analysis of the metal binding process using changes of free metal concentrations instead of changes of pH. It allows a resolution of small and significant differences in stabilities due to the wide range of free metal concentrations used in such investigations. In other words, one needs to use a competitor or a set of competitors that have the sensitivity and affinity toward the same metal ion to allow a monitoring of what happens with the protein when the free metal ion concentrations change systematically. To the best of our knowledge, such a procedure has been used only twice for MTs, and the results are highly significant because they show that the metal ions bind to MTs with a wide range of affinities. When highly sensitive fluorescent FluoZin-3 or RhodZin-3 probes were used, the affinities of Zn(II) to MT2 were demonstrated to differ and range from nanomolar (∼10–8 M) to picomolar (∼10–12 M). (73) With isothermal titration calorimetry (ITC), it was shown that Zn(II) and Pb(II) bind to MT3 with a wide range of affinities, similar to MT2; however, the investigations were performed at a lower pH. (137)
In other approaches to determine metal affinities, colorimetric (nonfluorescent) competitors were employed to monitor titrations of T with a metal ion. Fractional saturations─depending on reactant concentrations─are directly converted to the average Kd of MT based on a stoichiometric model and known affinities of the metal-chelating probes. An inherent issue with such an approach is the model’s assumption in treating all metal ions as indistinguishable and the narrow range of metal sensing of commonly employed chromophoric agents. (165−170) Focusing on narrow ranges only and not monitoring free metal ion concentrations results in an overestimation of the Kd because the contributions of weaker sites are not resolved. If all sites in ZnMT2 were to have the same affinities, all fractional saturations would report the same average value. With nano- to picomolar affinities of Zn(II) to MT2 (see below), the resulting Kd value reports average affinities of various sites. For instance, if the probe is added at a concentration that would allow a removal of ∼30% of Zn(II) from MT2, the average constant calculated will correspond to the Kd value of moderate sites because 30% of total Zn(II) corresponds to 2.1 metal equivalents. If the transfer is ∼45%, then the resulting Kd reports affinities from moderate to high-affinity sites, since 3.1 equiv of Zn(II) are transferred to the probe during equilibrium. Any higher fractional saturation mainly reports high affinities. To achieve detailed information on a weaker site, one would need to focus on only a few percent Zn(II) transfer, since the affinity of weak and moderate sites will be averaged above 14% transfer. Such a low Zn(II) transfer is usually not examined due to the limited response of the chelating probe, and therefore there is a high chance that the obtained value is significantly biased. A detailed analysis in this case would require several probes with various Zn(II) affinities and a presentation of total Zn(II) transfer against free metal ion concentrations.

3.3. Partially Metalated Zinc MT Species

3.3.1. Partially Metalated MT Species as Components of a Cellular Zinc Buffer

Although a function of MT as a zinc buffer was postulated repeatedly, the actual buffer would be T, which would have to be present in different states of metalation for effective buffering. The lack of further insight into MT metalation and speciation and the range in which cellular Zn(II) must be buffered led to the very unsatisfactory situation that research on MT remained phenomenological and correlative for at least 50 years. The then prevailing dogma that MT binds all seven Zn(II) with a low picomolar affinity was at odds with the increasing number of observations that eukaryotic cells keep free Zn(II) concentrations in the high picomolar to nanomolar range, far above the affinity of MT for Zn(II). (89,171) The assumed high cooperativity of Zn(II) binding and the then widely accepted overall dissociation constant would allow MTs to buffer free Zn(II) only in a narrow range around Kd (eqs 1 & 2; Figure 19, left panel). This range is far from the reported fluctuations of cellular free Zn(II) and the T/(T+MT) ratios found in many tissues (orange dashed box).
(1)
(2)

Figure 19

Figure 19. Speciation of zinc metallothionein (Zn7MT, MT) according to three thermodynamic models in the literature. (left) Model with all seven Zn(II) ions bound with the same affinity (Kd1–7 = 10–11.8 M). (141) (middle) Stepwise model with Kd values ranging from 10–11.8 to 10–7.8 M for human MT2. (73) (right) Stepwise model with Kd values close to each other in the range from 10–12.5 to 10–11.4 M for a modified human MT1a. (172) The upper panels show the relationship between ratios of the apo-form (T) over total protein and free Zn(II) concentrations (−log[Zn(II)]). (lower) Speciation of the apo-form, partially and fully Zn(II)-saturated species. The heuristic value of the models is discussed in the main text.

The situation drastically changed when human Zn(II)-MT2 was analyzed with the highly sensitive fluorescent zinc probes FluoZin-3 and RhodZin-3, and new insights into the affinity of MT for Zn(II) were gained. (73) It was discovered that the seven Zn(II) ions bind with various affinities ranging from picomolar to nanomolar. Four Zn(II) ions indeed bind with a low, highly similar, picomolar affinity forming a Zn4MT species. Whether or not the formation of this species is cooperative requires a high-resolution mapping and further insights into the metal-coupled protein folding. The binding of the remaining three Zn(II) ions results in the stepwise formation of Zn5MT, Zn6MT, and Zn7MT species with lower metal-to-protein affinity. The investigation with fluorescent probes and investigations performed later with other chelators and mass spectrometry showed that this process is reversible, meaning that dissociation (Kd7Kd1–4) of Zn(II) from Zn7MT also occurs stepwise as indicated in eqs 36.
(3)
(4)
(5)
(6)
The existence of several partially saturated species allows T to buffer Zn(II) in a much wider range (Figure 19, middle panel)─almost 4 orders of magnitude─than in the presumed case of MT binding all seven Zn(II) with the same affinity, as indeed would be required for the myriad of cellular functions of Zn(II) and its fluctuations as a signaling ion. The relationship between free Zn(II) and the concentration of particular species (eq 7) shows that free Zn(II) concentrations depend on the concentrations of not only the metal-free species but all partially metalated forms.
(7)
Using the above equations and the dissociation constants (Kd1–4 = 6.3 × 10–47.2, Kd5 = 3.5 × 10–11, Kd6 = 1.1 × 10–10, and Kd7 = 2.0 × 10–8 M) one can calculate the concentrations of any particular species as a function of free Zn(II) concentration(s). (73)
Taking into account the fluctuations of free Zn(II) established in numerous investigations of eukaryotic cell lines (143,144,171,173−176) it became clear that partially metal-saturated species such as Zn5MT or Zn6MT could serve as both Zn(II) donors and acceptors (orange box in the middle panel of Figure 19). Depending on Zn(II) concentrations, cell types, and the physiological or pathological state with increased or decreased concentrations of free Zn(II), the Zn7MT and Zn4MT species are expected to be present as well and to participate in buffering.
The different models of Zn(II) binding to MT are discussed in more depth because of their implications and the controversies they engendered. The state of the protein could be a simple reason for observed experimental differences. Without reconstitution and additional purification steps the protein is usually neither fully loaded with metal ions nor fully reduced when isolated from biological sources. A different starting point obviously would affect the determination of stability constants. (167) For a complicated system such as MT that binds seven Zn(II) ions to 20 sulfur donors, the determination of affinities is not a trivial matter, and it is critically important to employ highly sensitive methods that allow an investigation of affinities over a wide range. It is generally not achievable when using just one reagent because the signal resulting from Zn(II) binding/dissociation changes only 1 to 2 orders of magnitude in standard spectroscopic titrations at a constant pH. Highly sensitive fluorescent zinc probes made it possible to differentiate affinities for Zn(II) ions in MT2 for the first time. (73) UV-monitored pH titrations of MT do not have enough sensitivity for the resolution of stepwise changes of Zn(II) ion affinities. They are sensitive enough, however, in the case of Cd(II) ion affinities that are grouped according to the formation of the clusters and with log K differences reaching a value of 3. The application of electrospray ionization mass spectrometry (ESI-MS) to analyze the metal-to-thionein and metal-to-thionein + apo-carbonic anhydrase (apo-CA) systems led to the conclusion that all Zn(II) ions are bound tightly in human MT1a with very similar affinities (log K from 11.4 to 12.5, Figure 19, right panel). (172) In this approach, relative intensities of signals are treated quantitatively under the assumption that they reflect equilibria in solution when in fact protein species are measured as ions in the gas phase. The assumption of identical equilibria in both phases is inappropriate for several reasons. Similar to the spectroscopic studies the application of only one competitor for the analysis of the MT system and the resulting accuracy in the determination of signal intensity can overestimate affinities. When using apo-CA as a competitor, association/dissociation rates of Zn(II) between MT and CA are vastly different and can be modified in the gas phase. (172) The last but not least reason is a limitation inherent to ESI-MS-based determinations of affinities, namely, that Zn(II)-peptide complexes undergo metal deposition or supermetalation under conditions of ESI-MS. (177,178) At present, a comparison of the two models (Figure 19, middle and right panels) is not possible for severals reasons. Two different human MTs were used (MT1a and MT2). There could be an intrinsic difference between the two forms, or a difference could be due to the fact that the recombinant MT1a protein used had an additional 11 amino acids (72 instead of 61) as a result of the cloning and expression strategy. Given the pH dependence of thiolate coordination (Figure 17), the difference in pH of the MT solutions (pH 6.8 for the ESI-MS investigations and pH 7.4 for the fluorescence investigations) is expected to have a significant effect on affinities and speciation. Above all, there is no definition of pH in the gas phase, and the salts used in ESI-MS are usually not buffers in the physiological range.
As discussed above, analyses of tissues and cell lines indicated the presence of significant amounts of the apoprotein. Because of the low resolution of the differential labeling methods used for the detection of T/(T+MT) ratios, it was not possible to address partially metal-saturated species. Zn6MT, Zn5MT, and Zn4MT species are readily formed when aliquots of fully saturated MT (Zn7MT) and the metal-free form (T) are mixed and equilibrate. Depending on the ratios of reactants, different metal-saturated species form. For instance, the highest concentration of the Zn6MT species is formed when thionein over total protein is ∼14%, while Zn5MT and Zn4MT species predominate when the molar ratio reaches ∼29 and 43%, respectively. The species distribution matches exactly the results obtained from ABD-F differential labeling, indicating that partially metalated species of MT indeed form in vivo under normal physiological conditions. It is worth noting that an application of the differential labeling method for the determination of T/(MT+T) ratios provides information on freely available Zn(II) in tissues and cells. The biological significance became apparent only when independent measurements of free Zn(II) concentrations with fluorescent chelating agents became possible and established their range. One needs to keep in mind that the differential labeling measures all MTs present, whereas the above speciation is based on the properties of only one form, human MT2. Thus, the different MTs and the different amounts of MTs present in cells contribute to buffering and, if affinities of MTs other than MT2 for Zn(II) are different, the buffering range in addition to the buffering capacity will differ. Adding the number of inducers of differential MT gene expression, gene silencing, the redox and pH effects on the proteins, ligand interactions, and protein turnover, one begins to appreciate the complexity, subtlety, and possible fine-tuning of buffering cellular and subcellular Zn(II) and controlling signaling Zn(II) ions.

3.3.2. Structures of Partially Metalated MT Species

Metal-binding properties of MTs have been investigated extensively for several decades using a wide range of physicochemical methods. (73,179−182) Originally, the most informative ones were spectroscopic methods such as UV–vis absorption spectroscopy and spectropolarimetry (circular dichroism (CD)) to examine the number of metal ions that bind to MTs, for conformational studies that are associated with metal binding, kinetics of complex formation, and dissociation in the presence of numerous competing reagents and proteins. From the very beginning of the field, two processes were investigated: metal association with the apoprotein (T) and metal dissociation from the holo-protein (MT). Since Zn(II) has no signatures in most spectroscopic methods, other metal ions such as Cd(II) or Co(II) were used as probes for the Zn(II) binding processes and coordination modes. (183) Cd(II) complexes with Cys-rich proteins have conspicuous ligand-to-metal charge transfer bands with lower energy when compared to Zn(II), facilitating the interpretation of conformational changes. (184−186) Spectra of Co(II) complexes allow a direct observation of d-d bands and conclusions about coordination environment and geometries of the metal ions in partially and fully saturated MT species. (133,187) Fluorimetric methods were applied for structural studies of MT with attached FRET probes and to examine the metal association and dissociation and the determination of metal affinities. (73,151,154,188,189)113Cd and 111Cd-NMR spectroscopy was critical for the elucidation of the cluster formation in the α- and β-domains, cluster dynamics, and the kinetics of Cd(II) transfer. (190−193) However, there is a caveat: the Cd(II) metalation process differs from that for Zn(II), and despite the chemical similarity of the two cations information obtained for one cannot be transferred readily to the other. (98,154,163) Important steps in the characterization of MTs were the determination of NMR structures of isolated domains of human and rabbit MT2 as well as the solution of the crystal structure of rat MT2. (44,46−48) The MT structures characterized are the ones for the fully metal-saturated proteins. 3D structural investigations on partially metalated species were never performed. Metalation and demetalation pathways were investigated with various spectroscopies. However, because of the limited resolution of the physicochemical techniques, the absence of well-defined secondary structures, and significant coordination dynamics, structural details about partially saturated MT species remained elusive.
Advances in the structural characterization of dynamic and multiconformer, metal-depleted MT species came with the use of high-resolution mass spectrometric techniques. The pioneer in the application of mass spectrometry for the investigation of MT was Catherine Fenselau and her group. With ESI-MS as a high-resolution technique in the 1990s, it became possible to analyze MT alkylation with pharmaceuticals such as melphalan and chlorambucil, (194,195) metal ions in native and reconstituted MTs, and reactions with chelators. (196,197) In the latter experiments, numerous partially metal-saturated species were identified by using a direct infusion of solutions that contained either T with variable metal ion equivalents or MT with different reagents. ESI-MS became rather popular due to the ease of sample preparation and the rapid acquisition of a wealth of data. It is widely used for the determination of metal-to-protein stoichiometries as well as the characterization of the metalation processes in both mammalian and other MTs. (198−201) Different spectrometric profiles observed at various metal-to-apo-form molar ratios and intensities of signals that correspond to particular MxMT species provide information on the association of metal ions with the protein. While this information is rather useful for a protein characterization or a comparison of various metal ions or proteins, it is critical to understand its limitation. In particular, it is assumed that speciation in the aqueous, nonbuffered solution injected into the mass spectrometer corresponds quantitatively to that in the gas phase as represented by relative intensities of peaks in the mass spectrum. Under well-controlled conditions, native-like conformations of proteins can be retained during the ESI process. However, it does not apply readily to metal centers that underly their own rules in the ESI process. (202,203) In the case of MT, the structure of species is based almost entirely on metal networks and the intrinsically disordered character of the apoprotein, and the coupling of the folding and metalation processes must be considered. The dynamic character of the different metal binding sites may produce various profiles in mass spectra and is not exclusively linked to the thermodynamic stability of the binding sites. Several intriguing conclusions about MT metalation have been drawn from investigations of ESI-MS profiles of the whole protein and its isolated α- and β-domains. (138,163,204,205) One limitation is that the properties of the isolated domains are not fully additive in describing the whole protein. With such an approach, it has been proposed that Zn(II) and Cd(II) differ in the formation of clustered and independent sites at various pH values, notwithstanding that there is no definition of pH in the gas phase. (204,206) It is impossible to gain insights into the metalation mechanisms based on only relative changes of ESI-MS profiles. Other approaches with higher resolution must be used. For example, N-ethylmaleimide (NEM) is highly effective for a differential modification of free and Cd(II)-bound cysteine residues. (98,157) When combined with protein fragmentation or in-solution proteolytic digestion it can be employed to distinguish coordinated and free Cys residues and accurate and precise measurements of Cd(II) metalation, for example, to demonstrate that the Cd4Cys11 cluster is exlusively formed in the α-domain prior to a metalation of the β-domain. Likewise, iodoacetamide (IAM) was employed for the differential labeling of Cys residues in MT when Zn7MT was equilibrated with Zn(II)-depleted sorbitol dehydrogenase (apo-SDH). (153) With a subsequent top-down and bottom-up approach enhanced by a matrix-assisted laser desorption/ionization (MALDI) analysis, the importance of Cys21 in both Zn(II) association to and dissociation from the weakest Zn(II) binding site in the β-domain has been demonstrated. (153) The application of NEM and IAM together in a dual labeling method allowed further resolution of the Zn(II) metalation process. (98,207) It demonstrated that the first four Zn(II) ions bind to both α- and β-domains as suggested from the quenching of the reactivity of the thiols with Zn(II) ions and the analysis of ESI-MS profiles. (72,73,162,204)
MS-based investigations of MTs either confirmed conclusions drawn from previously performed spectroscopic investigations or further characterized the metalation-coupled protein folding. MS techniques are promising for those metal ions that do not form clusters cooperatively but sequentially, such as physiologically important Zn(II). The presence of multiple conformers in metal-depleted species with partially clustered and independent binding sites is especially challenging for a structure determination with mass spectrometry as well as analytical methods that are crucial for the obtainment of valuable solution-concurring freeze frames. One of the newer MS techniques adding a new dimension to MT research is ion mobility MS (IM-MS). Like ESI-MS it is based on characteristic m/z ratios, but in addition it can interrogate the possible heterogeneity of peptide/protein ions and resolve populations of conformers in an assembly. (155,208,209) Thus, MT conformations were first demonstrated for apo-MT2 and Cd7MT species. (210) It allows a distinguishing of final products, differently metalated states, and intermediates in the metalation reactions. (156) Experimental collision cross sections (CCS) can be correlated with those simulated by molecular dynamics (MD) to obtain information on the conformation of particular protein species. (136,211−214) The CCS profile analysis is also used to probe differences in gas-phase stabilities of partially metalated species upon collision-induced unfolding. (158,215)
Very recently a mass spectrometric investigation supported by molecular dynamics calculations characterized the coordination environments in all seven metalated species of human MT2 and demonstrated how the coordination changes upon successive metal association/dissociation. (216) Remarkably, the binding of Zn(II) is different from that of Cd(II). Thus, Zn(II)- and Cd(II)-MTs are isostructural only in the species with seven metal ions, limiting the use of Cd(II) for investigations of MTs in zinc metabolism. The work provides the highest resolution yet in terms of structure and reactivity of the protein with metal ions and sulfhydryl modifying agents. An important difference is that the first two Zn(II) and Cd(II) ions select different coordination sites and begin with mononuclear tetrathiolate coordination in the α-domain (Figure 20). The third and fourth metal ions then coordinate in the α-domain only for Cd(II) while they begin to populate the β-domain in the case of Zn(II), rendering the formation of clusters but one aspect of the coordination. In these metalation processes, the protein adapts many conformations depending on the metal load and the type of metal ion affecting the folding and generates a huge conformational space (“structural landscape”) to shape the functions of the protein for reactivity and interactions. The different binding properties of copper further expand this conformational space. The metalation process in the entire protein is different from that in the individual domain peptides because metalation in one domain affects sulfhydryl reactivity in the other. The investigation also provided evidence for the hyperreactivity of Cys21 and Cys29 in the β-domain of the zinc but not the cadmium protein. In the crystal structure of rat Cd5Zn2MT2, these sulfur atoms show S···HN hydrogen bonding to the peptide backbone. (47) How the suppression of such a reactivity by Cd(II) reflects on the biological function remains to be determined. With significance to the biological context, the investigation demonstrates that the structures and reactivities of MTs change depending on the availability of metal ions and illustrate the importance of other MT structures in addition to the only one determined with seven divalent metal ions by X-ray crystallography and NMR spectroscopy. This aspect of how the biological context dictates structures of MTs and how it relates to functions of structurally highly flexible MTs in metal buffering and interactions will be discussed further in section 4.

Figure 20

Figure 20. Order of the stepwise Zn(II) dissociation for the unfolding pathway for (A) Zn7MT2 and (B) Cd7MT2 obtained by constant-speed steered molecular dynamics simulations that involve Zn7MT2 and Cd7MT2 with the C-termini fixed. (216) The bars show the binding/unbinding of the metal ion for each metal-loaded species. Representative conformations of the protein when six, five, and four Zn(II) ions (C) or Cd(II) ions (D) are bound. Zn(II) and Cd(II) are represented by gray spheres, and the sulfur atoms are shown in yellow. (57) The figure is adopted from ref (216) with permission of the American Chemical Society.

3.4. Copper MT

Some copper was found in the first-ever preparation of MT from horse kidney. (2) A copper protein isolated from the liver of newborn calves and localized in heavy lysosomes showed an amino acid composition similar to that of MT. (7) Likewise, a major Cu(I)-binding protein from human fetal liver was identified as MT. (8) The Cu(I)-rich form is in an insoluble fraction, whereas the Zn(II)-rich form is in a soluble fraction. (217) When fetal and neonatal human livers were investigated, a linear correlation between cytosolic MT and Zn(II) but not Cu(I)/Cu(II) was noted, and an involvement of MT in copper and zinc metabolism was suggested. (218)
The variable metal content of MTs isolated from native tissues is summarized in at least two published tables. (181,219) The data indicate that native mammalian MTs contain Cu(I) in all instances when MT is not induced by Zn(II). The isolation of MT from tissues where its content is rather high, such as the liver and kidney, introduces a certain bias toward metal metabolism in these organs. In the eye, the stoichiometry of MT was found to be Zn6CuMT, which changed to Zn7MT after the induction of the protein with Zn(II). (220) The situation is different for the Cd(II) induction, where some Cu(I) can be found in mouse MT. (221) In Cd(II)-exposed rats, Cu(I) is found in plasma MT. (222) These observations suggest that Cd(II) competes with Cu(I) in copper proteins and that the displaced Cu(I) binds to MT. However, an indirect mechanism is also conceivable. Exposure to Cd(II) elicits an oxidative stress. Thus, the reactive (oxygen) species could release Cu(I) from its binding sites. The harsh procedures employed to isolate MT from tissues raise the issue of whether the presence of some Cu(I) in MT is an artifact of the isolation, namely, that MT, due to its high affinity for Cu(I), binds some Cu(I) that may become available when the tissue is homogenized. The substantial amounts of Cu(I) in MT3 and MT4 from natural sources seem to indicate that their Cu(I) content is not an artifact of isolation and that these proteins have a role in copper metabolism. MT4 was classified as a copper MT based on metal-binding properties and comparison with other MTs, although it binds Zn(II) as well. (223) It is not clear whether the amount of copper in MT is simply determined by the specifics of copper metabolism in a particular tissue. Yet another consideration is that subcellular differences in the metal metabolism may contribute to the different metal loads of MTs. For instance, in the rapidly growing liver of the bank vole, cytosolic ZnMT disappears while nuclear CuMT appears, suggesting the replacement of Zn(II) by Cu(I). (224) In cyanobacteria, it has been observed that the location where a protein folds influences its metal content. (225) A location-specific metalation of MT would lead to a mixed-metal composition of isolated MTs and add yet another level of complexity to the elemental speciation of MTs in vivo.

3.4.1. Cu(I) Binding Affinity of MT

When Cu(I)─in most cases as [Cu(MeCN)4]+─is titrated into Zn7MT, in which the Zn(II)-thiolate clusters determine its protein structure, different structures are obtained compared to when Cu(I) is titrated into T. (53,226) The stoichiometry of seven divalent metal ions does not apply to monovalent ions. Rabbit liver Zn(II)-MT2 binds 12 Cu(I) ions in one Cu6S9 and one Cu6S11 cluster. (53) Two distinct Cu(I)4-thiolate clusters are formed with 12–14 cysteine residues when T─prepared from rabbit liver─binds Cu(I) ions. (226) Cu8MT is a stable intermediate, as the phosphorescence titration demonstrates a breakpoint after the addition of eight metal ions before the binding of an additional four Cu(I) ions occurs. A Cu4S8–9 cluster instead of the typical Zn3S9 cluster has also been observed in human MT3. (227) A remarkable spectroscopic property is being exploited in the investigations of Cu(I) binding, namely, the luminescence of Cu(I) in MT, which is due to efficient MLCT in the lowest excited states and results in a thermally activated delayed fluorescence (TADF) combined with a short-life phosphorescence. Accordingly, the emission, depending on temperature, consists of two decay paths at variable relative intensities: direct phosphorescence and TADF-only. (228) Cu4S6 and Cu6S9 clusters in the β-domain were observed in titrations (followed with ESI-MS) before Cu4S6 and Cu7Sx clusters in the α-domain are formed with specific emission and CD properties. (229) There seems to be a domain preference for Cu(I) binding with the β-domain having a marginally higher affinity for Cu(I). (229,230) A stoichiometry of up to 20 Cu(I) ions can be reached as in the case with Hg(II) and Ag(I). A caveat is, however, that a human MT1a protein with 72 instead of the 61 amino acids of native MT1a was used in these investigations. Such “supermetalated/hypermetalated” species are expected not to exist under normal physiological conditions, as the cellular availability of Zn(II) and Cu(I) is strictly controlled and the synthesis of more MT would be induced to cope with a surplus of metal ions. Supermetalation can be a result of the nature of the mass spectrometric investigation. At this juncture, a reminder is necessary that the exact structures of the Cu(I)-thiolate clusters in mammalian MTs have not been determined.
Gaps in our knowledge about Cu(I) interactions with MTs remain for several reasons. One reason is related to the redox properties of copper. When Cu(II) reacts with MTs, Cu(II) is reduced to Cu(I), and the thiolate sulfurs in the protein are oxidized (the redox chemistry of Cu(I)/Cu(II) in MT is discussed in section 3.4.2). Investigations of Cu(I) interactions with T or MT require anaerobic conditions. Another reason is related to the coordination geometry of Cu(I), which forms diagonal, trigonal, and occasionally tetrahedral binding sites with cysteinyl sulfur. Because of the high number of sulfur donors in the MT clusters, complexes with various stoichiometries are formed, making Cu(I)/MT interactions difficult to investigate. The last but not least reason is the thermodynamics of the interactions of Cu(I)/Cu(II) with MTs. Cu(I) forms extremely tight complexes with the cysteinyl sulfurs in MTs resulting in stability constants that cannot be determined directly. To investigate the interaction, competing ligands, the choice for which has been much more limited compared to those for Zn(II) until recently, are required. (231−233) All approaches involve the determination of stability constants based on a reference to a competing reagent. In addition to the very high Cu(I)-MT stability, the instability toward disproportionation further complicates the measurements. Therefore, the Cu(I) ion needs to be stabilized by a low-affinity ligand complexation such as in the complex with acetonitrile. (234)
The first investigations of the affinity of Cu(I) to MT led to a formation constant (K) of at least 2 × 1016 M–1. (141) It is not an absolute value determined by any particular method but one estimated based on the known affinity of Zn(II) and Cd(II) to MT and the fact that Cu(I) displaces both metal ions from their MT complexes and, hence, must have a higher affinity. Taking into account that Zn(II) and Cd(II) bind to MTs with various affinities, the estimated value was corrected for the weaker Zn(II) and tighter Cd(II) sites in MT. Subsequently, the stability of Cu(I)-containing MTs was investigated with chromophoric chelating probes with a known Cu(I) affinity. These probes change their characteristic electronic absorption when Cu(I) binds, and they report the fractions of Cu(I) being in the complex and in the MT species. The application of bicinchoninate (Bca), a bidentate ligand with log β12 = 17.2, (235) demonstrated that the average Cu(I) affinity (Kd) in MTs is less than 10–17 M. (170) The application of another bidentate ligand, bathocuproine disulfonate (Bcs), with a higher Cu(I) affinity (log β12 = 19.8) (235) allowed the determination of the average stability constants of human MT2 and MT3 with Cu(I) as Kd = 4.3 × 10–19 M and Kd = 5.6 × 10–20 M, respectively, at pH 7.4 (25 mM Tris, 50 mM NaCl). (170) Using the stability constant of the Cu(Bcs)2 complex recently determined by cyclic voltammetry (log β12 = 20.81) and the above average Kd values of MT2 and MT3, one recalculates values of Kd = 4.2 × 10–20 M for MT2 and Kd = 5.5 × 10–21 M for MT3. (170,233) When correcting for pH 7.0, at which the constants for Bcs were redetermined, one may expect slightly lower Kd values of Cu(I) in MT2 and MT3. Importantly, in these competition experiments MT2 and MT3 species that are not fully Cu(I)-saturated were characterized, namely, Cu(I)4Zn4MT2 and Cu(I)4Zn4MT3 species that were obtained in redox reactions between Cu(II) and Zn7MTs. The presence of disulfide bonds due to redox reactions with Cu(II) and resulting different CuxSy clusters may change the average Cu(I) affinity for the β-cluster when compared to the protein with fully reduced cysteinyl sulfurs.
As with the spectroscopic approaches, competition with a ligand of known Cu(I) affinity is critical and serves as a reference for affinity when mass spectrometry (ESI-MS) is employed. Common ligands for competition are reduced glutathione (GSH), DL-dithiothreitol (DTT), or diethyldithiocarbamic acid (DETC). The literature on the affinity of Cu(I) for metalloproteins is replete with false assumptions, resulting in constants that are over- or underestimated. For instance, the average dissociation constant of the rabbit Cu10MT2 species characterized by ESI-MS and competition with DETC is reported as 4.1 × 10–16 M. (236) However, the Kd value of the reference Cu(I)-DETC complex had been determined in competition with DTT as 1.4 × 10–14 M under the assumption that the Kd of the Cu(I)-DTT species is in the range of 10–12 M (236) when in fact it is much lower, that is, ∼10–16 M. (237) When a different reference Kd value for Cu(I)-DTT (5.0 × 10–16 M, pH 7.3) based on spectroscopic competition with Bcs was used, the average Kd value for Cu(I)10MT2 turned out to be 2.6 × 10–20 M. (235,238) The use of the subsequently updated Cu(I)(Bcs)2 affinity (obtained from cyclic voltammetry) gave an average Kd value of 2.5 × 10–21 M for rabbit Cu(I)10MT2 (pH 7.0). It is expected to be slightly lower at pH 7.4. (233) Currently, it is unknown whether minor differences in the affinities of Cu(I) in Cu4Zn4MT2 (human) and in Cu10MT2 (rabbit) originate from methodological issues or are indeed related to the origin of the protein or the stoichiometry of the particular species. Similar values for both proteins indicate that Cu(I) binds, on average, several orders of magnitude more tightly than Zn(II) or Cd(II). More information was garnered from an investigation of human MT1a. (205) When apo-MT1a, that is, thionein (T), was titrated with [Cu(MeCN)4]PF6 and the reaction was monitored by ESI-MS, multiple Cu(I)1–20MT1a complexes with various relative signal intensities were detected. With the average Kd value of rabbit Cu(I)10MT2 (see above) as an affinity standard, average Kd values of particular Cu(I)1–20MT1a species varied from 3.3 × 10–21 M for Cu(I)4MT1a to 7.1 × 10–18 M for Cu(I)20MT1a. (205) When this analysis was applied to the α- and β-domain peptides of human MT1a and the same affinity standard was used, a much wider range of dissociation constants for Cu(I)20–xMT1a species was found. (54) It was suggested that only some of the CuxMT1a species have very high affinities. For instance, average Kd values for the stable complexes Cu(I)4MT1a, Cu(I)6MT1a, and Cu(I)10MT1a are 6.3 × 10–23, 2.5 × 10–21, and 2.5 × 10–20 M, respectively. A rather moderate affinity (Kd = 1.3 × 10–11 M) was found for the Cu(I)13MT species, while the affinities for other weak complexes vary between 10–4 and 10–10 M. (54) Bearing in mind that ESI-MS has some major limitations in a quantitative analysis and for the determination of equilibrium constants, these investigations show that the affinities of Cu(I) sites in MT vary by several orders of magnitude.

3.4.2. Cu(II) Interactions with MT

Currently, the exact mechanism of the interaction of MTs with Cu(II) is unknown. The reaction of one thiol with one Cu(II) ion results in the reduction to Cu(I) and the formation of a thiyl radical. When Cu(II) is added to Zn7MT2 or Zn7MT3, a cooperative formation of a Cu4Sx cluster in the β-domain together with dissociation of three Zn(II) ions and the formation of two disulfides was proposed; however, this was confirmed only by a shift of molecular mass in mass spectrometry. (239) The exact localization of the disulfide bridges is unknown and so is the potential interaction of the sulfur atoms in the disulfides with Cu(I). (240−242) This four-electron reaction occurs in situ and is believed to be part of the protection of the cell against any extracellular Cu(II). The addition of another equivalent of Cu(II) to Cu4MT2ox or Cu4MT3ox in vitro causes a similar oxidative dissociation of four Zn(II) from the α-domain and formation of the second Cu4Sy cluster and two disulfides. (239)
The redox chemistry and biology of copper MT is twofold. T or MT are reductants for Cu(II), but whether this reaction is ever important inside cells is unknown, because the main form of copper in the cell is Cu(I), and any Cu(II) would be readily reduced by glutathione and then sequestered by MT. Thiyl radicals can be formed in the one-electron oxidation of the thiolate(s). (242,243) MT3, as isolated, has the composition Cu4Zn3–4MT3. The Cu(I)4-thiolate cluster is in the β-domain and is stable against air oxidation. (227) However, a Cu4Zn4MT3 prepared in vitro has two homometallic clusters, a Cu4 thiolate cluster in the β-domain and a Zn4 thiolate cluster in the α-domain. The latter is air-sensitive, and one Zn(II) ion dissociates concomitant with the thiolate oxidation, resulting in a Zn(II)3-thiolate cluster in the α-domain. (223,244,245) Thus, both sulfur and copper redox chemistry occur in MTs. MTs can inhibit copper redox cycling, but they can also be the target of oxidants and electrophiles for a copper dissociation for yet to be defined purposes or, under an oxidative stress, to initiate cellular damage. (126)
In contrast to Cu(I), the chances are higher for any Cu(II) to escape from proteins because of its lower affinity (see the discussion in section 4.4), unless specific kinetic barriers evolved in proteins to prevent the dissociation of Cu(II) in the cell. Outside cells, Cu(II) can be scavenged and reduced by extracellular MT3. (246,247) Zn7MT3 suppresses the Cu(I)-catalyzed formation of hydroxyl radicals in the presence of ascorbate. (239) The finding could be interpreted as Zn(II)-MT controlling the redox activity of free Cu(II) ions. The growth inhibitory function of MT3, which is supported by both Zn7MT3 and native Cu4Zn3–4MT3, has not been explained in molecular terms, and thus, it is not clear whether it relates to copper metabolism. Because Cu(II) is needed for cell proliferation, the sequestration of Cu(II) as Cu(I) could be a function of Zn7MT3 with the native copper protein being the product of such a function.
Cu(II) is present extracellularly, not only in pathological states but also under normal conditions. Any MT secreted from cells is expected to encounter Cu(II). Where exactly and which MT protein interacts with Cu(II) and whether the same species as in vitro form is not known. The isolation of Cu(I)/Zn(II)-MTs demonstrates that species with less than four Cu(I) can form in vivo. These forms are likely partially oxidized (CuyZn7–xTo) and contain variable amounts of Zn(II). The location of Cu(I), Zn(II), and the disulfides in such forms are not known. The interaction of several Cu(II) complexes with MT has been investigated. (246,248,249) Although some of the Cu(II) ligands form stable complexes they react the same way as inorganic Cu(II) salts resulting in an oxidative formation of Cu(I)-thiolate clusters. However, complexes with a very high affinity for either Cu(I) or Cu(II) (with log K > 20) do not react with MT, demonstrating an important limitation of MT as a cellular Cu(I) acceptor and reducing agent: The competing molecule (protein or low-molecular-weight ligand) must bind Cu(I) more tightly than log K > 20 in order not to undergo a Cu(I) transfer or, in the case of Cu(II) complexes, in order not to be reduced to Cu(I) complexes. (249) The implication is that only copper proteins with these features can function independently and be resistant to competition with MTs. Proteins that do not fulfill these conditions are prone to react with MTs.
From investigations of the affinity of isolated MTs one could conclude that Cu(I)-MTs cannot coexist with Zn(II)-MTs in cells. One factor that allows both to occur in cells is the difference in subcellular localization: Cu(I)-MT in lysosomes, in particular, under conditions of high Cu(I) concentrations, and Zn(II)-MT in the cytosol. The higher chemical stability of the Cu(I)–S(thiolate) bond compared to the Zn(II)–S(thiolate) bond allows Cu(I)-MT to exist under the acidic conditions in lysosomes.

3.5. Cadmium MT

The discovery of MT as a cadmium protein from horse kidney as a result of searching for a function of Cd(II) in biology set a separate line of research into motion, namely, the putative role of MT in the detoxification of some metal ions. The discussion of cadmium MT here has another reason: Cd(II), either when used for an induction of MT in animals or when added during a heterologous expression of recombinant MT proteins, confers a higher stability on the protein. Therefore, and because its isotopes lend themselves for NMR investigations, it became the metal of choice for many biophysical investigations and consequently the basis for our knowledge about structure. The preoccupation with Cd(II)-MTs for decades has a very important educational consequence. It distracted from a focus on the physiological functions of these mammalian proteins in zinc and copper metabolism. Without doubt, it is one of the reasons why insights into the functions of MTs were delayed so much.
Cd(II) accumulates in the α-cluster, in contrast to Cu(I), for which a thermodynamic and kinetic preference for the β-domain was noted. (230) It is also different from Zn(II), which does not bind cooperatively to the α-domain and does not have a preference for either one of the domains. (98) Heterometallic Cd5Zn2MT can be formed in vivo upon induction with Cd(II), and five ions are the maximum number of Cd(II) to be incorporated. Homometallic Cd7MT can be produced only in vitro or in heterologous expression systems. The binding of an additional two Cd(II) ions per monomer induces dimerization. It is accompanied by a major rearrangement in the β-cluster, as only the four peaks of 113Cd in the α-cluster are conserved in NMR spectra. (250) Phosphate is involved in the dimerization. Two hydrogen phosphate anions (HPO42–) per dimer bind noncovalently with a binding constant of Kd = 23 μM at pH 8.0. (251)
The binding affinity of Cd(II) to its sites in MT is ∼3 orders of magnitude tighter than the strongest binding of Zn(II). It is typical for this group in the periodic system of the elements (PSE) that the kinetic lability decreases in the order Hg(II) > Cd(II) > Zn(II). Thus, intracluster exchange rates of Cd(II) are t1/2 = 0.5 s for the β-cluster (at 35 °C) as determined with 111Cd(II) saturation transfer NMR and 16 min for the α-cluster as determined with the radioisotope 109Cd(II). (58,252) However, the exchange rates of the metal ions in the β-cluster are different with half-lives differing by a factor of 4 and metal site 4 (Figure 5) with sulfur donors from Cys5, Cys7, Cys21, and Cys24 exchanging more slowly. Site 4 was identified recently as the site for the most weakly bound Zn(II) ion in human MT2 thus showing the highest kinetic lability. (153) The mechanism of this intramolecular self-exchange reaction is an intermolecular transfer with Cd(II) sharing sulfur donors from both β-domains in a dimeric intermediate. (252) An interdomain exchange is also possible and has been shown recently for Zn(II) in partially metalated species. (98,153) Thus, at least three types of dimers can form: noncovalent dimers during metal exchange or with additional Cd(II) ions and a phosphate ion bound and covalent dimers bridged by disulfides. (192) The cadmium protein was essential for structural work, especially for the determination of the cluster structures with 113Cd NMR (35) but also for the NMR solution and X-ray crystal structures. Protein dynamics also plays a role, as the Cd(II) exchange in the β-cluster of Cd7MT1 is faster than in Cd7MT2. (253) Cluster volumes are ∼20% higher for Cd(II) than for Zn(II). (48)
111Cd perturbed angular correlation of γ-rays (PAC) spectroscopy of rabbit liver MT provided evidence for Cd(II) in two different coordination environments. (254) A remarkable fact is that seven resonances are well-resolved for the seven sites in the 113Cd NMR spectra of MT (Figure 5). It signifies small differences in coordination environments despite each metal ion being in a tetrahedral geometry with four sulfur donors from cysteines.
Kinetics and thermodynamics determine the metal exchange. Ag(I) and Cu(I) have a preference for the β-domain, while Zn(II) and Cd(II) prefer the α-domain. Metal ions in one domain do not influence the reactivity of metal ions in the other domain. The mixing of Cd7MT and Zn7MT leads to a thermodynamics-based distribution in the clusters. With inorganic clusters, the mixing of homometallic clusters always results in a statistical distribution of metal ions, but in MT the formation of heterometallic clusters demonstrates “an appreciable selectivity in metal partitioning among and within the clusters” as afforded by the protein. (255) From a structural point of view, there is an interplay between coordination dynamics of the clusters and protein dynamics. In biological tissues, the availability of metal ions such as Cu(I) or Cd(II) and the occupancy of any Zn(II)-MT already existing will affect binding preferences. The different behavior of the two domains has led to the suggestion that the α-domain and β-domain have functions in storage or distribution of metal ions, respectively.

3.6. Metal Ion Selectivity

MTs bind the following ions: monovalent Cu(I), Ag(I), Au(I); divalent Zn(II), Cd(II), Co(II), Ni(II), Fe(II), Hg(II), Pt(II), Pb(II); trivalent In(III), Sb(III), Bi(III), As(III). (39) Even a derivative of rabbit MT1 containing 6 equiv of the TcO3+ ion has been prepared. (256) There are three issues with regard to physiological significance. First, the binding of many metal ions can be readily investigated in vitro. There is no a priori reason why any metal ion with a sufficient thiophilicity should not bind. The investigation of such binding can be a purely academic interest if the metal ion is never present in the organism. When a particular nonessential metal ion becomes available under occasions of environmental exposure, the question is how it will affect the function of MT in zinc (and copper) metabolism and whether it will be permanently sequestered or redistributed to harm other biomolecules. Second, in vivo foreign metal ions will never reach the high stoichiometries measured in vitro, for example, 20 Hg(II) bound to 20 cysteine residues. And third, the questions remain as to which relative concentrations of essential metal ions determine the native metal composition of MTs and whether the different MTs present in one cell have the same or different metal compositions. The translation of in vitro investigations to significance in biology faces several additional uncertainties: Which metal ions are available when T is made on the ribosome? How does metal composition and distribution change when the gene expression of MTs is induced, the redox state or reactive species changes, or when MTs translocate to subcellular compartments where metal concentrations differ? And, do other metal ions bind to the free coordination sites of MTs that are not fully saturated with Zn(II), and, if so, what are the effects on Zn(II) or Cu(I) availability in the cell?
Investigations of the interaction of iron with MTs are limited to only a few physicochemical reports. Fe(II) has a high similarity to other divalent metal ions in terms of binding to mammalian MTs with the same stoichiometry. The saturation of the T requires a higher excess of Fe(II) over the protein due to its corresponding lower affinity in the Irving-Williams series. (257) At a neutral pH, Fe(II)7MT is yellow and extremely air-sensitive, and it turns to a wine-red color when it is exposed to air. The characterization of Fe(II)-MT complexes by electronic absorption and magnetic circular dichroism spectra indicates that seven Fe(II) ions are bound to the protein in tetrahedral coordination environments. The absorption spectra show a 5E → 5T2 d-d transition in the near-infrared region at ∼1850 nm and a broad charge-transfer absorption in the UV region with a shoulder at 314 nm. Spectroscopic titrations demonstrate that the coordination environment of the first four equivalents is similar to that of Fe(II)/tetrathiolate in rubredoxin. The addition of more Fe(II) causes a red-shift of the electronic absorption until metal occupancy is reached with formation of Fe(II)-thiolate clusters that have either an S = 0 or S = 2 ground state as a result of antiferromagnetic coupling between high-spin Fe(II) ions. The clusters with S = 0 and S = 2 are attributed to tetranuclear and trinuclear centers, respectively. (258) Electron paramagnetic resonance (EPR) spectra of Fe(II)-MT complexes show a broad resonance. Their intensity increases up to 4 equiv, remaining almost constant between 4 and 6 equiv, and undergo a marked decrease between 6 and 7 equiv. Mössbauer spectroscopy shows the presence of paramagnetic and diamagnetic subspectra in the ratio of 3:4. (259) A diamagnetic component has been assigned to a four-metal cluster with antiferromagnetic coupling between high-spin Fe(II) ions. Magnetic susceptibility measurements show an average magnetic moment of 8.5 μB for the three Fe(II) ions responsible for the paramagnetic component. This value is slightly lower than for three completely uncoupled Fe(II) ions and suggests a three-metal cluster with a weak exchange coupling between adjacent Fe(II) ions. An analysis of the data demonstrated a pseudoplanar geometry rather than a chairlike geometry in the Fe3S9 cluster, indicating a significant difference to the β-cluster formed by Zn(II) and Cd(II).
Given the extensive Fe–S chemistry, one wonders why interactions of iron with MT in vivo have not been reported. Even though Fe(II)-MT complexes form in vitro, the occurrence of such a complex in vivo has not been documented. Yet, the reactions of Zn(II)-MT with iron complexes cannot be ruled out, especially under conditions of iron overload that may provide enough Fe(II) for a competition with Zn(II) or under conditions of low saturation of MT with Zn(II). Zn7MT induces iron release from the iron-storage protein ferritin, where iron is stored in the Fe(III) oxidation state. (260) Perhaps there is a thiolate/disulfide-based redox reaction involved in electron transfer through the ferritin shell. MT itself is too large to penetrate the ferritin channels for a direct contact with Fe(III). In another potentially biological significant interaction, MT may protect lysosomes by an iron sequestration. (261) Fe(II) does not displace Zn(II) from proteins due to its lower affinity compared to that of Zn(II). However, the addition of Fe(II) together with NO results in the formation of iron nitrosyl thiolates with a stoichiometry of Fe(NO)2(SR)2 as confirmed by EPR signals with g values of 2.013 and 2.039. (262) The same product was observed when Fe(II) and NO were added to apo-MT. In another investigation addressing reactivity, four Fe(II) ions were incorporated into an α-domain peptide of MT. The tetranuclear cluster was found to participate in one-electron and multielectron reductions of biological and artificial substrates. (263)

4. Relating Metallothionein Thermodynamics and Kinetics to Control of Cellular Zn(II) and Cu(I)

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4.1. Buffers for the Most Competitive Metal Ions

The Irving-Williams series describes the trend of affinities of the first d-series of divalent metal ions. (264,265) The late RJP Williams also suggested that the series is an overarching principle for the control of metal ions in biology. (266) The higher the affinity of the metal ion for the same coordination environment the lower is its availability, endowing the most competitive metals ions, Zn(II) and Cu(II), with the lowest available free metal ion concentrations. (267) From the affinities of biological metal complexes, he estimated the free metal ion concentrations theoretically available. The series can be employed to understand the metalation of metalloproteins. (268,269) Metalloproteins are in equilibrium with a pool of LMW ligands that remains largely undefined in terms of affinity, capacity, and coordination chemistry. Thus, for Zn(II), there is not a single equilibrium between only bound and unbound metal ions as in the original description of the series. Instead, one needs to consider the contribution of a LMW ligand pool of Zn(II):
Zn(II) metalloproteins (including MT) ⇌ Zn(II) LMW ligand pool ⇌ Zn(II) complexes such as aquo complexes and complexes with simple inorganic anions
In contrast, the Cu(I) transfer is thought to be mediated exclusively by metallchaperones without the intermediacy of an LMW ligand pool.
However, glutathione has been discussed in the binding of Cu(I) in the cell and in participating in the transfer of this ion to MT. (102)
The Irving-Williams series predicts the free concentrations of divalent metal ions from the set of the same ligand(s). The situation is different in biology, since the metal ions are in different coordination environments, and a myriad of additional equilibria pertain. One aspect that modifies the trend in d-block elements somewhat is the increase in thiophilicity of the metal ions and the decreases in the oxophilicity from left to right in the periodic table. (270) The employment of sulfur as a ligand for Cu(I) amplifies the affinity for the most competitive metal ions. (271) It emphasizes the significance of MTs by using sulfur ligand donors only. The series is a thermodynamic concept. Kinetic factors play a role as well. Kinetic trapping mechanisms are not considered in this equilibrium situation. The dissociation rates of metal ions in some proteins are so slow that, for equilibrium considerations, they are quasi irreversible. For example, in carbonic anhydrase t1/2 for the off-rate of Zn(II) has been estimated to be in the order of three years (4 °C, neutral pH). (272,273) In the series, there is a reversal for the most competitive metal ions: Ni(II) < Cu(II) > Zn(II), indicating that Zn(II) has a lower affinity than Cu(II) for the same ligand. If this would be the only determinant of metal distribution, then MT should always contain copper. Several additional factors explain how biology circumvented this restriction in the need for buffering in a specific range to avoid an overlap of functions. First, under the reducing condition in the cell, most of the copper is in the Cu(I) state, not the Cu(II) state considered in the series. The transition from Cu(II) to Cu(I) increases the affinity for copper in a sulfur environment even further. Second, while the Cu(II)/Zn(II) ratio outside cells is ∼1:1, there is ∼10 times more Zn(II) than Cu(I)/Cu(II) in the cell, and certain ratios of the total metal ions must be maintained. Third, membrane transporters participate in the control of cellular metal ions. They contribute a kinetic component to the buffering, which is called muffling. (274) Thus, the entire system of homeostatic control for the two essential metals zinc and copper must be considered in order to understand how Zn(II)-MT can exist in the presence of Cu(I)/Cu(II) in the cell. The phenomenon has been expressed with the term “the cell rules”, namely, that metalloproteins such as MTs do not have the ability for an absolute discrimination of these metal ions and that their metal preference is in part determined by other components of the metal buffering and muffling systems of cells. (275) Fourth, protein metallochaperones safeguard the trafficking of Cu(I) to a limited number of copper proteins, but apparently none exist for the distribution of Zn(II) to a few thousand proteins. Metallochaperones apparently obtain Cu(I) directly from its transporter, which itself safeguards Cu(I) as a pseudochaperone, minimizing the chance for Zn(II) instead of Cu(I) being bound to the metallochaperone.
Other, nonessential thiophilic metal ions challenge the system and compete on the basis of affinity, because there is no homeostatic system to control them. They piggyback on proteins for essential metal ions. Thus, the functions of MTs will depend on the burden of nonessential metals such as Cd(II), Hg(II), Au(I), Ag(I), and Bi(III), all of which have a higher affinity for MT than Zn(II). MT, as the name already foreshadowed, is different from other metalloenzymes that become metalated with the specific metal ion they need for catalytic function. MT is more promiscuous in this regard, arguing against a role of just one metal ion in the protein.
MTs participate in the control of the most competitive metal ions Zn(II) and Cu(I). With their affinities commensurate with the availability of free Zn(II) or Cu(I), which is so tightly bound that there is essentially no free Cu(I), they control potent effects that these metal ions have on proteins, and they control their cytotoxicity. Zn(II) in a copper site will not support any redox activity that copper has in an enzyme and inactivate it. The two competitive metal ions must be regulated in a way that Zn(II) does not end up in copper sites and Cu(I) does not end up in zinc sites. One way of avoiding a mismetalation is through a regulation of the availability of metal ions. (276) Zn(II) can end up in copper sites in heterologous expression systems, for example, when azurin is expressed in E. coli. (277) Cu(I), however, does not appear to end up in zinc sites in vivo, though it can bind in them, for example, copper alcohol dehydrogenase. (278) There is the possibility that MT has a role in keeping Zn(II) out of copper sites and also, perhaps, in fetal liver in keeping Cu(I) out of zinc sites and, of course, to ascertain that any Cd(II) present is not ending up in either zinc or copper sites. In addition, MT may remove directly the wrong metal from mismetalated proteins. This aspect is discussed further in section 4.7.2.
Zn(II) is buffered to picomolar, while Cu(I) is buffered to zeptomolar concentrations─a relatively new and very challenging area for bioanalytical chemistry (Figure 21). On the basis of the affinity to MT, cellular cadmium buffering occurs in a window between Zn(II) and Cu(I) (Figure 21). Any in vitro investigations of the effects of these metal ions on proteins, therefore, must consider these concentrations in the cellular context in order to have physiological relevance.

Figure 21

Figure 21. Free metal ion concentrations and buffering of the most competitive essential metal ions Zn(II) and Cu(I) and nonessential and toxic Cd(II). Estimated steady-state concentrations for Zn(II) and Cu(I) are picomolar and zeptomolar, respectively. Cd(II) interacts with the metabolism of both. Very low concentrations require stochastics when the volume limits the definition of concentrations. (279) Physiological and pathophysiological redox changes and other triggering events can increase the concentrations (arrows). Zn(II) fluctuations are used in cellular signaling at concentrations lower than those in calcium signaling. Recent work suggests a dynamic copper pool that is involved in copper signaling as further discussed in section 4.5.

4.2. Zinc Buffering

The exceptional scope of cellular zinc biochemistry should be recognized when possible roles of MTs are discussed. In parallel with the history of the MT field, the zinc field developed over the second half of the last century from a single zinc enzyme to the recognition that there are over 3000 human zinc proteins. (280−282) To satisfy these requirements of a huge number of proteins for Zn(II), the total Zn(II) concentration in cells is at least 1 order of magnitude higher than the one in blood. Two families of 24 zinc transporters altogether coordinate cellular uptake, extrusion, and supply to organelles. (283) The affinities of mammalian zinc proteins vary over a wide range from nanomolar for extracellular zinc sites to femtomolar for some intracellular sites (Table 1).
Table 1. Dissociation Constants (−log Kd) or Inhibitory Constants (Ki) of Zinc Proteins
protein/protein domainbinding amino acid residueszinc site function–log Kd or Kiconditionsa (method of determination)ref
Aminopeptidase-B (Rattus norvegicus)HHECatalytic12.450 mM Tris pH 7.4, 75 mM NaCl (EAMB) (284)
Angiotensin converting enzyme (Oryctolagus cuniculus - rabbit)HHECatalytic8.250 mM HEPES pH 7.5, 300 mM NaCl (EADT) (285)
Carbonic anhydrase (Homo sapiens)HHHCatalytic12.015 mM MOPS pH 7.0 (EAMB) (286,287)
   11.4  
    15 mM phosphate pH 7 
Carboxypeptidase A (Bos taurus)HEHCatalytic10.550 mM Tris-HCl pH 8, 1 M NaCl (EQD) (288)
Dipeptidyl peptidase III (Rattus norvegicus)HHECatalytic12.350 mM phosphate pH 7.4 (EAMB) (289)
Erythrocyte glyoxalase I (Homo sapiens)QEHECatalytic10.6100 mM Tris-HCl pH 8.5, 100 mM NaCl (EAMB) (290)
Leucine aminopeptidase (Bos taurus)BinuclearCatalytic9–11100 mM Tris-HCl pH 7.5, 1 M KCl (EQD) (291)
Porphobilinogen synthase (Homo sapiens)CCCCCatalytic1.6 pMpH 7.2 I = 0.1 M (EAC) (292)
Human sonic hedgehog (Homo sapiens)HDH <10100 mM HEPES pH 7.5, 150 mM NaCl (FLDT) (293)
Sorbitol dehydrogenase (Ovis aries)HECCatalytic11.250 mM HEPES pH 7.4, 100 mM KNO3 (294)
Human serum albumin (Homo sapiens)HDHDTransport7.530 mM HEPES pH 7.0, 250 mM NaCl (EQD) (295)
Bovine serum albumin (Bos taurus)HDHDTransport7.330 mM HEPES pH 7.0, 250 mM NaCl (EQD) (295)
DNA-binding domains (DBD) of nuclear hormone receptors (Homo sapiens)2 sites: CCCCStructural9.3, 10.0100 mM bis-Tris pH 7.4 (RT) (296)
 hERα-DBD 9.5, 9.7  
 GR-DBD    
Keap1 (Mus musculus)CCCCStructural11.0(PAR) (297)
Mammalian serum retinol-binding protein (Sus domesticus)HHHStructural11.720 mM HEPES pH 7.5, 50 mM K2SO4 (EQD) (298)
MTF-1 (Mus musculus)6 sites: CCHHStructural10.5 (average value)100 mM HEPES, pH 7.0, 50 mM NaCl (RT) (299,300)
 first zinc finger    
    50 mM HEPES, pH 7.0, 100 mM NaClO4 (CDC) 
   11.6  
PDZ and LIM domain protein 1 – LIM domain (Homo sapiens)2 sites: CCHC, CCCHStructural14.5 (average value)50 mM Tris pH 7.4, 150 mM NaCl (CDC) (301)
Rad50 protein (Homo sapiens)CC+CCStructural∼19b50 mM HEPES pH 7.4, 150 mM NaCl (FLC) (302)
Superoxide dismutase (Homo sapiens)HHHDStructural13.4100 mM phosphate pH, 7.4 (PAR) (303)
Transcription factor Sp1 (Homo sapiens)CCHH (third zinc finger)Structural9.250 mM pH 7.0, NaCl (RT) (302,304)
   12.750 mM pH 7.0, NaClO4 (CDC) 
Tristetraprolin (Mus musculus)2 sites: CCCHStructural10.2200 mM HEPES pH, 100 mM NaCl (RT) (305)
Xeroderma pigmentosum group A complementing protein XPAzf (Homo sapiens)CCCCStructural9.850 mM phosphate pH 7.4 (RT) (306)
CD4-Lck complex, zinc clasp (Homo sapiens)CC+CC (interprotein site, heterodimer)Structural, regulatory18.6b50 mM HEPES pH 7.4, 100 mM KNO3 (FLC) (307,308)
Ca2+ ATPase (Homo sapiens)N.D.Regulatory80 pM20 mM HEPES-Tris pH 7.4 (EAMB) (309)
Caspase 3KEHRegulatory6.9 nM100 mM HEPES pH 7.5, 100 mM NaCl (EAMB) (310)
Caspase 6  2.6 nM  
Caspase 7  76 nM  
Caspase 8 (Homo sapiens)  4.3 nM  
CathepsinsHC for cathepsin SRegulatoryIC50 ∼160 nM  (311)
Dimethylarginine dimethylaminohydrolase-1 (Bos taurus)HCRegulatory4.2 nM25 mM HEPES pH 7.4, 50 mM NaCl (EAMB) (312)
KallikreinsHH or HERegulatory10 nM-10 μM  (313)
NMDA receptorHHEDRegulatory10 nM10 mM tricine pH 7.3 (EAMB) (314)
Phosphoglucomutase (rabbit)SDDDRegulatory11.625 mM histidine-Tris pH 7.5, 1.5 mM Mg2+ (EAMB) (315)
Protein tyrosine phosphatase 1B (Homo sapiens)N.D.Regulatory7.850 mM HEPES pH 7.4, 100 mM KNO3 (EAMB) (294)
Receptor protein tyrosine phosphatase β (Homo sapiens)N.D.Regulatory21 pM50 mM HEPES pH 7.4 (EAMB) (316)
a

EQD – equilibrium dialysis, EADT – enzyme activity: direct titration, EAMB – enzyme activity in metal buffers, EAC – enzyme activity measured in the cells, PAR – competition with PAR [4-(2-pyridylazo)resorcinol], CDC – CD-monitored competition with chelating agents, FLDT- fluorescence monitored direct titration, FLC – fluorescence monitored competition with chelating agents, RT – reverse titration.

b

Dissociation constant of the homo- or heterodimeric interprotein Zn(II) complex–constant is defined by [A][B][Zn(II)]/[ZnAB], where A and B are components of interprotein binding sites. The constant has the unit M2.

An important conclusion is that the buffering discussed for MTs (Figure 19, middle panel) covers this range of affinities. Therefore, MTs can have a dynamic buffering role in cellular Zn(II) redistribution, in controlling the metalation (“zincation”) of zinc proteins, and in reprogramming the system in such a way that cells in different states adjust to different pZn (−log[Zn(II)]free) values─the zinc potential─and zinc buffering capacities. (89) Zinc buffering by MTs is reduced when MTF-1, the zinc-sensing transcription factor, is ablated and the sensitivity of the non-MTF1-controlled Zn(II)-responsive transcriptome increases. (317) Adjustments of the “MT/T system” to various pZn values have been observed in the human HT-29 colon cancer cell line. (89) Cells incubated with an increasing concentration of ZnSO4 (0–200 μM) demonstrated a decreased pZn value from 9.1 to 8.3, corresponding to the range from 0.8 pM to 5.2 nM free Zn(II) concentrations. Simultaneously, the zinc buffering capacity, the surplus of cellular ligands binding Zn(II) tightly, and the MT/T molar ratio changed from 65 to 219 μM, from 28.2 to 22.6 μM, and from 2.3 to 3.6, respectively. Under this paradigm of increased total cellular zinc, free Zn(II) and the concentrations of MTs increase, while the zinc buffering properties of the cell decrease. (89,318) In addition to serving as a steady-state buffer to maintain homeostasis, the dynamic regulation of MTs allows a resetting of the zinc buffering to satisfy Zn(II) requirements when the state of the cell changes, for example, in differentiation. (89) When mouse mammary epithelial cells differentiate into secretory cells zinc homeostasis is remodelled by an induction of MT1 and MT2 through the glucocorticoid receptor pathway, concomitant with an increase in cytosolic free Zn(II) to maintain the lactating phenotype. (319) Increases of free Zn(II) modulate gene expression of proteins that are important for specific processes, for example, neurite expansion and synaptic growth. (320)

4.3. Zinc Signaling

A remarkable development, which followed the advent of the synthesis of fluorescent Zn(II)-chelating probes and sensors similar to the ones employed in the calcium signaling field, was the recognition of intra- and extracellular zinc signals with important effector roles. (117) In contrast to micromolar to nanomolar changes for Ca(II) ions, the nanomolar to picomolar changes of Zn(II) ions and the coordination chemistry make investigations more challenging. The occurrence of signaling Zn(II) ion transients that variously have been referred to zinc waves, sparks, or spikes means that Zn(II) ions need to be buffered not only at a steady state but temporal changes in their availability also need to be controlled. Thus, buffering must allow zinc signals to occur, reach their targets, and then restore concentrations to steady-state levels. On the one hand, MT is a source of signaling Zn(II) ions when it transduces redox signals into zinc signals or possibly as result of local pH changes due to an inherent pH dependence of the Zn(II) affinity. (86,321) T, on the other hand, removes Zn(II) ions from sites that are targets of the signaling Zn(II) ions.

4.4. Copper Buffering

In contrast to the few thousand zinc proteins, copper usage is much more limited. Estimates are that the human copper proteome consists of only 54 human copper proteins. (322) The reducing environment of the cell comes at a cost. Cu(I) is even more potent than Fe(II) in catalyzing Fenton chemistry, and it can disproportionate into Cu0 and Cu(II). MTs sequester Cu(I) in such a way that Cu(I) is no longer solvent-accessible once it is bound and that there is basically no free Cu(I). Since the volume of a typical cell is in the range of a few picoliters (10–12 L), femtomolar concentrations of free Cu(I) (Figure 21) amount to only approximately a single ion per cell, thus essentially solving the issue of how Fenton chemistry can be avoided. In MT3, Cu(I) is redox-stable in the presence of oxygen. (227) However, it does not mean that Cu(I)-MT does not participate in redox reactions. In vitro, Cu(I)-MT reacts with cytochrome c, and a role in a redox chain was proposed. (323) Furthermore, copper chaperones safeguard Cu(I) in direct transfer to the limited number of proteins. These proteins and copper transporters use sulfur donors in their coordination environments. The copper transfer from the copper chaperone of superoxide dismutase (CCS) to the active site of superoxide dismutase (SOD) involves the trigonal sulfur coordination of Cu(I) in CCS and redox reactions in a sequence of events that has turned out to be remarkably complex and recently shown to even involve the copper transporter Ctr1. (324,325) Such sulfur chemistry could apply to a Cu(I) transfer from MT. A “catalyzed” transfer is necessary. With diffusion-limited association rates, copper dissociation from sites with attomolar binding constants would take years. The use of metallochaperones for copper would seem to make a copper buffer unnecessary. Indeed, in many accounts of copper metabolism, MTs do not have a central role and are suggested to function in storage or salvage only. One model for cellular copper homeostasis is based on the observation that significant amounts of Cu(I) have been postulated to be in the glutathione pool (102,326) and that this high-capacity pool has a role in copper buffering. Glutathione forms preferentially a tetranuclear [Cu4(GS)6]2– complex. Since such a complex forms superoxide, it is likely not the copper buffering species. On the basis of its affinity of ∼1016 M–1 the actually free Cu(I) concentration is sub-femtomolar, that is, attomolar. (327) With a ratiometric two-photon probe, crips-17 with a Kd of 8 aM, it was possible to demonstrate that Cu(I) indeed is buffered to ∼1 aM free Cu(I), in agreement with dissociation constants of copper chaperones. (145) A role of glutathione in copper metabolism is further supported by the fact that induced redox changes in the glutathione pool affect free Cu(I) concentrations as measured with the ratiometric FRET probe FCP-1. (328) A possible conclusion from these studies is that ZnMT is needed as a high-affinity and low-capacity pool for scavenging Cu(I) in order to avoid the Fenton chemistry of Cu(I) buffered by the glutathione pool.
In order to discuss a role of MTs in copper metabolism, one must examine how their affinities and binding mechanisms are related to those of copper proteins. Copper enzymes are sometimes described as Cu(II) enzymes, for example, copper superoxide dismutase, though the major valence state of cellular copper is Cu(I). The estimated Cu(I) binding constant for yeast copper superoxide dismutase (1020 M–1) is 5 orders of magnitude higher than the one for Cu(II) (∼1015 M–1). (329) Murine S-adenosylhomocysteine hydrolase has a K of 3.8 × 1014 M–1 for Cu(II) in the same range. (330) Thus, on the basis of equilibrium concentrations only, Cu(II) is more likely to dissociate than Cu(I) from ligand environments with nitrogen or oxygen donors, rendering cellular concentrations of free Cu(II) potentially significantly higher than those of Cu(I) with implications not only for competition with Zn(II) but also redox biology, where any free Cu(II) in the cell is expected to be reduced to Cu(I) immediately. Thus, free Cu(II) in the cell is unlikely to be of physiological relevance. Its contribution to pathophysiological processes is a different matter, though.
With the exception of MT3, MT4, and MTs in neonatal liver, highly Cu(I)-loaded MT species in human cells have not been reported, and all the forms described contain Zn(II) as well. (181,219) However, there are large variations of metal content in different species. For example, variable Cu/Zn ratios from 0.1 to 20 were found in calf livers, with a total metal content from 7 to 15 g-atoms/mol of protein. (331) We anticipate that the formation of Cu(I)-MT species may occur by different mechanisms. One mechanism involves a direct Cu(I) binding to Zn(II)-depleted species present under conditions of low free Zn(II) concentrations (eq 8) and occurring without Zn(II) dissociation. A reorganization of metals sites is expected to take place. In this case, ZnxMT may have role as a “copper sponge”, and the CuyZnxMT species formed could serve as a Cu(I) acceptor and donor similar to the Zn(II) buffering process. In such a mechanism Zn(II) and Cu(I) buffering would occur independently; that is, Zn(II) metabolism is not affected.
(8)
Alternatively, Cu(I)/Zn(II)MT species could form via a metal ion exchange when Cu(I) with its higher affinity for MT causes a Zn(II) dissociation (eq 9). The equilibrium constant describing this exchange constant (Kex) is different from the one describing the process in eq 8, and it will be yet different if other species such as Zn6MT are involved (eq 10).
(9)
(10)
In this process, Cu(I) binding impacts the Zn(II) availability significantly, and the relative concentrations of both metal ions are linked. Increases in Cu(I) concentrations would increase the Cu(I) load of MT, while free Zn(II) concentrations would increase simultaneously. Likewise the cellular Zn(II) influx could cause a Cu(I) dissociation from MT and increase free Cu(I) concentrations. This scenario is potentially very important, since the buffering of both Zn(II) and Cu(I) converge in the MT molecule, which could serve as a rheostat for the ratio and indicate a metabolic link between the two essential metal ions. The actual affinities of both metal ions in mixed CuyZn7–xMT species remain unknown. They likely differ from those in the binary systems. Eq 11, rearranged from eq 10, expresses how MT species may control the concentrations of both essential metal ions simultaneously.
(11)
Figure 22 summarizes the two described hypothetical modes of action for CuyZnxMT in an independent or linked buffering of the two metal ions.

Figure 22

Figure 22. Two modes of action in which MT buffers Cu(I) either independent of Zn(II) or dependent on Zn(II).

Another variable in this buffering is that mixed Cu(I)/Zn(II) MT complexes may be oxidized either during their formation involving Cu(II) or in reactions with oxidizing agents (Figure 23). Sulfur oxidation in CuyZn7–xMT will change the buffering properties of the system due to either a dissociation of one metal ion or a change of affinity toward a particular metal ion.

Figure 23

Figure 23. Pathways for the formation of mixed CuyZn7–xMT in reduced and oxidized forms.

4.5. Copper Signaling

Evidence for copper signaling accumulated recently, not only in the brain for olfaction and circadian rhythm but also generally for proliferation, autophagy, and fat metabolism. (328,332) Like in the case of zinc, it appears to involve both copper secretion from cells by exocytosis (extracellular signaling) and the release of labile Cu(I) ions in the cell (intracellular signaling). Besides Zn(II)-containing vesicles (zincosomes), copper storage vesicles (CSV) have been identified in the brain and in other tissues. (333−336) The copper concentration in these vesicles is estimated to be 100 mM. (333) The CSV are considered a dynamic copper pool that is regulated for signaling with labile copper ions. Targets are, for example, phosphodiesterase 3B (PDE3B), which is involved in cAMP-dependent lipolysis, (337) or β-secretase-1 (BACE), (338) an enzyme that cleaves amyloid precursor protein (APP) to generate amyloidβ (Aβ). Copper signaling requires a control of the transients in time and space and the state of cupric and cuprous ions, and neither the chemistry nor the biology is presently known. The chemical nature of the labile copper pool remains to be defined. Because free Cu(I) is virtually absent in cells─and needs to be extremely low due to its toxicity─it remains unknown how copper signaling can operate. The cellular biology where in the cell such labile copper pools exist also needs to be addressed. Major unresolved issues are how the dynamic pools of the two most competitive ions are regulated separately─or whether they interact through the linked buffering discussed for MT─what the exact role of glutathione is and how the released ions can target specific proteins in signaling. And then there is the overarching question of why signaling with both metal ions became advantageous in evolution.

4.6. Controlling Zn(II)/Cu(I) Ratios

It is an unproductive state of affairs that scientific communities became so specialized that they address either zinc or copper biology but rarely both. Focusing on a single metal ion will provide only incomplete answers. Regrettably, it also applies to research on MT and glutathione, both of which are discussed in either zinc or copper metabolism. With the exception of MT3, mixed Cu(I)/Zn(II) species have received almost no attention. The capacity of the MT/T and GSH/GSSG systems to buffer essential metal ions can be challenged by nonessential metal ions such as Cd(II). Metal buffering in these thiolate coordination environments further depends on the redox state of the system, relating it to redox buffering. How the glutathione and metallothionein redox systems buffer Zn(II) and Cu(I), assisted by kinetic muffling by transporters and dynamic regulation at multiple levels, how kinetic lability is incorporated into thermodynamic stability, and how the two systems interact are most remarkable aspects of biological inorganic chemistry. Answers about the physiological function will not be found by investigations only in vitro. Needed are determinations of the in situ metal compositions of these pools and correlations with total metal concentrations and respective zinc or copper metalloproteins in the same cellular compartment.
In the absence of bona fide metallochaperones for Zn(II), MT can have an active role in cellular Zn(II) redistribution, which will be discussed now for metal transfer reactions.

4.7. Metal Transfer and Exchange Reactions

In addition to metal exchange between MTs and between MTs and proteins, there is metal transfer. Metal transfer reactions have been discussed as presumed function of MTs. How and when a protein acquires Zn(II) for function remains an unresolved question in biochemistry. Metal transfer reactions have been investigated in both directions: MT as a Zn(II) donor to the apoforms of zinc proteins and T as a Zn(II) acceptor from zinc proteins. While the investigations to be discussed show metal transfer in principle, it must be acknowledged that the conditions under which the experiments were performed do not reflect the conditions in vivo. With the knowledge we have now about partially metal-saturated MT species, neither the fully Zn(II)-loaded protein (Zn7MT) nor the Zn(II)-free protein (T) are expected to exist under normal conditions, and hence the investigations must be reinterpreted by considering ZnxMT species under various cellular conditions.

4.7.1. Metal Transfer from MT to Proteins

Zn(II)-MT can deliver Zn(II) to several apoforms of zinc enzymes restoring their enzymatic activity. (339) Zn(II)-MT (log K7.4 from 8 to 12) is a much better Zn(II) donor than ZnEDTA (apparent log K7.4 = 13.6) to apo-carbonic anhydrase (CA) (log K7.0 = 12). (287,340) Accordingly, it was suggested already 40 years ago that metal transfer by an associative mechanism is the physiological function of MT. (341) For the interaction of EDTA with zinc finger peptides it was shown that an associative ligand exchange mechanism gives a 6 orders of magnitude faster Zn(II) transfer compared to a dissociative mechanism, emphasizing the power of kinetic mechanisms in controlling metal transfer. (273) However, MT is not a genuine metal transferase because metal transfer occurs at stoichiometric amounts and there is no evidence for catalysis, making MT a carrier protein similar to metallochaperones in this context.
Not only the apoforms of enzymes but also those of zinc finger peptides are acceptors of Zn(II) from MT. The preferential transfer of a single Zn(II) ion from Zn7MT to a zinc finger peptide requires interprotein contact. (135,342) The absence of metal transfer when the proteins are separated by a dialysis membrane supports an associative mechanism. When the Zn(II) transfer potential of the chemically synthesized individual MT domain peptides was examined, the N-terminal β-domain was the better Zn(II) donor to apo-SDH, but the cumulative properties of the two individual domains do not describe the properties of the whole protein. (75) This result is consistent with a weaker Zn(II) binding in the β-domain and observations that investigations with the individual domains do not recapitulate all the properties of intact MT.
An associative process does not necessarily mean a ligand-exchange mechanism with the ligand donors in the active site. MT may simply bind to the surface of the Zn(II) acceptor protein, and the interaction then triggers metal transfer. Some active sites are buried and not readily accessible for chelating agents, but there can be pathways for the migration of the metal ion from a binding site on the surface to the active site. (343) A direct interaction of MT with a protein is not the only mechanism of transfer. The GSH/GSSG couple mediates transfer, suggesting that the state of specific redox couples is a driving force for the Zn(II) distribution. (92) With apo-SDH as a Zn(II) acceptor, GSH was found to inhibit the transfer in the absence of GSSG but to enhance it in its presence. In the absence of either GSH or GSSG a stoichiometry of 1:1 (MT/apo-SDH) restored the activity, indicating that one Zn(II) ion was transferred. In order to determine whether the transfer is kinetically or thermodynamically controlled, carbonic anhydrase was also examined. It did not serve as a Zn(II) donor to apo-SDH (log K = 11.2). (294) Since the prevailing opinion at that time was that MT has a Kd of 1.4 × 10–13 M (pH 7) for Zn(II) while CA has a Kd of 1 × 10–12 M (pH 7) for Zn(II), it was concluded that the transfer from MT must be kinetically controlled. Observations of metal transfer from an apparently stronger to a weaker Zn(II) binding site were reconciled by postulating a kinetic lability in thermodynamically stable Zn(II)/thiolate clusters in MT. The results must now be re-evaluated in view of the different affinities of MT for Zn(II), which make thermodynamically controlled transfer possible. Indeed, different molar MT/T ratios modulate the activity of apo-SDH. Below a midpoint ratio of 0.4 [T/(T+MT)] Zn(II) transfer occurs, and the enzyme is activated. Above this ratio, Zn(II) transfer does not occur. (294)
Using stable isotope-labeled 67Zn3Cd4MT2 to examine metal transfer to apo-CA, it was noted that, in the absence of GSH or GSSG, both Zn(II) and Cd(II) are transferred to apo-CA and that, in the presence of GSH, the transfer of Zn(II) was increased while that of Cd(II) was reduced. Thus, GSH protects against a Cd(II) mismetalation. (344) A metal transfer by an exchange of different metal ions has been proposed as a mechanism of rescuing proteins that inadvertently have been metalated with Cd(II). Thus, the higher affinity of MT for Cd(II) compared to Zn(II) allows Zn(II)-MT to bind Cd(II) from a protein mismetalated with Cd(II) and then to donate Zn(II) to form the native zinc protein. (345) It suggests a function of MTs as a “clean sweeper in metalloprotein quality control.”

4.7.2. Metal Transfer from Proteins to Thionein

In vitro, T removes Zn(II) from Zn(II) binding sites in proteins in a process mediated by other Zn(II) chelating agents such as GSH. (72) With the multiple arrangements of its cysteines and their flexibility, T is a chelating agent par excellence and has the potential to remove Zn(II) from numerous, structurally different sites. However, on the basis of affinity, it would not be able to remove Zn(II) from sites in proteins where Zn(II) binds with femtomolar affinities. Clearly, the presence of such a strong chelating agent would be detrimental to many Zn(II)-dependent cellular processes. The present view delineated above, therefore, is that T does not exist in the cell. Upon induction, it will bind any surplus of readily available Zn(II) and re-equilibrate with any existing MTs. Nevertheless, the induction of T will increase the chelating capacity in the cell and adjust it to affinities needed to restore steady-state concentrations of free Zn(II) ions once zinc transients (signals) have been induced and have targeted regulatory Zn(II)-binding sites, which are a third category of binding sites in addition to catalytic and structural sites in proteins (Table 1). For example, a Zn(II)-binding site is present in mitochondrial aconitase, inhibiting its activity. (346) When heart extracts of MT-null mice were incubated with radioisotope-labeled 65Zn(II)-MT, Zn(II) transfer to m-aconitase but not c-aconitase was demonstrated. (347) The affinities of Zn(II) for regulatory zinc sites in proteins are often lower than those for catalytic or structural sites (Table 1). The regulation of proteins by reversible Zn(II) binding is not widely acknowledged but sufficiently documented. (348) Once inhibited, the proteins likely need to be activated again at some point in cellular time. T removes Zn(II) from Zn(II)-inhibited enzymes. (349) Other examples are protein tyrosine phosphatases, enzymes that are not zinc enzymes when they are active, but they are tightly inhibited by Zn(II) ions. (316,350) The MT/T ratio also modulates their inhibition. Protein tyrosine phosphatase 1B with a log K value of 7.8 for Zn(II) binding has a midpoint ratio [T/(T+MT)] of 0.08 for inhibition. (294) These results demonstrate a role of the MT/T system in regulating enzymatic and other protein activities via controlling Zn(II) availability. Also, MT/T would remove any Zn(II) bound adventitiously on the surface of proteins. If loosely bound Zn(II) on proteins were to contribute to buffering, the control of Zn(II) transients for signaling would not be possible. Zn(II) in catalytic and many structural sites in zinc proteins is kinetically trapped and not in fast exchange and does not contribute to buffering. These boundary conditions determine the range for MT/T buffering and zinc regulation.
The presence of a second chelator can accelerate the rate of metal transfer from a metalloenzyme to T. T removes Zn(II) from the active site of carboxypeptidase A ∼400-fold faster in the presence of D-penicillamine (β,β′-dimethyl-d-cysteine). (351,352) The effect is because T has a higher affinity for Zn(II) than D-penicillamine and consequently by transferring Zn(II) from D-penicillamine to T the removal of Zn(II) from the enzyme becomes quasi-irreversible. The process has been referred to as catalytic chelation and could well present a way of how an LMW ligand such as glutathione kinetically controls metal transfer involving the MT system.
T also abolishes DNA binding of the zinc finger transcription factor Sp1. (353) Likewise, Zn(II) is transferred from the zinc finger transcription factor TFIIIA to T. (354) Another system investigated is the estrogen receptor, the DNA binding domain of which requires two structural Zn(II) ions in tetrathiolate coordination environments. T abrogates and MT restores estrogen receptor binding to DNA. (355) The observations of MT transferring Zn(II) to the apo-forms of zinc transcription factors and the latter transferring Zn(II) to T suggest that the MT/T ratio could also modulate gene expression, including the induction or repression of MT genes. (356) With the exception of MTF-1, knowledge about the zinc regulation of Zn(II)-binding domains of transcription factors is sparse. There are many types of transcription factors with different zinc finger domains and different Zn(II) affinities. (135,357) Some of these Zn(II)-binding domains are involved directly in DNA interaction while others are not. (358,359) Thus, whether MT/T regulates Zn(II)-dependent transcription factors requires further investigations with knowledge of which ZnxMT species are present in the nucleus and with a consideration of different Zn(II) affinities and Zn(II) coordination environments of the transcription factors.
To address the issue of metal transfer with a more “realistic” experiment, a zinc protein (SDH) or a Zn(II)-inhibited protein (PTP1B) were mixed with different MT/T ratios, more closely mimicking the distribution of ZnxMT species. The result of these experiments shows that the MT/T ratio determines the Zn(II) availability, (294) indicating that the MT system is indeed a determinant of biological activity. (78)The important point is that regulation with Zn(II) ions would not be possible if they were freely available. MT as a zinc homeostatic gene product is a regulated zinc buffer that serves as a rheostat for the availability of Zn(II) ions and their role as cellular signaling ions.

4.7.3. A Role of ZnATP

In addition to MT/glutathione interactions, there is an interaction of MT with nucleotides. On the basis of an earlier observation of GTP binding to MT, (360) the binding of ATP and GTP was investigated in more detail. (361) For ATP, a 1:1 complex with a Kd = 176 μM at pH 7.4 was observed for rabbit Cd,ZnMT2. ATP enhances the Zn(II) transfer from human Zn7MT2 to apo-SDH and the reaction of MT with DTNB. These phenomena can now be explained by ATP initially binding to MT and then binding Zn(II) to form a ZnATP complex. The removal of Zn(II) will increase the reactivity with DTNB as free thiols become available. With the poorly hydrolyzable analogue adenosine 5′-[β,y-imido]triphosphate it was shown that the hydrolysis of ATP is not involved. GTP elicited virtually identical effects, but adenosine diphosphate (ADP) had no effect on the Zn(II) transfer. One group of investigators found no evidence for MT binding ATP. (362) Subsequently, the binding was confirmed with a Kd value of 310 μM at pH 7.6 when a kinetic method was employed to follow the reaction of MT with DTNB. (363) In this assay, rabbit MT1, MT2, and MT3 had similar effects. ADP affects the reactivity of the thiols in MT, but adenosine monophosphate (AMP) does not. It was then shown that chloride binds to MT, allowing 35Cl NMR to be employed in a competition assay with ATP to demonstrate the interaction of MT with ATP. (57) The estimated binding constant is 993 μM at pH 7.4 for Cd7MT. Furthermore, scanning tunneling microscopy, which was earlier employed to visualize the shape of single MT molecules, (56) demonstrated a change in the shape of MT upon interacting with ATP, as further corroborated by changes of the N- and C-terminal amino acids in 1H NMR total correlation spectroscopy (TOCSY) spectra. (57) The change of shape is due to either the binding of ATP or the removal of Zn(II). Given the typical ATP concentrations in a cell, MT is expected to interact with ATP.
ZnATP rather than MgATP is the preferred substrate for some kinases involved in cofactor biosynthesis: pyridoxal kinase, riboflavin kinase, and NAD kinase. (364−367) X-ray structures of the first two enzymes indeed show the interaction with a ZnATP complex. (368,369) Noteworthy, MT activates pyridoxal kinase. (370) An equilibrium dialysis showed that ATP removes Zn(II) from MT. This observation was confirmed in competition experiments with the Zn(II) probe FluoZin-3. (73) These experiments provide a unifying explanation for the observed MT/ATP interaction, namely, binding of ATP to MT followed by the removal of Zn(II) and the formation of a ZnATP complex that activates pyridoxal kinase. ATP forms a stronger complex with Zn(II) than ADP, and it has a stronger tendency to form ternary complexes with other LMW ligands. (371) The MT/ZnATP interaction could channel Zn(II) into specific reactions that require ZnATP as a cofactor. The very low free Zn(II) concentrations would not be sufficient to form a ZnATP complex. Therefore, Zn(II)-MT is thought to be the limiting molecule in the activation of pyridoxal kinase and probably other specific kinases that ulilize ZnATP as the substrate. It is a case of metal transfer to a cofactor rather than an enzyme and possibly a fundamental metabolic link between energy metabolism and Zn(II)-dependent cofactor biosynthesis (Figure 24).

Figure 24

Figure 24. Interaction of pyridoxal kinase with ZnATP (PDB code: 1LHR). The crystal structure of pyridoxal kinase (PK) shows complexation with ZnATP. (368) There is not enough free Zn(II) available to form the ZnATP needed to activate PK directly. However, the interaction of Zn(II)-MT with ATP could provide the ZnATP to activate PK. (370)

5. Regulation of Metallothionein in Biological Space and Time

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5.1. Multiplicity of MT Genes and Their Regulation

Molecular and cellular biology and molecular genetics provided new powerful tools for MT research. However, while they added important biological context, they did not answer the question about the function(s) of MTs either, primarily because the state of the protein is not addressed. The advent of the polymerase chain reaction (PCR) and rapid nucleic acid sequencing generated a wealth of information on sequences of additional MTs and their expression in many cells, previously not amenable to otherwise laborious MT isolation. It demonstrated a rather ubiquitous expression of MT1/2, cell-specific expression patterns of MTs that change in many diseases as investigated widely in cancer, (372) and rather restricted expression of MT3 in the brain and MT4 in squamous epithelia. With regard to transcription profiling, there is a caveat, however. The correspondence between mRNA and protein levels is generally poor for MTs. (373) Multiple mutations in MT genes were detected, and their associations with different health conditions widened the functional implications of MTs even further. (374)
The human MT gene cluster on chromosome 16q13 was defined. (19,375) With the cloning of six additional genes and MT3 and MT4, the number of expressed human proteins is at least 11. (182,376) There are yet additional genes; some are likely genuine pseudogenes, but for others the translation into stable or “functional” proteins has not been demonstrated yet. Referring to the different gene products as isoproteins implies similar structures and functions─an assumption of perceived redundancy rather than a fact. The variation of a significant number of amino acids in different MT1 proteins and their differential regulation underscores qualitative as well as quantitative differences.
The HUGO Gene Nomenclature Committee (HGNC) (www.genenames.org) has listed symbols and names for human MT genes (Table 2). Among these 19 genes, 7 are pseudogenes, leaving 12 genes for possible expression of MTs. Remarkably, one gene, MT1HL1─inferred from homology to be related to MT1h─has been retrotransposed to chromosome 1. The classification as a “pseudogene” is based on structural faults or atypical amino acids. It is a supposition that MTs with atypical amino acids are not expressed, as such amino acids have been observed in MTs from other species. MT1HL1 and MT1b are not thought to be pseudogenes. The putative proteins have 18 and 21 cysteine residues, respectively. In MT1HL1, each domain has one cysteine residue less. In addition, it has a tyrosine. The incorporation of the additional Cys into human MT2 at the corresponding position found in MT1b leads to a functional MT. (189) The expression of MT4 and MT1b is very restricted to a specific tissue or condition.
Table 2. Human MT Genes and Pseudogenes in Alphabetical Order Based on HGNC (HUGO Gene Nomenclature Committee)
HGNC ID (gene)approved symbolapproved nameprevious symbolsaliaseschromosome
HGNC:7393MT1Ametallothionein 1AMT1, MT1S 16q13
HGNC:7394MT1Bmetallothionein 1BMT1, MT1Q 16q13
HGNC:7395MT1CPmetallothionein 1C, pseudogene  16q13
HGNC:7396MT1DPmetallothionein 1D, pseudogene MTM16q13
HGNC:7397MT1Emetallothionein 1EMT1MTD16q13
HGNC:7398MT1Fmetallothionein 1FMT1 16q13
HGNC:7399MT1Gmetallothionein 1GMT1MT1K16q13
HGNC:7400MT1Hmetallothionein 1HMT1 16q13
HGNC:31864MT1HL1metallothionein 1H like 1MT1P2 1q43
HGNC:7401MT1IPmetallothionein 1I, pseudogeneMT1, MT1IMTE16q13
HGNC:7402MT1JPmetallothionein 1J, pseudogeneMT1, MT1NP, MT1JMTB16q13
HGNC:7404MT1Lmetallothionein 1L, pseudogeneMT1MTF, MT1R16q13
HGNC:14296MT1Mmetallothionein 1MMT1, MT1K 16q13
HGNC:23681MT1P1metallothionein 1 pseudogene 1 bA435O5.39q22.32
HGNC:16120MT1P3metallothionein 1 pseudogene 3C20orf127, MTL4dJ614O4.620q11.22
HGNC:7405MT1Xmetallothionein 1XMT1MT1l16q13
HGNC:7406MT2Ametallothionein 2AMT2 16q13
HGNC:7408MT3metallothionein 3 GIF16q13
HGNC:18705MT4metallothionein 4 MTIV16q13
In addition to defining the extent of the MT family, the second most important contribution of molecular biology was insights into the regulation of basal and induced MT gene expression. It linked MTs and the cellular signal transduction pathways and networks. Induction is remarkably complex and dynamic and includes many environmental factors and hormones: heavy metal ions (essential ones, Zn(II) and Cu(I)/Cu(II), and toxic ones, Cd(II) and others), glucocorticoids, bacterial endotoxins (lipopolysaccharides, LPS), physical and chemical stress, high-energy radiation (UV, X-rays, and γ-rays), heat shock, tissue damage, infection, and inflammation (pro-inflammatory cytokines: IL1β, IL6, TNFα), that is, factors that demonstrate a specific role in the acute phase response, chemotherapeutic and other drugs, changes in oxygen concentration, that is, hypoxia and reoxygenation, interferons related to viral infections, phorbol 12-myristate 13-acetate (PMA)─an activator of protein kinase C, a key signaling enzyme, cold induction, and even psychological stress, such as physical restraint or social isolation. Overall, a major area of MT function is the biology of stress, defense, and inflammation. Protein kinase C has also been shown to interact with MT2a. (377) Moreover, the enzyme phosphorylates rat MT1 at Ser32, a highly conserved residue, and affects the Zn(II) regulation of gene expression. (378)
The many pathways of induction give the impression of pleiotropic actions of MT. However, the question remains: Are they all part of one primary molecular function, or do they reflect different functions of MTs? Some of the pathways are linked to zinc metabolism, but for others the link is either not obvious or investigations on how the inducers affect distribution or redistribution of cellular Zn(II) or Cu(I) under either changes of the cellular state or exposure to stress have not been reported.
In a way, the generally investigated bioinorganic chemistry of MT can be seen with the metaphor of the protein being the tip of the iceberg. One needs to look at what lies underneath: how the regulatory network of the expression of MT genes relates to the functions of the protein in health and disease. The complex control of the gene expression of MTs by a slew of inducers and the differential expression of the MT genes is an essential biological feature and contains crucial and yet to be fully delineated information about the specific function(s) of the proteins. The regulation of gene expression through multiple and pivotal signal transduction cascades targeting cis-regulatory elements (CREs) has left the impression that MTs are multifunctional and multipurpose proteins. However, molecular biology does not address protein chemistry or function directly. We suggest that the diverse conditions of induction, most of which are Zn(II)-dependent themselves, are a reflection of the many facets of zinc metabolism and that they converge into a major function in buffering and redistributing Zn(II). The critical observation that the Zn(II)-sensing transcription factor MTF-1, which binds at multiple MREs and requires Zn(II) for activation, is required for both basal and induced expression of MT1/2 genes in combination with coactivators or other transcription factors, and that other proteins are repressors of gene expression, supports this concept. Such control is well-fitting to adjust the buffering capacity for Zn(II) at the gene level with further control of the pZn of buffering afforded by redox reactions and other interactions at the protein level. The pathways leading to gene expression of MTs pinpoint the different scenarios where Zn(II) is involved. Understanding what MT does requires understanding the roles of its induction in specific aspects of zinc biology, the knowledge of which is still evolving and was not contemporaneous with the discovery of the pathways for MT induction, which started over 40 years ago. An interpretation of how these scientific fields inform each other has much to offer. With the knowledge about the inorganic biochemistry of MTs available now, it becomes possible to attempt a synthesis of the myriad of observations and integrate MTs in the cellular signal transduction and metal homeostatic networks. A synthesis underscores the complexity of functions of MTs in multiple and principal cellular processes. When leaving out the rest of the iceberg, the protein itself can be investigated ad infinitum without ever defining biological functions.
An even more copious literature discusses the involvement of MTs in diseases. However, without a functional context that links the gene expression and the state of the protein an interpretation is tenuous or impossible. Generally, investigations are limited to changes in gene expression of MTs, and the findings are advanced by an emphasis on the potential for a diagnosis and prognosis. Mutations in MTs also have been linked to specific diseases. (374) With a focus on transcriptomics only, pursued widely because of a lack of specific antibodies for the different gene products or metamorphic forms of MT, a molecular mechanism of the protein is seldom addressed, and therefore the exact roles of MTs in the diseases remain enigmatic. MT induction is often seen as generating an “antioxidant” rather than reflecting on the implications for changes in metal metabolism. In our opinion, statements that try to relate the protein to general observations of redox changes are superficial without identifying targets and should address changes in metal metabolism and resolving the ambiguity of whether the induced protein restricts metal ions or makes them available as would be the case in an antioxidant function when the protein thiols are oxidized. We will discuss general pathways that underlie many diseases and not discuss in detail noncommunicable diseases such as cancer, diabetes, and neurodegeneration, for which a lot of work regarding a positive or negative effect of MTs and a role of MT3 in the brain has been published as apparent from the cited reviews. (372,379−383) With specific reference to the healthy, diseased, or injured brain, MT3 is mainly in neurons, while MT1/2 are mainly in astrocytes. (384) Various forms of stress, inflammation, hypoxia, bacterial and viral infections, and aspects of cell fate (cell cycle/proliferation, differentiation/development, cell death/apoptosis) and cell adhesion/migration are such general processes linked to pathways of MT induction. Translating phenomenology into teleology, that is, what the protein does specifically, will reveal the implications for metal metabolism. Overall, such consequences of changed metal metabolism have not caught the interest of the wider biomedical community despite the prospect that knowledge about specific functions of MTs would open the field for therapeutic interventions. Thus, not surprisingly, not a single protein in the family has been approached for drug discovery so far.
We will pursue the discussion of induction through CREs, including the link with Zn(II) when known, but the discussion is not comprehensive, as there are additional inducers that transactivate the gene expression of MTs with roles yet to be established. MT3 is also different in terms of its induction from MT1/2. Hypoxia induces MT3 in adipocytes. (385) Only a modest induction by metal ions was noted in human neuronal cells. (386) In macrophages the interleukins IL4 and IL13 induce MT3. (387)
An in silico analysis of the complex promoters of MT genes provides information on the different binding sites of transcription factors and the differential expression of the MT genes. (388) In many cases, transcription factors heterodimerize and establish crosstalk between several pathways in gene expression. Pro-inflammatory cytokines are inducers with specific response elements for interleukin 6 (IL-6RE), interferons (ISREs), and signal tranducers and activators of transcription (STAT) transcription factors. However, MTs also have anti-inflammatory effects, probably mediated through ARE alone or in combination with an upstream stimulatory factor/major late transcription factor (USF/MLTF). Specific inductions of particular MTs and cell-specific inductions then need to be related to the specific properties of the proteins, once they become known, and to the coordinate expression of zinc transporters or other Zn(II)-dependent proteins.
MRE: Metal response element (RE). Among several proteins discussed in the metal-dependent induction of MTs, MRE-binding transcription factor-1 (MTF-1) is a key transcription factor. (389,390) It regulates the expression of MT1/2 genes. The induction requires Zn(II). The induction of MTF-1-mediated MT transcription by Cd(II) or Cu(I) occurs through the displacement of Zn(II) by these metals. (391) The responsiveness of MTs differs, as there are between one and five MRE sites in the promoters of different MT genes. When Zn(II) concentrations are elevated, MTF-1 induces T to bind Zn(II). It also induces ZnT1, the major cellular zinc exporter. It is certainly a mechanism to deal with increased metal concentrations. But MTF-1 can also serve as a transcriptional repressor, for example, for the zinc importer Zip10, suggesting an additional regulatory loop to control Zn(II) import. In this latter case, the MRE is downstream of the transcription start site, and the interaction with MTF-1 prevents the RNA polymerase from initiating the transcription. (392) While MTF-1 is a key factor in increasing T, that is, increasing the chelating capacity for excess concentrations of Zn(II), the question remains as to what happens under a zinc deficiency. In a mouse fibroblast cell line that has adapted to an extreme zinc deprivation, an amplification of the MT gene locus was noted, suggesting a role of MT in scavenging Zn(II) from the environment under these conditions. (393)
GRE: Glucocorticoid RE. It was the first specific induction pathway found for MT and is related to the response to various forms of stress that produce steroid hormones. (394) There is also a response to other ligands targeting the transcription factors of the nuclear hormone receptor family to which the glucocorticoid receptor, a zinc protein, belongs, for example, the hormones vitamin D and retinoic acid formed from vitamin A, but also ligands of the arylhydrocarbon receptor (AhR).
HRE: Hypoxia RE. Human MT2A mRNA increases under hypoxia, and evidence for an HRE was found, thus identifying this particular MT as an oxygen-regulated protein. (395) The transcription factor involved is hypoxia-inducible factor Hif1α. Hif1α interacts with MTF-1, (396) is stabilized by MT, (397) activated by phosphorylation, and interacts also with Hif1β (aryl hydrocarbon receptor nuclear translocator (ARNT)). Hypoxia responsiveness links MT and the important process of oxygen regulation, inflammation and angiogenesis, the formation of blood vessels under normal conditions but also critically involved in cancer metastasis, kidney, lung and heart disease, diabetes and acute respiratory syndrome as experienced in the Covid-19 pandemic due to SARS-CoV-2 infection.
XRE: Xenobiotic RE. The only directly ligand-activated transcription factor AhR, which heterodimerizes with ARNT, binds at this cis element. Xenobiotics such as halogenated aromatic hydrocarbons activate it, but in contrast to what the name indicates, also many endogenous substances important for stem cell maintenance and differentiation are activators. AhR interacts with the GR to activate human MT2A. (398) Furthermore, the induction of MT gene expression by 2,3,7,8-tetrachlorodibenzodioxin (TCDD, an AhR ligand) and the presence of MREs in close proximity of XREs has been observed and further discussed. (399)
ARE: Antioxidant RE. This response element was first described for the mouse MT1 gene (400) and, either alone or together with the USF/MLTF, is responsible for the induction of MT by oxidative stress. Oxidative stress can also activate MTF-1, presumably through Zn(II) released during oxidative stress. Like XRE, the ARE is activated by many substances such as electrophiles that pose a potential threat to the cell, and it is a major factor in the transcriptional response of MT to so many substances. The transcription factor is nuclear factor erythroid 2-related factor 2 (nrf2), which is not known to be a zinc protein. However, it interacts with Kelch-like ECH-associated protein1 (Keap1), which is a zinc protein, in which electrophiles react with the Zn(II)/thiolates and eject Zn(II). (297)
In addition, MT modulates other important signal transduction pathways for which no CREs on MTs have been identified. One is the tumor necrosis factor alpha (TNFα) pathway in inflammation. The transcription factor is nuclear factor kappaB (NF-κB), a dimer of p50/p65. p50 has been suggested to interact with MT (401) while MTF-1 interacts with p65 under conditions of hypoxia. (402) Altogether, at least 24 zinc sites in proteins involved in the pathway have been identified. (403) Controversies about the role of MT in this pathway have been reviewed. (404)
The other transcription factor discussed in interactions with MT is p53. It is the product of the TP53 gene and is called the guardian of the genome. Mutations in this gene are responsible for approximately half of cancers. p53 is a zinc protein. The zinc site has a structural function and is essential for its transcriptional activity, as the protein will misfold if it is devoid of Zn(II), for example, mutation in the zinc site (R175H), leading to a 1000-fold weaker Zn(II) binding. A very short exposure to Zn(II) (<30 min) rescues the protein and leads to cancer cell death, adducing to the unique concept that zinc metallochaperone therapy is a viable drug approach to rescue mutant p53. (405) It has been suggested that MT modulates Zn(II) binding to p53 and hence the transcriptional activity of p53 and that the two proteins interact. (406,407)
Numerous reviews have discussed the regulation of MT genes. The effects are often cell-, metal-, and MT-specific. Some MT1 and MT2 genes are constitutively expressed in most tissues, and their expression can be induced, in particular, by metal ions. In contrast, MT3 and MT4 are expressed only in some specific tissues and are genererally not directly induced by metal ions. Some of the transcription factors and their DNA recognition elements are specific for individual MT genes. The information has not been fully exploited to provide comprehensive insights into how specific signal transduction pathways and metal and redox metabolism are linked. In summary, instead of looking at MTs in isolation one needs to look at both levels of regulation, the processes upstream in its induction─the input─and those downstream in its activities in metal and redox metabolism─the output─to understand how the MT system works in cells (Figure 25).

Figure 25

Figure 25. Combined gene and protein regulation of the MT system. (left) Many signal transduction pathways converge at the MT promoters to induce the expression of at least 11 human MT genes. Various steps in these pathways depend on Zn(II). A Zn(II)-sensing transcription factor (MTF-1) controls the metal-dependent transcription at metal regulatory elements. Among the other inducers are interferons, glucocorticoids, and redox signals acting on the respective regulatory elements (ISRE, GRE, HRE, XRE, and ARE). DNA methylation can silence the effects of these cis-acting factors. (right) Newly synthesized thioneins bind metal ions to form metallothioneins. The availability of metal ions from MT proteins is linked to redox changes and redox signaling. Protein modifications, interactions with low molecular ligands and other proteins, and protein degradation afford additional layers of regulation as discussed in the main text. With regard to the induction of gene expression, it is a generalized scheme, as not all the respective regulator elements are present in a given MT gene.

When the MT protein is looked at in isolation, an important aspect is missed, namely, that Zn(II) coordination is part of some of the transcription factors for a constitutive and induced expression of MTs and that additional steps in the signal transduction pathways leading to expression are Zn(II)-regulated. The zinc transcription factors include proteins with coordination of the CCCC type, CCHH finger type (Sp1, retinoic acid receptor, Ikaros), and other types such as in Churchill, a neural inducing factor with at least three zinc sites, including a thiolate sulfur μ2-bridge (Figure 26).

Figure 26

Figure 26. Structures of mammalian proteins containing Znx(Cys/His)y clusters. (top left and right) Human embryonic neural inducing factor churchill (2JOX) (408) and dimerization domain (1RMD) (409) of mouse V(D)J recombination-activating protein RAG1, respectively. In both structures unique Zn2HisCys6 clusters with one bridging thiolate sulfur are present. (bottom) Structure of human euchromatic histone methyltransferase 2 containing a Zn3Cys6 cluster. (410)

There is virtually no information on a possible regulation of Zn(II)-containing transcription factors via their Zn(II) content, with the exception of the in vitro work of T removing Zn(II) from transcription factors.
In contrast to the induction of the MT gene expression, negative regulators have received comparatively less attention. (411) A negative regulation includes the silencing of genes by a DNA methylation on their cis-elements, that is, the DNA sequences recognized by transcription factors, and several transcription factors that are repressors. Among those, zinc finger transcription factors feature as well, for example, PZ120 with a CCHH zinc finger (POZ) motif. (412)
Epigenetic effects include histone modifications that affect chromatin structure and hence transcription factor access to DNA. Posttranscriptional control at the mRNA level of MTs has also been observed. (413,414)
Regarding proteolysis, mainly the lysosomal enzymes cathepsin B and L degrade MT with cell-, metal-, and MT-specific differences. (62) While cathepsins are not zinc enzymes, cathepsin B is strongly Zn(II)-inhibited with a half-maximal inhibitory concentration (IC50) of 150 nM (pH 7.4). (127) A Zn(II) ion has been found in the active site of cathepsin L. (415) The interaction of Zn(II) with cathepsins indicates a role of Zn(II) in controlling MT degradation by these enzymes in addition to the effect of the metal load of MT on degradation. It is generally accepted that T is more susceptible to degradation than MT. Further investigations are now warranted to determine whether the partially metalated MT species have different rates of degradation.

5.2. Spatiotemporal Distribution of MT in Cells

After having discussed the dynamics of the protein in terms of reactivity and metal coordination and the exquisite regulation of their genes, yet another significant biological aspect of dynamics is the distribution of the protein in cellular space and the changes in biological time. It involves additional interactions with proteins and important activities of MTs in the redistribution of metal ions and controlling metal metabolism in subcellular organelles. Investigations have addressed translocations to mitochondria, lysosomes, and the nucleus.
Despite lacking a mitochondrial targeting sequence, Zn7MT1/MT2/MT3 are translocated into the intermembrane space of liver, but not heart, mitochondria. Translocation results in a dissociation of Zn(II), which inhibits mitochondrial respiration. (416) Vice versa, T binds Zn(II) from the inhibitory site and activates respiration. The effect seems to be specific for the β-domain, indicating either a Zn(II) dissociation from this domain or a disulfide formation with proteins of the mitochondrial import machinery. Modifying the lysines of MT abolishes translocation. Investigations with Cu(I)-containing MTs regarding mitochondrial biology have not been reported.
MTs do not possess a nuclear localization sequence either. In the developing, but not adult, rat liver and kidney, MT was detected in the nucleus and cytoplasm. (417) In cultured primary hepatocytes of adult rats, however, MT was found in nuclei, but only in the early S phase and not in the G0 or G1 phases of the cell cycle, indicating that the localization is linked to proliferation and the cell cycle. (418) Nuclear MT was found in many tumors and suggested as a marker of the proliferative capacity of cells. (419) With a monoclonal antibody raised against monomeric rat liver MT1, cell cycle-specific nuclear localization of MT was shown in proliferating human HT-29 colon cancer cells. (420) A transient nuclear localization was noted early in the cell cycle when cells differentiated and were shown to depend on mitogenic signaling cascades. (421) Several attributes of the nucleocytoplasmic shuttling have been reported: requirement for energy (ATP) and dependence on phosphorylation signaling, the oxidation of a cytosolic factor, and the state of the cell. A perinuclear localization of the MT1 mRNA and association with the cytoskeleton mediated by the 3′ untranslated region is deemed necessary for nuclear import. (422) The translocation to the nucleus was also suggested to require the small GTPase Ran, an interaction of MT with a cytosolic protein that must be oxidized for nuclear translocation, and dependence on energy. (423−425) Nitric oxide was also proposed to be an essential signal for nuclear translocation and Zn(II) dissociation in the nucleus. (120) The observation that the decrease of cytoplasmic Zn(II)MT correlates with an increase of Cu(I)MT in the nucleus complicates matters further, as it could indicate that a metal exchange process occurs in the cytoplasm and then Cu(I)-MT translocates to the nucleus. (224) The function of MT in the nucleus is presently unknown.
While MTs are found in lysosomes, not much information is available on the translocation to lysosomes. A case has been made for functions of MT3 in lysosomes. MT3 is thought to participate in the process of bringing Zn(II) into lysosomes, controlling the lysosomal pH and activities of proteins, resulting in a role in autophagy and cell death in cortical astrocytes. (426)
The implications of these findings for investigating the functions of MT are huge, as they suggest that MTs need to be investigated with regard to subcellular localization as a function of specific cellular stages in addition to their identity and metal load.

5.3. Extracellular MT

In addition to intracellular redistribution, MTs have been found outside cells. The transfer of Cd(II)MT from liver to kidney requires secretion and uptake. (427−429) Under Cd(II) exposure, extracellular MT contains mainly Cd(II) and copper. (222) Extracellular MT has been determined in blood, urine, or bile. In blood, it is a zinc, copper, or cadmium protein, and degradation products of the protein were also found. (430) Reference ranges of 32 ± 16 ng/mL in plasma and 10 ± 6 ng per micromole creatinine in urine were determined with antibodies specific against human MT1. (431) After surgery, a significant increase in blood was noted, suggesting that MT is an acute phase protein. Since Zn(II) decreases in blood while Cu(II) increases during the acute phase response, it remains a pressing issue to determine whether the increase of MT is linked to both metal ions or only one of them. (432) There is a general lack of knowledge about whether specific MT forms are extracellular or all forms can be found in the extracellular fluid and what the metal load and oxidation state are in the extracellular environment, which is much more oxidizing than the intracellular environment. MT3 is slightly different in this regard. MT3 was identified originally in an assay investigating its effect on inhibiting neuronal growth when added extracellularly. (13) The growth inhibitory factor activity is maintained with Zn(II)-MT3 or Zn(II)/Cu(I)-MT3, the native form, but not other MTs. MT3 appears to be involved in clearing the synaptic cleft from Zn(II), which is a neurotransmitter/neuromodulator of specific neurons, and the reloading of Zn(II) into their synaptic vesicles. (433)
Neither uptake nor secretion mechanisms of MTs are known in any particular detail. For uptake, there is some information, namely, that it occurs through endocytosis via the lipoprotein receptor megalin/LRP2. MT1/2 secreted from astrocytes is found in the extracellular fluid of the injured brain and taken up by a megalin receptor on neurons where it affects axon regeneration. (434) The MT binding on human astrocytes was characterized with a Kd value of 0.84 nM and a Bmax of 99.82 fmol/mg protein to an unidentified binding site. (435) In proximal tubule cells, the multiligand receptor complex megalin: cubulin: amnionless is involved in uptake, but the 24p3/NGAL/lipocalin-2 (LCN2) receptor (SLC22A17) has a 1000-fold higher affinity for MT. (436,437) Human hepatocytes (HepG2 cell line) take up MT by a lipid-raft-dependent endocytosis. (311) MT colocalizes with albumin but not transferrin, also supporting a pathway of nonclassical endocytosis. After uptake in liver cells, MT ends up in an endosomal cellular compartment, from which the metal is released. These experiments were not yet performed with copper MT. For secretion, there is no known mechanism. MT does not have sequence motifs for export or any glycosylation sites. MT secretion is not due to leakage from damaged cells, though. It was described in the intestine, (438) white adipose tissue, (439) astrocytes, (434) and immune cells. (440) Of particular interest is that MT2 concentrations were found to be rather high in exosomes from mesenchymal stromal cells and are required to suppress the inflammatory response in the colon. (441) Taken together, MTs are part of intraluminal vesicles in multivesicular bodies (endosomes), and there seems to be a role for them in metal uptake and in biological communication, in particular, between immune cells. (440)

5.4. Interaction of MT with Proteins

In addition to the membrane receptors involved in cellular uptake of MT numerous interactions with cellular and extracellular proteins have been reported. (442) However, none of these protein–protein interactions (PPIs) has been investigated with structural methods that would reveal the molecular features of the presumed interactions, and none of them fulfill the criteria set out for firmly establishing stable PPIs by employing different techniques. Since MTs are relatively small and metamorphic proteins, nonspecific interactions through covalent or ionic interactions can occur. With its 20 cysteine residues MT is prone to form mixed disulfides. Also conspicuous for interactions are the eight lysine residues, which provide positive charges on the surface of the protein. Transient or stable interactions between MTs and proteins could also be mediated by metal ions. For example, PPIs of other proteins are mediated by Zn(II). Such interactions will depend on pZn and thus on MTs. (171) Implications about functions of interactions are also tenuous. Most of the interactions do not seem to have any obvious or direct connection to zinc, copper, or redox metabolism. The exceptions are some metal transfer reactions, which were found not to occur when proteins were separated by a dialysis membrane, indicating that a direct interaction is necessary for transfer.
With ZnT3, the zinc exporter that loads secretory vesicles with Zn(II), MT3 is part of a synaptic vesicle cycle. MT3 interacts with the small GTPase Rab3a. (433) Further support for such a role of MT3 comes from additional interactions with many other proteins participating in the synaptic vesicle cycle. (443) It was pointed out that such a specific role of MT3 would be different from a general one as a metal buffer.
An assumption in all these PPIs is that we know what the structure of MT is in vivo─by no means a foregone conclusion. Interactions have been examined with metal-reconstituted MTs that may not be relevant physiologically. When antibodies are employed for MT detection, their specificity is fraught with uncertainty, including which MT they detect. Future work has to identify the particular forms of MT involved in any putative interaction. Interactions have focused on MT only, and hence another unresolved issue remains whether there are PPIs with T. With these caveats, any information gleaned from the databases dealing with interactomes and reactomes deserves much scepticism.

6. From Metallothionein Structure to Functions, Roles, and Purposes

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6.1. MTs in Other Organisms

On the basis of sequence information, MTs were divided into three classes: Class I MTs related to mammalian MTs, class II MTs with no or only a seemingly distant relationship to mammalian MTs, and class III MTs as nontranslationally synthesized polypeptides that bind metal ions with cysteine thiolates. (18) These definitions were made by committees of scientists who convened at international meetings on metallothioneins. The problem with these definitions is arbitrariness. For class II, a criterion is lacking how the degree of similarity should be defined, while for class III MTs there is no reason why these peptides should be considered MTs in the first place.
Phylogenetic trees were constructed when many sequences of MTs became available. MTs were divided into 15 families. (444) However, it was noted that these trees are unrooted, suggesting that MTs did not evolve from a common ancestor. The heterogeneity of MTs lumped in class II increased continuously. Thousands of MTs bear no evolutionary relationship to class I MTs, again emphasizing their polyphyletic origin. (445) Phylogenetic and synteny analyses of vertebrate MTs identified ortholog and paralog genes, established a root for the fish clade, and support the functional divergence of MT3 and MT4 relative to MT1&2. (446) The enormous diversity of MTs may have evolved for different functions in different organisms. Class II MTs bind different metal ions (Zn(II), Cu(I), Cd(II)), and indeed some are Cu(I)- and others Cd(II)-binding proteins. They can have only one or even three domains, mononuclear zinc finger-type of structures or binuclear Zn2Cys6 clusters; they can be shorter, for example, fungi, Neurospora crassa MT with 25 amino acids including 7 Cys, or much longer, for example, flatworm MT with 227 amino acids with 34 Cys, than mammalian MTs, and they have atypical amino acids such as tyrosine residues. Histidine residues are also quite common in some and participate in metal binding. There are 91 Cys in Trichomonas vaginalis MT. Tetrahymena thermophile MTT1 has several CCC motifs. Cys motifs are not unique for MTs, nor are Zn(II)/thiolate clusters. A Zn3Cys9 cluster as in the β-domain of class I MTs was found in fungal and fly proteins and in human histone methyltransferases (Figure 26). (41,42,410) This variability demonstrates that all the definitions set out initially for equine renal cortex MT, (18) the prototype “MT”, do not hold when considering class II MTs: low molecular weight proteins, high metal content, characteristic amino acid composition (high cysteine, no aromatic amino acids or histidine), unique amino acid sequence, and a characteristic distribution of tetrahedral metal–thiolate complexes and metal–thiolate clusters.
What is a metallothionein then? The name for the protein was coined at a time when the sulfur binding of metal ions in biology was a curiosity. Looking at the heterogeneity of proteins and peptides classified as MTs, the use of the word “MT” does not mean much beyond the statement inherent in the name that it defines a protein that binds metals with the sulfur donor atoms from cysteines. It describes a property that is not unique to MT and is therefore trite and trivial. Taking it ad absurdum, the definition would apply to some zinc finger type of proteins or even iron–sulfur proteins. Nevertheless, the word MT is constantly used to indicate function. It is a most unsatisfactory situation, as the word cannot be used both generically and specifically at the same time as discussed here for the specific meaning related to the structures and functions of mammalian MTs. Even for mammalian MTs many additional investigations are required to explain sequence variations in terms of metal usage and metabolism in each species. The consequence of these uncertainties is that MTs are being called multifunctional and multipurpose proteins. In the worst-case scenario, it can become a pretense for investigations under an assumption of function. A clear understanding of what the word MT means hinders progress and gives a license for unfounded conclusions. For each MT, a possible function needs to be investigated in the biological context of zinc and copper metabolism, redox metabolism, and the environmental exposure of the organism to essential and nonessential elements. Chemical studies alone are not sufficient. To overcome this conundrum, the word MT should be understood as a generic term only when it is applied to proteins that do not bear any relationship to mammalian MTs, and further conclusions about function need a combination of chemical and biological investigations and not an a priori assumption of a perceived classification as an MT. To paraphrase the problem of epistemology with a quote from Voltaire (François-Marie Arouet): "Everything that is not clear is not right." Unclear terminology does not advance science.

6.2. The Dilemma of MT’s Biological Functions

In chemistry, the concept of “function” is relatively straightforward. Functional groups determine reactivity. Pure chemistry gains functions in applications, for example, the combination of Zn(s)/Zn(II) and Cus/Cu(II) in the Daniell cell─a battery. In biology, functions are restricted to the chemistry that is possible in the biological milieu─the very foundation of biological inorganic chemistry as opposed to the entire field of bioinorganic chemistry, which also addresses the chemistry of biomolecules regardless of physiological significance. As functional groups, the thiols of the cysteines in MT are involved in metal binding and in the reactivity with oxidants or electrophiles, providing a chemical basis for the functional potential of MT. While the interaction of the zinc and copper electrodes and their respective electrolytes defines a battery, one molecule alone does not define biological function but rather its interaction(s) with other biomolecules.
We believe that a general discussion of the meaning of function in biology will be helpful in order to recognize how chemical structure relates to chemical reactivity in biology. Specifically, we follow a history of philosophical and pragmatic discussions in the context of gene ontology, where the concept of biological function is “not as straightforward as it might seem” because function is divided into causal role function and selected effect function. (447) A causal role is connected to how a part has a role in a system, that is, an “activity” that is part of a more complex “mechanism”. A selected effect gives the ultimate answer why a part exists in the first place. It is linked to teleology: Why was the part selected in evolution? The necessity to pursue investigations about the origin of MTs has been pointed out. (448) An answer to this question imposes constraints on the myriad of causal functions and distinguishes a proper function from an accidental or adventitious function, an issue that is critically important when analyzing the many functions proposed for MTs. Molecular biologists conceive of function as specific, coordinated activities that serve a purpose. But it remains a candidate function until the evolved function has been related to past survival and reproduction. “Function” has two distinct aspects. The first is how the protein works at the mechanistic, molecular level, and the second is the functional role at the mechanistic, cellular level. It is this second aspect that purely chemical studies of MTs fail to address, as it requires relating the molecular properties to cellular biology. These definitions and the hierarchy of functions from molecules to cells to tissues to organisms needs to inform and direct any discussion about the functions of MTs. Working toward a molecular mechanism can be seen as a reductionist approach. But a holistic approach in the context of the operation of a cell is needed as well.
Gene ontology (GO) terms are categorized under Molecular Function, Cellular Component, Biological Process (www.geneontology.org). Gene ontology states for the molecular function of human MT2a (www.genecards.org): protein, drug, Zn(II) ion, metal ion binding. It is listing properties, not necessarily biological functions. The entry for biological process is left blank for the protein. Attempts to define the overall function of MT based on its chemical properties and existing data largely failed because the biological context was not considered. Likewise, defining functions solely on its biology also failed because the chemical properties were not considered.
The inducers of gene expression of MTs are seen as making “metallothionein” when in fact it is “thionein” that is synthesized, perhaps the central misrepresentation leading to confusion. What the induced protein does and where it gets its metal from and whether or not it is induced for additional functions, such as reducing another substrate, is largely left unanswered. One issue persisting in the field is that there is no name for the uniquely biological principle that MT embodies. Without a name, the biological principle is difficult to fathom and to communicate. One could coin a new name, but we will refrain from doing so, because the term “buffer” with the appropriate qualifications, we believe, may suffice for the time being. There are also semantic issues because the name metallothionein is used for many types of other proteins that are sulfur- and metal-rich and with reference to a mammalian protein with a given stoichiometry of seven divalent metal ions. After more than 60 years, it is time to get at the gist of the matter and discuss what the words used to describe or indicate functions of MTs mean, that is, buffer, scavenger, storage/reservoir, homeostatic protein, chaperone, detoxification. Chaperone means a specific protein to deliver Zn(II) or Cu(I) by direct protein–protein interactions. The word is fitting for genuine copper metallochaperones. For Zn(II), the situation is different, as there is free Zn(II) and no bona fide metallochaperone. A dozen MTs are unlikely to serve a role as specific chaperones to provide Zn(II) to ∼3000 proteins by direct interactions. For Cu(I), the term scavenger seems to be appropriate, as MTs sequester Cu(I) when the concentrations are high. For Zn(II), the opposite proposition was made, namely, that MT is a scavenger of Zn(II) at low Zn(II) concentrations, (393) serving as a temporary storage and reservoir. For Zn(II), we prefer the term buffer, as the protein is not saturated and the “MT/T” ratio is related to the free Zn(II) concentrations like a metal buffer, albeit with the notion that there are interactions with other biomolecules and mechanism for accepting and donating metal ions in specific processes. When discussing MTs as metal buffers, the first issue again is a semantic one, namely, naming the protein only metallothionein and not naming it after the ligand, that is, thionein. A metal buffer exists in a metal-free and a metal-bound form. Hence, the buffer would be MT/T. Neither term specifies the degree of saturation with metal ions, which is needed to define the different pKd values for buffering. In chemistry, applications of metal buffers are relatively rare, but in biology metal buffering is essential. Metals need to be homeostatically controlled in order not to interfere with other biological functions of the cell, AND each metal ion needs to be controlled relative to the other metal ions. The circumstances of chemical buffering are specific for the biological context. The biological metal buffer is dynamic and changes its buffering capacity through changes in gene expression of the protein AND its ratio to effect changes in pM (−log[Mn+]free). The protein does not buffer at one constant pM, and it remains to be shown whether the different MTs differ in their buffering properties. Buffering by MTs is affected by changes of pH and operates on specific biological time scales. And, importantly, MT is not inert─a property for which a chemical buffer is usually chosen─because it interacts with the system, being modified by reactions that change metal composition, thiols, and perhaps other groups. Last but not least, its metal buffering is linked to redox buffering. Thus, when MT is widely discussed as an antioxidant and as a cytoprotective agent without reference to the linked properties as a metal buffer, it must be understood that the reactivity of the thiol(ate)s with oxidants and electrophiles affects metal equilibria and that the ensuing changes of metal ion concentrations can have either a pro-antioxidant or a pro-oxidant effect. A role in detoxification after intoxication also relates to buffering essential metals and functions in redox biology. A longstanding issue is whether or not mammals possess a detoxification system for Cd(II). The late professor H.A.O. Hill asked a critical question in a discussion section during one of the earlier International Metallothionein Symposia: If it is detoxification, why do we not get rid of it? Subsequent research weakened the argument that a primary function of MT is the detoxification of Cd(II). Cd(II) is not an inducer of MTF-1 controlled gene transcription but, rather, displaces Zn(II) in MT, which is the actual inducer. (391) Cd(II) is not safely sequestered in MT, as the exchange rates are higher than those of Zn(II) and it can dissociate by a reaction of MT with NO. (125) The argument in favor of a role in detoxification is based on higher amounts of MT protecting against cadmium toxicity, MT1/MT2 null mice being hypersensitive to Cd(II), and genetic polymorphisms of MTs modulating cadmium toxicity. (449) All these observations indicate that MT has a role when exposed to Cd(II), but they do not refute a primary function in zinc and copper metabolism as supported by the complexity of the system, its regulation, and its integration into cellular signaling networks. The question remains what the capacity of the MT system is to accumulate Cd(II) before interfering with its functions in zinc or copper metabolism.
Chemical approaches to characterize the structure of the protein have advanced, but ultimately the function of the protein is a biological issue, which will not be resolved by purely chemical approaches that do not consider the biological context of the expression of MT genes controlled by so many factors. Combining knowledge from different scientific disciplines provides untapped opportunities for determining functions. Defining a structure of MT has been problematic, as it depends on the type of metal, metal load, and redox conditions, resulting in some purely chemical observations of a biological macromolecule with limited relevance to biology. Comprehensive attempts to combine the information about the gene expression of MTs and the cellular redistribution of metal ions or changes in redox potential are largely missing. Primarily, we need to look at the reason for the induction of T as a metal acceptor to restrict metal ions and, secondarily, at a donor of metal ions from the produced MT when metal ions are needed. The time scales are different: gene induction is slow; protein chemistry is fast.
With Zn(II) and Cu(I) as cargo, MT functions are inevitably linked to the numerous global functions of Zn(II) and the more specific functions of Cu(I). Compared to zinc MT, the literature is even less clear about the functions of copper MT. (450) Further investigations are necessary to clarify whether MT is involved in the storage, scavenging, or buffering of Cu(I). Either donating or accepting Zn(II) can activate or inhibit biological processes. An understanding is necessary how Zn(II) is redistributed to where it is required for function. Both actions have been described: MT delivering Zn(II) and T removing Zn(II). In the acquisition of Zn(II), there can be a huge number of protein–protein interactions, as T is an intrinsically disordered protein─maybe exactly what is required, namely, having a vast conformational space for serving as a versatile and multidentate chelating agent. Having one molecule performing the function of an otherwise large number of single chelating agents would again be a uniquely biological activity. Likewise, one can argue that the metamorphic states of MT with different metal load and oxidation state provide a vast conformational space for the recognition of Zn(II) acceptor proteins and for interactions with LMW ligands.
The discussion of MT function provides unparalleled lessons for protein science and biology, in general, and for control of metal metabolism, in particular. Why have chemical, biochemical, and genetic approaches not answered the question of MTs’ primary functions, and why has the situation persisted for 60 years? One reason is the limitations of each of the approaches and how the merger of them has been attempted with an insufficient integration of the different disciplines. Cysteine coordination chemistry became prominent in biochemistry only with the discovery of zinc finger and related proteins. Until that time MTs, with few exceptions, were rather unique with their sulfur coordination environments. Despite all the work, the redox connection was not considered as a major aspect of MT’s function in zinc metabolism until 40 years after its discovery, and all investigations were performed under the assumption of very tight binding of the metal ions, which was not challenged until 50 years after the discovery of MT. Tight binding suggested a role in storage, which was already inconsistent with the kinetic lability observed in metal transfer reactions. Also, on the basis of the tight binding of metal ions, a structural model based on seven divalent metal ions bound was considered THE metallothionein, and the structures of the metal/thiolate cluster, which turn out to be just one state of the metal coordination in the protein, preoccupied the imagination of many investigators.
Reasons for the failure to agree on the functions of MT thus are at least twofold, operational and a lack of considering the biological context. At least two reasons are operational. First, there is no method for probing the state of MT in the cell. Accordingly, the protein was isolated, and since heterogeneous either intrinsically or due to the procedures for isolation, metal ions were removed and the proteins were reconstituted with a chosen equivalent of metal ions. Structural investigations then led to one 3D structure. Second, determinations of metal affinities relied on methods that did not have a sufficient resolution to reveal differences. Thus, a critical insight later was that the metals do not bind with the same affinities and that there are weaker binding sites. Thus, the affinities of some sites are not stronger than those for other zinc or copper proteins, assigning MT a more dynamic function in metal metabolism. More robust methods for measuring affinities together with experiments showing the presence of T challenged the dogma that it is a protein saturated with seven divalent metal ions. As to the biological context, retrospectively, the failure to define a function has yet other reasons. The biological information on how metal ions are controlled was inadequate or even nonexistent, and therefore it was not understood how the chemical properties of MT could play a role in biology. But equally important, coming to the overarching question of the role in zinc and copper metabolism, insight into the quantitative aspects of how cellular Zn(II) and Cu(I) ions need to be buffered and are controlled and how these metal ions can become regulatory and signaling ions did not exist. Hence, it was not clear why a molecule such as MT would be needed. Therefore, its chemical activities could not be converted to biological activities, and the chemical properties did not match a biological need for them. Accordingly, no specific functional tests could have been applied to investigate the phenotype of the genetically modified animals, in which MT1/2 were ablated. Retrospectively, one can understand the confusion about role, function, and purpose in the many discussions about functions that are the result of its primary functions.
Some scientists had the vision and steadfastness to emphasize the importance of MTs for zinc metabolism. “Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms.” (emphasis added). (451) Or with regard to the role of MT in intermolecular metal transfer: “It may represent the mechanistic basis for the chaperoning function that MT was suggested to have in channeling and regulating the flow of essential metals, in particular of zinc, to and from their many sites of action”. (36) MT is the “keystone that has been endowed with all the necessary attributes (emphasis added) to safeguard and regulate zinc homeostasis.” (452) Such and similar statements have been made repeatedly, and they were in essence correct. However, the characterization of MT was incomplete to put the function on a firm molecular basis. Only now it seems possible to relate the uniquely biological principles of MTs described here to a central role in the control of the homeostasis of two essential metal ions. It provides a new and different perspective how the molecular functions and activities of MTs translate into biological activities: MT has arolein zinc, copper, and redox metabolism with afunctionof buffering these metals ions for thepurposeof controlling their availability for control of biological processes.
Since a primary function had not been assigned to MT unambiguously, some investigators called it a multifunctional or multipurpose protein because of the pleiotropic effects observed. It blurred the focus on its mechanism of action and perpetuated a conundrum, in which functions, roles, purposes, properties, and chemical and biological activities became mixed up. Without a clear understanding of what MT does work remains phenomenological, and past work begs for an interpretation.
Starting with chemistry, the major functional group in the protein is the bifunctional sulfhydryl group: it binds metal ions, and it is redox-active. The complexity increases when the MT protein is considered. Two metal clusters in two domains are important for its function and behave differently. At least the three-metal cluster is not unique for MTs, though. The properties indicate two additional functions in the molecule─one for “storage” of metal ions in the C-terminal domain and one for “delivery” of metal ions in the N-terminal domain. The properties of the clusters are the sum of the different affinities for metal ions─and likely different redox activities─of the 20 cysteines. Multifunctionality starts with these chemical activities. We then need to ask how these chemical properties become biological properties. Here, an understanding of the biological context is necessary, as biology controls metal concentrations and redox potentials and, therefore, limits the number of molecular states in which MT can exist. In other words, not all chemical properties are necessarily biological properties. Chemistry focused on the structure to solve the issue of function, but even structure underlies the constraints of biology. Biology focused on function with insufficient reference to structure and reactivity. Moving on to biology, several purely biological factors need to be added to the chemical properties, in particular, the biological systems that regulate expression of MTs and control the proteins. In biology, it is not just MT but the whole system and its control under different conditions. Do the different MT proteins have different functions or purposes or is there redundancy such that they have the same functions or purposes under different circumstances? The multitude of inducers of gene expression of MTs could be the basis for different purposes of MT but not necessarily different functions. For this aspect of the MT system, a plausibility argument was put forward: “proteins whose synthesis is subject to such complex and fine regulation as MTs are likely to occupy a central role in cellular metabolism”. (66) This role was then related to normal metabolism and to conditions of stress. However, the function of MTs under normal physiological conditions was considered elusive based on genetic experiments that did not provide evidence for an essential function in the reproduction, development, and maintenance of mice. They epitomize the difficulties of functional genomics. (453) The interpretation of the knockout (ko) experiments is based on an operational failure at the time, namely, a limited examination of the phenotype. The ko mice, which were engineered by two independent groups and in which only MT1 and MT2 were ablated, were examined for cadmium toxicity, which was enhanced. (64,64) Originally, the mice were not subjected to meaningful tests that would have examined specific roles of Zn(II). Later, these mice showed deficiencies under many experimental challenges such as being sensitive to high and low Zn(II). Crosses of a murine model of Menkes disease (ATP7a mutation) and Mt1/Mt2 ko mice support a physiological “function” of MT in ameliorating copper toxicity through Cu(I) sequestration. (454) MT is also involved in the Cu(I)-dependent trafficking of ATP7a from the trans Golgi to the plasma membrane. (455) These findings indeed show a biological function in copper metabolism. There is an important implication, namely, that the Zn(II) induction of MT restricts the Cu(I) availability, thus demonstrating a role of zinc in copper metabolism. Changes in the expression of MT have a multitude of effects, and the lack of a phenotype under laboratory conditions is apparent only.
The functioning of MT in zinc and copper metabolism obviously affects the many functions that these metal ions have. It appears that the roles of MTs in zinc and copper metabolism for various purposes can be reduced to a definite primary function, that is, its molecular mechanism. To examine this hypothesis, we discuss the relationship of its primary function in a hierarchy of roles and purposes at four levels (Table 3). The number of levels could be increased, of course, but we apply Ockham’s razor.
Table 3. Levels of Biological “Functions” of MTs
Primary LevelSecondary LevelTertiary LevelQuaternary Level
Function & PurposeFunction & PurposeRoleRole
Metal metabolismRedox metabolismProtection, natural compounds, and radiationProtection, man-made compounds, and radiation
Metal donation and chelation  Drug resistance cytotoxic agents, based on thiol/thiolate reactivity only
Zinc “buffer”? Redox control in Zn(II) and Cu(I)/Cu(II) metabolismRedox “buffer”? Zn(II) control in redox metabolismScavenger: Cd(II), other metal ions/metalloids, free radicals 
Specifically, we posit:
-Primary (physiological) function. It is based on the chemical activity of a biologically controlled and highly dynamic metal buffer that controls the availability and biological activitites of Zn(II) and Cu(I) ions. MT ascertains the right balance between these two most competitive metal ions. As a biological metal buffer, MT has at least three additional biological features. Its molecular state depends on the redox state, it is regulated by many factors and interactions, and it is translocated to where it functions inside and outside cells, making it highly dynamic in biological time and space. The biological terms chaperone, storage, transport, and receptor binding are all additional roles but remain linked to its primary function. Whether it is a genuine metallochaperone for Zn(II) or Cu(I) will depend on the demonstration of specific mechanisms of metal loading and unloading in interactions with specific targets. There may be some specialization in terms of roles for some MTs such as MT3 and MT4, perhaps being more involved in copper metabolism. With regard to purpose, we see an involvement in cell proliferation, differentiation, and apoptosis under normal conditions but also under specific types of stress such as in the acute phase response and viral defense. Under all these conditions the characteristics of the buffer changes so that the availability of Zn(II) ions is increased or decreased. The functions of MTs seem to become essential under conditions of Zn(II) deficiency and overload. It reflects exactly the behavior of a buffer, where larger fluctuations are observed if the buffer capacity is diminished.
-Secondary (physiological) functions. There is the possibility that MT/T is a redox buffer only, and one that is metal-controlled, but the evidence is much weaker. Many studies that consider its antioxidant role do not address the targets and functions of the dissociated metal ions. Unless T has redox functions in the absence of metal ions, there is no need to postulate a secondary function.
-Tertiary functions. MT provides some protection against naturally occurring toxins such as toxic metal ions under conditions of exposure, for example, Cd(II), and overproduction of reactive species/radicals in oxidative distress and physical and psychological stress. It can have two consequences, namely, that the Zn(II) made available is needed for cytoprotection, clearly a purpose, or that the agents interfere with the functions of Zn(II) or Cu(I) when they overwhelm the buffering capacity. Again, these tertiary functions are linked to the primary (or secondary) function. Protection against radiation is also a tertiary function, which is linked to protective functions of Zn(II) and its involvement in cellular and molecular repair processes. These functions clearly have a role in pathology and toxicology.
-Quaternary functions. It includes the role in quenching natural and man-made chemicals that are sulfhydryl-reactive, hence lowering their toxicity or leading to resistance in the case of drugs. It is not a purpose but a role.
For the field to advance, a clear understanding is necessary of what defines a protein as a metallothionein based on a molecular mechanism and on biological functions. How these attributes are modified in other organisms and correspond to modifications in zinc and copper metabolism remains to be defined.

6.3. Challenges for Future Research

A major impediment for progress is that investigations have not addressed─notwithstanding the difficulties in analytical chemistry─the metal occupancy of MTs in vivo and correlated it with specific parameters of zinc, copper, and redox metabolism. This aim should be pursued with a speciation of the different MT proteins. Such analytics will be very challenging, as they would need to include the oxidation state of the cysteinyl sulfur in the proteins and any other modifications, such as phosphorylation, that may have occurred. Once such high-resolution and sensitive methods have been developed and verified, the changes over time and in cellular space need to be addressed to understand the dynamics of the protein. Since the type of metal ion and occupancy determine the protein’s structure, such investigations are crucial when both direct and metal-mediated transient or stable protein–protein interactions are addressed. There was no incentive to look at mixed-metal compositions because it was thought to be either Zn(II) or Cu(I) but not both. Insights about the intricacies of zinc and copper metabolism in terms of their regulation, interaction, and regulatory functions such as dynamic pools involved in signaling is still developing. There was also no incentive to look at properties of MT1 isoforms, because the name “iso” is often understood to indicate that they are the same proteins. The differential gene regulation and changes of up to 10 amino acid residues in the protein structure of MT1 “isoforms” certainly are expected to endow these proteins with different properties. Thus,
1.

What is the metal composition and metal load in vivo for all the different MT proteins present in a tissue? It requires new techniques that address proteins, redox state, metal saturation, and modifications. For cell biological investigations there is the need for specific antibodies to the different gene products, although the availability of such antibodies would not yet solve the analytical challenge of addressing the state of the protein. Presently, most investigations focus on mRNA levels only, for which the correlation with the corresponding proteins is uncertain.

2.

What are the structures of the proteins with different metal occupancies, including mixed-metal species and their physical properties, including different properties of the individual MT proteins?

3.

Extracellular MT, secretion and uptake pathways, including receptors, and mechanisms of intracellular translocation need to be characterized with the possibility that these processes are accompanied by changes in metalation.

4.

What are the dynamic changes of specific MTs in specific biological events such as proliferation, differentiation, and apoptosis and under conditions of various types of stress?

5.

Investigations should switch from a mere focus on toxic metals to how toxic metals affect zinc and copper metabolism.

6.

The interaction of MTs with other proteins needs to be characterized structurally, including investigations that address whether the metamorphic or multimorphic nature of MTs is a factor in the selection of binding partners.

7.

Once the analytical chemistry for determining specific MTs, their metal load, redox state, ligand interactions, and modifications is available, the dynamics of the proteins in the cell needs to be addressed. It is the most challenging part─and a gargantuan task─as it requires a subcellular resolution of the coordination dynamics and reactions of MTs and a methodology for simultaneously resolving metal ion fluctuations and signals spatiotemporally.

6.4. Conclusions

One of the mysteries in biochemistry for over 60 years has been the function of the gene family of MTs. The quest is sometimes a tale of red herrings and wild goose chases and illustrates the tantalizing difficulties of defining biological functions of proteins and metal ions. The pleiotropic effects of MTs observed with their gene expression under the control of so many pathways would seem to indicate a fundamentally important system. With reference to the immense structural variability of MTs in other organisms, it should be obvious why it did not make sense to treat all MTs in a review that tries to link structures and functions. The issues discussed here need to be the starting point and guiding principle for investigations of other proteins that are referred to as MTs. It is a huge task because one will need to understand zinc and copper metabolism in each organism and relate it to the variations in MT structures. An impediment for a wider understanding of function is the use of a generic name for many different types of MT proteins and the lack of a word that expresses the uniquely biological activities of MTs in metal and redox metabolism. A consensus about the relationship between the chemistry and biology of MTs is necessary to make progress in this important aspect of metallobiochemistry, which is at the center of how Zn(II) and Cu(I)─and Cu(II)─are controlled and what the consequences are for health if such control is perturbed. Biology needs a definition of molecular mechanisms of MTs that lead to biological functions, roles, and purposes rather than just an operational definition of these proteins being cysteine-rich and binding metal ions. After an exegesis of the literature, this article summarized and critically evaluated advances in the chemistry of MTs to provide interpretative guidance for ontology. Pivotal was the realization that MT is a highly dynamic multimorphic protein with coordination and protein dynamics as reflected in different metal loads, oxidation states, coordination environments, and protein conformations. It binds several Zn(II) and Cu(I) ions with different affinities dependent on the biological context. It will also bind nonessential, thiophilic metal ions, the binding of which interferes with its functions. The framework for the exclusive use of sulfur coordination chemistry in mammalian MTs is the redox chemistry, high discrimination afforded for metal ions, and the pH dependence of affinity for metal ions and reactivity. While this article focused primarily on affinities, the area of kinetics remains to be fully explored. The exquisite control of cellular zinc and copper homeostasis and the dynamic balance of these ions, buffering Zn(II) and Cu(I) to very low concentrations and yet serving as signaling ions in cellular regulation, suggests a role of MT as a regulated, uniquely biological, interacting buffer for these highly competitive metal ions. Such an interpretation was not possible until recently because critical insights about the molecular and cellular regulation of Zn(II) and Cu(I) were lacking. The establishment of a 3D structure was a landmark in MT research, but, unfortunately, it gave the wrong impression that a fully metal-loaded form is THE state of the protein. A myriad of publications are based on assumptions about both structure and function, describe phenomena such as changes in the proteins’ gene expression, and discuss no clear molecular relationship of the protein to either redox or metal metabolism. The theme in this article is how chemical activity informs biological roles and how biology informs chemistry by limiting what is possible in the biological milieu. It serves as a reminder that the overall problem is one of biology and not chemistry, though both are necessary, and that function(s) need(s) to be interpreted with a consideration of the availability of metal ions and the redox state in the biological system. MT affords some protection against Cd(II) and other toxic metal ions, metalloids, and chemicals, but it is a matter of natural or man-made exposure and cannot be the primary function of mammalian MTs; one must consider the extent to which these scavenging functions interfere with the primary function of MTs in metal metabolism. One would hope that refocusing on the essential questions and bridging the disjunct scientific communities that focus on Zn(II), Cu(I), toxic metal ions, or redox biology will lead to performing the right experiments to test specific hypotheses instead of continuing phenomenological research. The results of such research will have a tremendous impact on many disciplines and for the health and disease of living systems. Our attempt of a synthesis of the present knowledge, of course, does not exclude the possibility that yet additional functions, roles, and purposes of MTs may be discovered in the future.

Author Information

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  • Notes
    The authors declare no competing financial interest.

Biographies

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Artur Krężel received his diploma in chemistry (master of science, 2000) and a PhD degree in bioinorganic chemistry (2004) on the topic of glutathione coordination chemistry from the University of Wrocław, Poland, under the supervision of Prof. Wojciech Bal. During his postdoctoral training (2004–2007) at the University of Texas Medical Branch in Galveston, he worked with Prof. Wolfgang Maret on mechanisms of cellular zinc homeostasis, which bore fruit in the discovery of metallothioneins as physiological zinc ion buffers. He then accepted a position as an assistant professor at the Faculty of Biotechnology of his alma mater. In 2011, he received a D.Sc. degree from his university. Currently, he is a full professor and head of the Department of Chemical Biology. His research work concentrates on several areas at the interfaces of inorganic biochemistry, biophysics, and chemical biology, in particular, an understanding of the molecular bases of zinc and copper metabolism. Examples of this endeavor are structure–function relationships, the stability of metalloproteins, protein folding and thermodynamics, and the development of new analytical methods, such as fluorescent probes for selective protein modifications and metal sensing.

Wolfgang Maret FRSC is the Professor of Metallomics in the Departments of Nutritional Sciences (research) and Biochemistry (teaching) at King’s College London, London, UK. He obtained his master of science (Dipl. Chem.) in chemistry and his PhD in Natural Sciences from Saarland University, Saarbrücken, Germany. His academic career includes postdoctoral research at The University of Chicago (Department of Biophysics & Theoretical Biology), assistant professor in the Center for Biochemical and Biophysical Sciences and Medicine at Harvard Medical School with an additional teaching appointment at The Bouvé College of Pharmacy, Northeastern University, Boston, MA, and associate professor at the University of Texas Medical Branch in Galveston, Texas (Departments of Preventive Medicine & Community Health and Anesthesiology). His research interests began with the catalytic mechanisms of metalloenzymes as investigated with spectroscopic and kinetic methods and continued with the molecular and cellular mechanisms of how metal ions control protein structure and function and how proteins control nutritionally essential elements and mitigate the effects of nonessential and toxic elements. His interest in the role of metal and redox biology in health and disease extends to the etiology of liver disease, diabetes, traumatic brain injury, skin disease, and arthritis. From 2012 to 2014, he served as the president of the International Society for Zinc Biology and from 2012 to 2016 as chair of the editorial board of the journal Metallomics of the Royal Society of Chemistry.

Acknowledgments

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We recognize the centennial of the birthday of Bert L. Vallee and the passing of Milan Vašák in 2019. Research in A.K.’s laboratory was supported by the National Science Center of Poland under Opus Grant No. 2018/31/B/NZ1/00567. Publication of this article was supported financially by the Excellence Initiative - Research University (IDUB) program for the University of Wrocław. W.M. thanks his colleagues at King’s College London, Professors C. Hogstrand and S. Sturzenbaum, Professor N. Bury, University of Suffolk, and Professor P. Kille, Cardiff University, for engaging discussions on all matters of metallothioneinology over the past decade.

Abbreviations

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MT

metallothionein

T

thionein

DTNB

5,5′-dithiobis(2-nitrobenzoic acid)

GSH

glutathione

GSSG

glutathione disulfide

References

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