Preparation and 113Cd NMR studies of homogeneous reconstituted metallothionein: reaffirmation of the two-cluster arrangement of metals.

113Cd NMR analysis of rabbit liver metallothionein 2 reconstituted with 113Cd at all seven binding sites has previously indicated that the metals are arranged in two metal-thiolate clusters [Otvos, J.D., & Armitage, I.M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. Spectra of the protein always contained more than seven resonances, however, suggesting the samples were in some way heterogeneous. Results of a recent study of 113Cd metallothionein reconstituted in a different manner but also giving spectra with more than seven resonances have been interpreted as arguing against the two-cluster model of metal binding and in favor of a model in which structural flexibility of the protein allows many configurational substates of the cluster(s) to coexist [Vasak, M., Hawkes, G.E., Nicholson, J.K., & Sadler, P.J. (1985) Biochemistry 24, 740-747]. Data are presented here that indicate that dimers and larger oligomers of metallothionein formed as byproducts of metal reconstitution are the likely source of at least some of the 113Cd resonances attributed by these workers to configurational substrates. Removal of the contaminating oligomers by gel filtration yields a verifiably homogeneous protein whose 113Cd spectrum consists of seven resonances of comparable intensity. Unambiguous confirmation of the existence and structures of the two previously proposed metal-thiolate clusters was obtained by two-dimensional chemical shift correlation spectroscopy and spectral simulation of the 113Cd-113Cd splitting patterns of the individual resonances.