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We have previously established that ATP binds to mammalian metallothionein-2 (MT). The interaction between ATP and MT and the associated conformational change of the protein affect the sulfhydryl reactivity and zinc transfer potential of MT [Jiang, L.-J., Maret, W., and Vallee, B. L. (1998) The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U.S.A. 95, 9146-9149]. NMR spectroscopic investigations have now provided further evidence for the interaction. (35)Cl NMR spectroscopy has further identified chloride as an additional biological MT ligand, which can interfere with the interaction of ATP with MT. (1)H NMR/TOCSY spectra demonstrate that ATP binding affects the N- and C-terminal amino acids of the MT molecule. Scanning tunneling microscopy recorded images of single MT molecules in buffered solutions. Moreover, this technique demonstrates that the otherwise nearly linear MT molecule bends by about 20 degrees at its central hinge region between the domains in the presence of ATP. These results may bear on the development of mild obesity in MT null mice and the role of MT in the regulation of energy balance. The interaction suggests a mechanism for the cellular translocation, retention, and reactivity of the ATP*MT complex in the mitochondrial intermembrane space. Both MT and ATP are localized there, and MT and thionein alternately bind and release zinc, thereby affecting mitochondrial respiration.

We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT ( K d = 176 ± 33 μM, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol–disulfide interchange with Ellman’s reagent [5,5′-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di- or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two critical cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding constant and the cellular concentration of ATP indicate that in the cell MT could be essentially saturated with ATP at low concentrations of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.