Metal substitution of Neurospora copper metallothionein

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Abstract
The binding of diamagnetic Zn(II), Cd(II) and Hg(II), and paramagnetic Co(II) and Ni(II) ions to the apo form of Neurospora metallothionein (MT) was investigated by various spectroscopic techniques. In contrast to native copper MT, which binds 6 mol of Cu(I)/mol of protein (Lerch, 1980), all substituted forms reveal an overall metal to protein stoichiometry of 3. The charge-transfer (CT) transitions of the complexes containing diamagnetic metal ions as well as the d-d transitions of those with paramagnetic metal ions are indicative of a distorted Td coordination. EPR and absorption measurements of the Co(II) derivative are in agreement with the presence of a metal-thiolate cluster in this protein. Metal titration studies of the apoprotein reveal characteristic spectral features for the derivatives containing 2 metal equivalents as compared to those with a full complement of 3 metal ions. The former features are indicative of an exclusive Td type of metal-sulfur coordination whereas the latter suggest that the 3rd metal ion is coordinated in a different fashion. This finding is in agreement with the presence of only 7 cysteine residues in Neurospora MT as opposed to 9 cysteine residues in the 3-metal cluster of mammalian MT (Winge and Miklossy, 1982).