CHARACTERIZATION AND SEQUENTIAL LOCALIZATION OF THE METAL-CLUSTERS IN SEA-URCHIN METALLOTHIONEIN

The mode of metal binding in sea urchin metallothionein (MT) was explored by electronic absorption, chiroptical, NMR, and mass spectroscopic methods. Recombinant sea urchin MT containing 7 equiv of the natural mixture of Cd isotopes was stripped of the metal by exposure to low pH and reconstituted with Cd-113 (>95% enriched). Comparison of the electronic spectroscopic and chiroptical features and the Cd-113 NMR spectra of the reconstituted material with those of the native recombinant material indicated that the reconstituted material had regained the native conformation. The shoulder at 250 nm in the electronic absorption spectrum, the biphasic circular dichroism profile centered at 250 nm, and the chemical shift positions (605-695 ppm) of the seven Cd-113 NMR resonances all strongly suggested that sea urchin MT like all other well characterized MTs contains clusters made up of tetrahedral Cd-thiolate units. The Cd-113 chemical shift correlation spectrum of the reconstituted protein proved the existence of such metal clusters and allowed the unambiguous assignment of some of the metal connectivities. Homonuclear decoupling experiments in which Cd resonances were selectively saturated indicated moreover a partitioning of the metal complement into two separate clusters containing three and four Cd ions. The same proposition was supported by the selective reduction of three Cd-113 resonances upon partial metal depletion following exposure of the protein to EDTA. Thus, the three-metal cluster is energetically less stable than the four-metal cluster. That the two clusters are separate entities was also demonstrated by the isolation of a protein fragment containing the four-metal cluster resulting from partial proteolysis of sea urchin MT by subtilisin in the presence of EDTA. Amino acid sequence and electrospray mass spectroscopic analysis identified this fragment as the N-terminal portion of the whole protein. This is in marked contrast to the known mammalian forms where the more stable four-metal cluster is associated with the C-terminal domain. One can conclude therefore that the sea urchin MT contains the same type of metal-thiolate clusters as those found in mammalian MTs, but that they are interchanged in their location along the polypeptide chain.