Determination of the Cd/S cluster stoichiometry in Fucus vesiculosus metallothionein.

The seaweed Fucus vesiculosus is unusual when compared with other algal species, in that it can survive in toxic-metal-contaminated aquatic environments. The metallothionein gene has been identified in F. vesiculosus by Kille and co-workers (Morris, C. A., Nicolaus, B., Sampson, V., Harwood, J. L., and Kille, P. (1999) Biochem. J. 338, 553), which suggests a possible protective mechanism against toxic metals for this species. We report the first detailed study of the metal binding properties of F. vesiculosus metallothionein using UV absorption, circular dichroism (CD), and electrospray mass spectral techniques. The overall metal-to-sulfur ratios of this novel algal protein when bound to divalent cadmium and zinc were determined to be Cd(6)S(16) and Zn(6)S(16), respectively. Mixed Cd/Zn species were also formed when Cd(2+) was added to the Zn-containing Fucus metallothionein. Only one conformation was identified at low pH for the native protein. Analysis of the UV absorption, CD, and ESI-MS spectral data recorded during stepwise, acid-induced demetalation supports a two-domain structure for the protein, with two 3-metal binding sites. The data suggest that one of the domains is significantly less stable than the other, and we tentatively propose from the arrangement of cysteines in the sequence that the two domains are M(3)S(7) and M(3)S(9) (where M = Cd(2+) or Zn(2+)). While the M(3)S(9) cluster is known in the beta clusters of crab, lobster, and mammalian metallothioneins, the M(3)S(7) is a hitherto unknown cluster structure. Metallothionein in F. vesiculosus is thought to act as a protective mechanism against incoming toxic metals. The metal binding studies reported are a putative model for metal binding in vivo.