Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4

The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases ( k _fast ≃ 5000 min ⁻¹ ·M ⁻¹ ; k _slow ≃ 200 min ⁻¹ ·M ⁻¹ , pH 8.6, 25°C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein ( k ≃ 800 min ⁻¹ ·M ⁻¹ , pH 8.0, 25°C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.