CHARACTERIZATION AND SEQUENTIAL LOCALIZATION OF THE METAL-CLUSTERS INSEA-URCHIN METALLOTHIONEIN

The mode of metal binding in sea urchin metallothionein (MT) was explo red 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 elec tronic spectroscopic and chiroptical features and the Cd-113 NMR spect ra of the reconstituted material with those of the native recombinant material indicated that the reconstituted material had regained the na tive 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 NM R resonances all strongly suggested that sea urchin MT like all other well characterized MTs contains clusters made up of tetrahedral Cd-thi olate units. The Cd-113 chemical shift correlation spectrum of the rec onstituted protein proved the existence of such metal clusters and all owed the unambiguous assignment of some of the metal connectivities. H omonuclear decoupling experiments in which Cd resonances were selectiv ely saturated indicated moreover a partitioning of the metal complemen t into two separate clusters containing three and four Cd ions. The sa me proposition was supported by the selective reduction of three Cd-11 3 resonances upon partial metal depletion following exposure of the pr otein to EDTA. Thus, the three-metal cluster is energetically less sta ble than the four-metal cluster. That the two clusters are separate en tities was also demonstrated by the isolation of a protein fragment co ntaining the four-metal cluster resulting from partial proteolysis of sea urchin MT by subtilisin in the presence of EDTA. Amino acid sequen ce and electrospray mass spectroscopic analysis identified this fragme nt as the N-terminal portion of the whole protein. This is in marked c ontrast to the known mammalian forms where the more stable four-metal cluster is associated with the C-terminal domain. One can conclude the refore that the sea urchin MT contains the same type of metal-thiolate clusters as those found in mammalian MTs, but that they are interchan ged in their location along the polypeptide chain.