Reconstitution of Cu,Zn-superoxide dismutase by the Cu(I).glutathione complex.
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-over-loaded 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.
Ciriolo
M R
MR
Department of Biology, Tor Vergata University of Rome, Italy.
Desideri
A
A
Paci
M
M
Rotilio
G
G
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000201:enzymology; D005978:Glutathione / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013482:Superoxide Dismutase / Q000378:metabolism*
D003300:Copper; D013482:Superoxide Dismutase; D005978:Glutathione