Cobalt substitution studies on bovine erythrocyte superoxide dismutase: evidence for a novel cobalt-superoxide dismutase derivative.
Three cobalt derivatives of bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) have been prepared under different pH conditions using a cobalt-thiocyanate complex which has already proved to yield specific substitutions on other copper proteins. The cobalt-protein derivatives have been characterized by optical, circular dichroism and fluorescence spectroscopies. One derivative, referred to as Co2Co2-protein, contains Co(II) ions specifically bound at both Zn(II) and Cu(II) sites. On the basis of their spectroscopic properties, the other two derivatives can be referred as E2Co2- and Co2E2-superoxide dismutase, with cobalt substituting, respectively, at the zinc and the copper sites leaving the contiguous site empty (E). The Co2E2-protein complex represents a novel derivative, since it has never been described in literature. The optical spectrum in the visible region of Co2-Co2-protein well corresponds to the sum of the spectra of the other two derivatives. The circular dichroism spectrum of Co2Co2-derivative, however, is not the sum of individual E2Co2- and Co2E2-proteins, suggesting that the presence of Co(II) in one site strongly affects the geometry of the neighbouring site. Some discrepancies between our spectroscopic data and those reported in literature are discussed. The results obtained from fluorescence experiments indicate that Co(II) ions exert a different quenching effect on the tyrosine emission, depending on whether they are located in the Zn(II) or in the Cu(II) site. The fluorescence quenching can be attributed to a 'heavy atom' and 'paramagnetic ion' effect by Co(II) ions.
Salvato
B
B
Department of Biology, University of Padova, Italy.
Beltramini
M
M
Ricchelli
F
F
Tallandini
L
L
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D003035:Cobalt / Q000097:blood*; D003300:Copper / Q000097:blood; D004912:Erythrocytes / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000097:blood* / Q000138:chemical synthesis; D014443:Tyrosine; D015032:Zinc / Q000097:blood
D003035:Cobalt; D014443:Tyrosine; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc