Spectroscopic properties of the cobalt(II)-substituted .alpha.-fragment of rabbit liver metallothionein

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Abstract
The C-terminal segment of rabbit liver metallothionein 1 (.alpha.-fragment) containing four paramagnetic Co(II) ions was obtained by stoichiometric replacement of the originally bound diamagnetic Cd(II) ions. The latter form was prepared by limited proteolysis with subtilisin as described previously [ Winge, D. R., and MIklossy, K. A. (1982) J. Biol. Chem. 257, 3471-3476]. Electronic absorption, magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) measurements were employed to monitor the stepwise incorporation of Co(II) ions into the metal-free fragment. Absorption and MCD spectra of the apofragment containing the first 3 Co(II) equiv show the typical features of tetrahedral tetrathiolate Co(II) coordination. However, in the d-d region only small changes in the visible and no apparent change in the near-infrared region are discernible when the fourth Co(II) is bound. This unusual spectral behavior was not seen in Co(II) substitution of native metallothionein [Vasak, M., and Kagi, J.H. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6709-6713] and may indicate a different cluster geometry. In the charge-transfer region, the binding of all 4 Co(II) equiv is accompanied by characteristic increments of the thiolate S .fwdarw. Co(II) bands. As in the formation of Co(II)7-metallothionein, the development of the the charge-transfer and EPR spectral properties upon binding of the first 2 Co(II) equiv to the apofragment is indicative of isolated, noninteracting tetrahedral tetrathiolate Co(II) complexes. The binding of the additional Co(II) ion is accompanied by a red shift in the charge-transfer region and by the dramatic loss of paramagnetism in the EPR spectra, both diagnostic of the formation of metal-thiolate cluster structures. Thus, these data suggest that the four-metal cluster in the isolated .alpha.-fragment and in Co(II)7-metallothionein is similar but not identical.