Spectroscopic Studies of Cobalt(ii) Thiolates and Cobalt(ii) - Substituted Metallothioneins.

Abstract

Metallothioneins (MT) are widely occurring proteins characterized by low molecular weights and extremely high metal and sulfur content. Mammalian forms usually contain seven diamagnetic metal ions such as Zn(II) and/or Cd(II) and 20 cysteine residues. An understanding of the physiological role(s) of MT requires the elucidation of the nature and type of metal clusters, and the mechanism of assembly in MT. The stepwise incorporation of Co(II) ions into apo-metallothionein-2 from rabbit liver has been monitored by UV-visible absorption, low temperature magnetic circular dichroism (MCD), and EPR spectroscopies. Interpretation of the results has been facilitated by parallel spectroscopic studies of synthetic cobalt thiolate complexes, (i.e. (${\rm Co(S}\sb2$-o-xyl)$\sb2$) $\sp{2-}$, (Co(SR)$\sb4$) $\sp{2-}$, (Co$\sb4$(SR)$\sb{10}$) $\sp{2-}$; R = Et ot Ph) and cobalt(II)-substituted rubredoxins. Low temperature MCD spectroscopy has enabled selective investigation of the electronic and magnetic properties of the constituent cobalt thiolate centers in MT. These results for fully metal saturated Co(II) - MT are consistent with both tetranuclear and trinuclear cobalt clusters having S = 0 and S = 3/2 ground states respectively. Since only the trinuclear cobalt thiolate cluster exhibits temperature dependent MCD bands, this technique provides a method of selectively investigating the magnetic properties and monitoring the assembly of this type of center. Based on comparison of the electronic and magnetic properties of the cobalt thiolate clusters in metallothionein with crystallographically-defined model complexes, it is suggested that the tetranuclear center has an adamantane type structure. Detailed studies of the temperature dependence of MCD bands for the adamantane type cluster, (Co$\sb4$(SEt)$\sb{10}$) $\sp{2-}$, indicate an S = 0 ground state, resulting from antiferromagnetic coupling between four S = 3/2 Co(II) ions, with an S = 1 state 66 cm$\sp{-1}$ higher in energy. Low temperature MCD and EPR data for samples of MT with between zero and seven equivalents of cobalt show that cluster formation (i.e. utilization of bridging thiolates) ensues after the addition of approximately three equivalents of cobalt. Significant formation of the paramagnetic trinuclear clusters only occurs on addition of more than five equivalents of cobalt. The mechanism of assembly of Co(II)-MT is a multistep process.