The reconstitution of Cu,Zn-superoxide dismutase from the copper-free protein by the Cu(I)·GSH complex was monitored by: (a) EPR and optical spectroscopy upon reoxidation of the enzyme-bound copper; (b) NMR spectroscopy following the broadening of the resonances of the Cu(I)·GSH complex after addition of Cu-free,Zn-superoxide dismutase; and (c) NMR spectroscopy of the Cu-free,Co(II) enzyme following the appearance of the isotropically shifted resonances of the Cu(I),Co enzyme. Cu(I)·GSH was found to be a very stable complex in the presence of oxygen and a more efficient copper donor to the copper-free enzyme than other low molecular weight Cu(II) complexes. In particular, 100% reconstitution was obtained with stoichiometric copper at any GSH:copper ratio between 2 and 500. Evidence was obtained for the occurrence of a Cu(I)·GSH·protein intermediate in the reconstitution process. In view of the inability of copper-thionein to reconstitute Cu,Zn-superoxide dismutase and of the detection of copper·GSH complexes in copper-overloaded hepatoma cells (Freedman, J. H., Ciriolo, M. R., and Peisach, J. (1989) J. Biol. Chem. 264, 5598-5605), Cu(I)·GSH is proposed as a likely candidate for copper donation to Cu-free,Zn-superoxide dismutase in vivo.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jul 5 1990|
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