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Wolfgang Maret received his diploma in chemistry (1977) and his Ph.D. in natural sciences (1980) from the Saarland University, Saarbruecken, Germany. The mentor for his Ph.D. thesis on metal substitution in liver alcohol dehydrogenase was Prof. Michael Zeppezauer. During his postdoctoral years in Prof. Marvin W. Makinen's laboratory at the University of Chicago (1980-1982), he continued his education in mechanistic enzymology and spectroscopy. He joined Prof. Bert L. Vallee's Center for Biochemical and Biophysical Sciences and Medicine at Harvard Medical School as an assistant professor in 1986 and began to work on the chemical and biochemical mechanisms of cellular zinc homeostasis. Since 2003, Dr. Maret has been an associate professor in the Division of Human Nutrition (Department of Preventive Medicine and Community Health) at the University of Texas Medical Branch in Galveston, TX. His work focuses on molecular mechanisms of cellular metal homeostasis, sulfur redox chemistry, structure and function of metalloenzymes, and functions of micronutrients in chronic and degenerative diseases.

The nutritional essentiality of zinc for the growth of living organisms had been recognized long before zinc biochemistry began with the discovery of zinc in carbonic anhydrase in 1939. Painstaking analytical work then demonstrated the presence of zinc as a catalytic and structural cofactor in a few hundred enzymes. In the 1980s, the field again gained momentum with the new principle of "zinc finger" proteins, in which zinc has structural functions in domains that interact with other biomolecules. Advances in structural biology and a rapid increase in the availability of gene/protein databases now made it possible to predict zinc-binding sites from metal-binding motifs detected in sequences. This procedure resulted in the definition of zinc proteomes and the remarkable estimate that the human genome encodes ∼3000 zinc proteins. More recent developments focus on the regulatory functions of zinc(II) ions in intra- and intercellular information transfer and have tantalizing implications for yet additional functions of zinc in signal transduction and cellular control. At least three dozen proteins homeostatically control the vesicular storage and subcellular distribution of zinc and the concentrations of zinc(II) ions. Novel principles emerge from quantitative investigations on how strongly zinc interacts with proteins and how it is buffered to control the remarkably low cellular and subcellular concentrations of free zinc(II) ions. It is fair to conclude that the impact of zinc for health and disease will be at least as far-reaching as that of iron.

Our knowledge of the molecular mechanisms of intracellular homeostatic control of zinc ions is now firmly grounded on experimental findings gleaned from the study of zinc proteomes and metallomes, zinc transporters, and insights from the use of computational approaches. A cell's repertoire of zinc homeostatic molecules includes cytosolic zinc-binding proteins, transporters localized to cytoplasmic and organellar membranes, and sensors of cytoplasmic free zinc ions. Under steady state conditions, a primary function of cytosolic zinc-binding proteins is to buffer the relatively large zinc content found in most cells to a cytosolic zinc(ii) ion concentration in the picomolar range. Under non-steady state conditions, zinc-binding proteins and transporters act in concert to modulate transient changes in cytosolic zinc ion concentration in a process that is called zinc muffling. For example, if a cell is challenged by an influx of zinc ions, muffling reactions will dampen the resulting rise in cytosolic zinc ion concentration and eventually restore the cytosolic zinc ion concentration to its original value by shuttling zinc ions into subcellular stores or by removing zinc ions from the cell. In addition, muffling reactions provide a potential means to control changes in cytosolic zinc ion concentrations for purposes of cell signalling in what would otherwise be considered a buffered environment not conducive for signalling. Such intracellular zinc ion signals are known to derive from redox modifications of zinc-thiolate coordination environments, release from subcellular zinc stores, and zinc ion influx via channels. Recently, it has been discovered that metallothionein binds its seven zinc ions with different affinities. This property makes metallothionein particularly well positioned to participate in zinc buffering and muffling reactions. In addition, it is well established that metallothionein is a source of zinc ions under conditions of redox signalling. We suggest that the biological functions of transient changes in cytosolic zinc ion concentrations (presumptive zinc signals) complement those of calcium ions in both spatial and temporal dimensions.

The solution and complexation chemistry of zinc ions is the basis for zinc biology. In living organisms, zinc is redox-inert and has only one valence state: Zn(II). Its coordination environment in proteins is limited by oxygen, nitrogen, and sulfur donors from the side chains of a few amino acids. In an estimated 10% of all human proteins, zinc has a catalytic or structural function and remains bound during the lifetime of the protein. However, in other proteins zinc ions bind reversibly with dissociation and association rates commensurate with the requirements in regulation, transport, transfer, sensing, signalling, and storage. In contrast to the extensive knowledge about zinc proteins, the coordination chemistry of the “mobile” zinc ions in these processes, i.e. when not bound to proteins, is virtually unexplored and the mechanisms of ligand exchange are poorly understood. Knowledge of the biological inorganic chemistry of zinc ions is essential for understanding its cellular biology and for designing complexes that deliver zinc to proteins and chelating agents that remove zinc from proteins, for detecting zinc ion species by qualitative and quantitative analysis, and for proper planning and execution of experiments involving zinc ions and nanoparticles such as zinc oxide (ZnO). In most investigations, reference is made to zinc or Zn2+ without full appreciation of how biological zinc ions are buffered and how the d-block cation Zn2+ differs from s-block cations such as Ca2+ with regard to significantly higher affinity for ligands, preference for the donor atoms of ligands, and coordination dynamics. Zinc needs to be tightly controlled. The interaction with low molecular weight ligands such as water and inorganic and organic anions is highly relevant to its biology but in contrast to its coordination in proteins has not been discussed in the biochemical literature. From the discussion in this article, it is becoming evident that zinc ion speciation is important in zinc biochemistry and for biological recognition as a variety of low molecular weight zinc complexes have already been implicated in biological processes, e.g. with ATP, glutathione, citrate, ethylenediaminedisuccinic acid, nicotianamine, or bacillithiol.

We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins. Thus, the present, an earlier, and accompanying manuscripts Metallothionein (MT) is a unique metalloprotein in which cysteine constitutes one-third of its amino acids and histidine and aromatic amino acids all are completely absent. All 20 cysteines bind seven zinc atoms such that each metal atom has a complement of four cysteine ligands. It is important to emphasize that the by now well known multinuclear cluster network, identified by both x-ray diffraction and NMR spectroscopy only less than a decade ago (1,2), shows the exceptional structural arrangement that was hitherto unknown in zinc/sulfur chemistry and has thus far been encountered solely in biological material such as MT. The zinc/cysteinyl interactions in the two clusters are of two different types: they are either bridging or terminal cysteine thiolates. In the ␤-domain cluster, three bridging and six terminal cysteine thiolates provide a coordination environment that is formally identical for each of the three zinc atoms. In the ␣-domain cluster, there are two different zinc sites; two of them have one terminal ligand and three bridging ligands, respectively, while the other two have two terminal and two bridging ligands.We have performed and advocated experiments to relate the structure of MT to its possible function(s) on the basis of the nature of zinc coordination in MT (3-5). We have suggested that the characteristics of the cluster motif might be the key to the mode of cellular zinc distribution (6). MT binds zinc with high thermodynamic stability [K d ϭ 1.4 ϫ 10 Ϫ13 M for human MT at pH 7.0 (7)] while simultaneously providing a mechanism for kinetic lability whereby zinc can be released at rates that are orders of magnitude greater than those observed for zinc metalloenzymes. Zinc, as well as cadmium, is known to undergo rapid inter-and intracluster exchange (8, 9).MT apparently binds zinc with higher affinity than do many other proteins. For MT to serve as a source for the distribution of zinc, mechanisms would be required that could regulate the binding and release of the metal. It has been shown that an interaction of MT with glutathione disulfide (GSSG) or other biological disulfides releases zinc (10, 11) and that the combination of reduced glutathione (GSH) and GSSG enhances transfer of zinc from MT to an apoenzyme (12). This has led us to infer that the reactivity and redox behavior of the sulfur ligands in the MT clusters are crucial for the dynamic state of zinc. We proposed that the zinc-sulfur cluster chemistry might be sensitive to changes of the cellular redox state and that oxidizing conditions induce the transfer of zinc from its binding sites in MT to those of lower affinity in other proteins.We here provide additional support for this concept and show that a number of compounds, including some of potential biological importance, can oxidize the ...

Metallothionein (MT), despite its high metal binding constant (K Zn ‫؍‬ 3.2 ؋ 10 13 M ؊1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability constants. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions-i.e., from the enzymes to thionein (the apoform of MT) and from MT to the apoenzymes. Agents that mediate or enhance zinc transfer have been identified that provide kinetic pathways in either direction. MT does not transfer all of its seven zinc atoms to an apoenzyme, but apparently contains at least one that is more prone to transfer than the others. Modification of thiol ligands in MT zinc clusters increases the total number of zinc ions released and, hence, the extent of transfer. Aside from disulfide reagents, we show that selenium compounds are potential cellular enhancers of zinc transfer from MT to apoenzymes. Zinc transfer from zinc enzymes to thionein, on the other hand, is mediated by zinc-chelating agents such as Tris buffer, citrate, or glutathione. Redox agents are asymmetrically involved in both directions of zinc transfer. For example, reduced glutathione mediates zinc transfer from enzymes to thionein, whereas glutathione disulfide oxidizes MT with enhanced release of zinc and transfer of zinc to apoenzymes. Therefore, the cellular redox state as well as the concentration of other biological chelating agents might well determine the direction of zinc transfer and ultimately affect zinc distribution.Metallothionein (MT) is a protein that has long been in search of a function. Its composition and structure (1-3) have clearly identified it as a biological metal-chelating agent, yet it does not resemble conventional proteins and its two distinct zincsulfur clusters are unlike any known inorganic zinc(II) complexes. Two properties of MT might reveal aspects of its cellular function(s). Its binding of zinc is exceptionally strong owing to the exclusive coordination of the metal with cysteine sulfur ligands [stability constant of Zn 7 MT-2 ϭ 3.2 ϫ 10 13 M Ϫ1 at pH 7.4 (4)]. Thionein (T), the apoform, is a potent zinc acceptor. On the other hand, the sulfur ligands are highly reactive and determine not only the binding of zinc to T but also its release from MT (5, 6), which then becomes a zinc donor. This raises questions as to the circumstances under which T removes zinc from proteins and͞or MT donates it to apoproteins. Zinc would be expected to be transferred from the protein with the lower stability constant to that with the higher one (reaction 1), which in most cases would determine unidirectional zinc flow from the zinc protein to T. Zn-Donor ϩ T^Zn-MT^T ϩ Zn-Acceptor [1]Indeed, T has been shown to block the action of zincdependent transcription factors-e.g., TFIIIA and Sp1-presumably by means of metal chelation (7,8). MT, on the other hand, transfers at least some of its complexed zinc ions to a number of apoproteins (9). The process by which the latter is ac...

Zinc/cysteine coordination environments in proteins are redox-active. Oxidation of the sulfur ligands mobilizes zinc, while reduction of the oxidized ligands enhances zinc binding, providing redox control over the availability of zinc ions. Some zinc proteins are redox sensors, in which zinc release is coupled to conformational changes that control varied functions such as enzymatic activity, binding interactions, and molecular chaperone activity. Whereas the released zinc ion in redox sensors has no known function, the redox signal is transduced to specific and sensitive zinc signals in redox transducers. Released zinc can bind to sites on other proteins and modulate signal transduction, generation of metabolic energy, mitochondrial function, and gene expression. The paradigm of such redox transducers is the zinc protein metallothionein, which, together with its apoprotein, thionein, functions at a central node in cellular signaling by redistributing cellular zinc, presiding over the availability of zinc, and interconverting redox and zinc signals. In this regard, the transduction of nitric oxide (NO) signals into zinc signals by metallothionein has received particular attention. It appears that redox-inert zinc has been chosen to control some aspects of cellular thiol/disulfide redox metabolism. Tight control of zinc is essential for redox homeostasis because both increases and decreases of cellular zinc elicit oxidative stress. Depending on its availability, zinc can be cytoprotective as a pro-antioxidant or cytotoxic as a pro-oxidant. Any condition with acute or chronic oxidative stress is expected to perturb zinc homeostasis.