Sulfide ions as modulators of metal–thiolate cluster size in a plant metallothionein

Tamara Huber,Eva Freisinger

doi:10.1039/c3dt32438a

Abstract

Metallothioneins are small cysteine-rich proteins coordinating various transition metal ions preferably with the electron configuration d(10). They are ubiquitously present in all phyla, and next to phytochelatins they represent a successful molecular concept for high-capacity metal ion binding. Recent studies showed the incorporation of sulfide ions into the metal-thiolate cluster of metallothionein 2 from the plant Cicer arietinum (cicMT2) increasing the cadmium binding capacity and stability of the cluster. In the present work, the sulfide-induced structural changes accompanying the cluster formation and the sulfide-modulated increase in cluster size are analyzed in detail with a variety of analytical and spectroscopic techniques. Evaluation of the mechanism of sulfide containing Cd(II)-thiolate cluster formation in cicMT2 reveals a strong dependence on the sequence of metal and sulfide additions for successful sulfide incorporation. To probe the general ability of metallothioneins to form sulfide containing larger metal-thiolate clusters, analogous experiments were performed with a mammalian metallothionein. The observation that the cadmium binding ability of rabbit liver MT2A was only slightly increased led to the development of a hypothesis in which the long cysteine-free linker regions present in certain plant metallothioneins may contribute to the accommodation of the respective larger cluster assemblies.

Highlights

Metallothioneins (MTs) comprise a diverse class of metal binding proteins and their occurrence is reported throughout the animal kingdom, in plants, several eukaryotic microorganisms, as well as in some prokaryotes.[1,2,3] MTs are low molecular mass proteins (
The dithiobis(2nitro-benzoic acid) (DTNB) assay indicates that within the error range of the method all Cys residues of the protein are in the reduced state, which was confirmed by Electronic supplementary information (ESI)-MS at acidic pH (ESI†)
Building up on our previous results that the size of the CdII– thiolate cluster from the plant MT cicMT2 can be increased in the presence of sulfide ions resulting in a monomeric protein form with the average composition Cd9S7cicMT2,30 we investigated the increase in cluster size for the ZnII-form, the ability of cicMT2 to form and host even larger cluster structures when more sulfide ions are supplied, and tried to shed light on a possible mechanism of CdII/S-thiolate cluster formation

Summary

Introduction

Metallothioneins (MTs) comprise a diverse class of metal binding proteins and their occurrence is reported throughout the animal kingdom, in plants, several eukaryotic microorganisms, as well as in some prokaryotes.[1,2,3] MTs are low molecular mass proteins (

Methods

Results

Conclusion