The plant metallothionein 2 from Cicer arietinum forms a single metal-thiolate cluster

The plant metallothionein 2 from Cicer arietinum (chickpea; cicMT2) is a typical member of this subfamily and features two cysteine-rich regions containing eight and six cysteine residues, respectively, separated by a linker region 41 amino acids in length. This metallothionein thus differs significantly from the well-studied vertebrate forms. A synthetic gene encoding cicMT2 was designed, cloned into a suitable vector, and the protein was over-expressed in Escherichia coli. For the first time, an in-depth spectroscopic characterization of cicMT2 in the presence of divalent metal ions is performed showing a binding capacity for five Zn(II), Cd(II), or Co(II) ions and the typical features of metal-thiolate clusters. Based on proteolytic digestion experiments, the cluster arrangement formed by the divalent metal ions and the cysteine thiolate groups connects the amino-terminal with the carboxy-terminal cysteine-rich region. The cluster formation process, put into effect with the addition of the fourth metal ion to the apo protein, was investigated using the characteristic shift of absorption bands observed in the UV/Vis spectra upon titration with Co(II). The pH-dependent Zn(II)- and Cd(II)-thiolate cluster stability is one of the highest observed for plant MTs so far, but lower than that usually found in vertebrate metallothioneins. The dependence of the pH stability on the ionic strength of the solution is more pronounced for the Cd(II)- than for the Zn(II)-form of the protein.