The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase

Abstract

The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four.65 Znlabeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG. In vivo, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.The possible functional implications of the zinc cluster structure of metallothionein (MT) have been the subject of much speculation (1-3). In contrast with other zinc proteins, MT is remarkable in that it binds zinc with high thermodynamic stability [K d ϭ 1.4 ϫ 10 Ϫ13 M at pH 7.0 for human MT (4)] while exhibiting a kinetic lability that results in facile zinc exchange reactions (5). This unusual combination appears to be a characteristic property of the clusters and, likely, a critical element in their hypothetical function to ''[provide] zinc where and when needed and for whatever role'' (6). Thus, in MT the protein plays a role in the biological function of zinc, a paradigm quite different from that in most other zinc proteins where zinc plays a role in the biological function of the protein. The tight binding of zinc to MT raises questions of how it is released and whether or not the release is controlled. In this regard, we have identified glutathione disulfide (GSSG) as a cellular ligand that reacts with MT and mobilizes zinc, resulting in the suggestion that the zinc content of MT is linked to the redox state of glutathione in the cell in such a manner that zinc remains bound to MT as long as high thiol reducing power prevails and is released once the redox balance becomes more oxidizing (7). We here extend these findings by studying the role of glutathione-mediated zinc release in the presence of a zinc acceptor such as an apoenzyme. In particular, we show that only one of the seven zinc atoms i...

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“…Indeed, NO has been shown previously to trigger zinc release from metallothionein (Pearce et al, 2000), a metalloprotein that also releases zinc after DTDP oxidation (Jiang et al, 1998;Maret and Vallee, 1998). We also find evidence that the intracellular signaling cascades activated by DTDP appear to be similar to those observed by other researchers after exposure to extracellular zinc.…”

Catalytic selenols couple the redox cycles of metallothionein and glutathione

“…3, trace b). If the fact that one zinc ion is easily extracted by PAR from MT is taken into account [3], the percentage of regenerated MT is close to 100%.…”

“…Zinc release is elicited by oxidation of the cysteine ligands, a process in which the glutathione system has been implicated [3]. Glutathione is the most abundant cellular thiol, with concentrations of 0.1±10 mm [25].…”

“…the reduction of thionin by GSH. The GSH/GSSG couple was chosen because it modulates zinc transfer from MT and is a major determinant of the cellular thiol/disulfide state [3]. Selenocystamine was chosen as a catalyst because it is a precursor of selenocysteamine, a selenol (R-SeH) akin to selenocysteine, the catalytic residue in selenoenzymes, some of which participate in sulfur redox reactions [12].…”

“…The low redox potential of the clusters allows oxidation by a number of mild cellular oxidants, among which disulfides such as glutathione disulfide (GSSG) and compounds with selenium in its lower oxidation states feature prominently. In vitro experiments have shown that the GSH/GSSG redox couple modulates zinc transfer from MT to zinc-depleted enzymes [3]. Based on this, it has been suggested that changes in the redox state serve as a driving force and signal for zinc distribution from its cellular reservoir in MT when zinc is needed.…”