ESTABLISHMENT OF THE METAL-TO-CYSTEINE CONNECTIVITIES IN SILVER-SUBSTITUTED YEAST METALLOTHIONEIN

To elucidate the three-dimensional solution structure of yeast copper-metallothionein (MT) from Saccharomyces cerevisiae, silver-substituted yeast metallothionein has been prepared as an isomorphic, NMR active, metal ion derivative. Using Ag-MT, H-1-Ag-109 heteronuclear multiple quantum coherence transfer (HMQC) experiments have been performed to identify the individual Ag-109 resonances and their associated cysteine ligands, respectively. Specific factors associated with the optimized execution of the H-1-Ag-109 HMQC experiments are identified and discussed. Analysis of the HMQC data has established the specific connectivities between 10 of the 12 cysteine residues and seven bound Ag-109(I) metal ions. The data confirm the exclusive involvement of cysteine thiolates in metal coordination and indicate that a minimum of eight cysteines are involved as bridging, shared ligands. Additionally, the present data suggest the existence of a mixed coordination number (2 and 3) for the seven bound Ag(I) ions.