Formation and spectroscopic characterization of a novel monomeric cadmium- and phosphate-containing form of metallothionein

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Abstract
The occurrence of a new monomeric cadmium and phosphate containing metallothionein (MT) form, i.e., Cd13-(Pi)2-MT, is reported. The preferential formation of this MT form from Cd7-MT has been shown to occur in the presence of phosphate and free cadmium at low protein concentration and elevated temperatures. This form displays in gel filtration an apparent molecular weight of 8,900 as opposed to 12,000 for Cd7-MT, suggesting the presence of a more globular structure. This new MT form was characterized by electronic absorption, by circular dichroism (CD), and by 1H, 31P, and 113Cd NMR spectroscopy. The Cd13-(Pi)2-MT form displays at least 24 113Cd signals between 240 and 520 ppm indicating (i) the absence of the original cluster structure of Cd7-MT, (ii) the participation of oxygen and/or nitrogen ligands besides thiolates in metal coordination, and (iii) the presence of more than one stable MT form in the sample. From homonuclear 113Cd COSY and CD studies, evidence for the existence of a cluster structure was obtained. It has been demonstrated that in the Cd13-(Pi)2-MT form two phosphate molecules are bound noncovalently and shown that a very slow exchange with [32P]phosphate in solution (half-life of approximately 56 h) takes place. In the 31P NMR studies, three 31P signals from protein-bound phosphate between 3.5 and 6 ppm have been observed. The 31P signal at 5.7 ppm displayed a heteronuclear 31P-113Cd coupling (J2 = 56 Hz) which provides evidence for direct metal-phosphate coordination. The structural and NMR features of this new MT form are discussed.

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