Superoxide dismutase. Examination of the metal binding sites by electron spin echo spectroscopy
Journal of Biological Chemistry
We have studied the local environment of Cu(I1) in bovine superoxide dismutase using the nuclear modulation effect in pulsed EPR spectroscopy. The echo envelope spectrum contains lines arising from the interaction of the spin of the Cu(I1) with the remote 14Natoms of the ligated imidazoles. In the zinc-free protein, we observe lines of 0.7, 1.5, and 4 MHz, comparable to those seen in a Cu(I1) model compound that contains an imidazole ligand. In the holoprotein at either neutral or elevated pH,
... al or elevated pH, or in a protein preparation where the Zn(I1) has been substituted by Cd(II), the low frequency lines are split, indicating that the interaction of the nitrogen with the copper has been altered by the presence of a divalent metal linked by an imidazolate bridge to the Cu(I1). For the holoprotein at low pH, this splitting is not observed suggesting that the Zn(I1) is no longer coordinated to the bridging imidazole. The binding of azide and cyanide to both the holoprotein and the protein lacking Zn(I1) have been studied. The "N-azide complex of holosuperoxide dismutase exhibits an echo envelope spectrum very similar to that of the untreated holoprotein, suggesting that the Zn(I1)imidazolate structure is not appreciably altered. For the Zn(II)-free protein, the echo envelope spectrum of the "N-azide complex is similar to that observed in the absence of azide. These results are to be contrasted with those obtained for the cyano complexes of both protein preparations. For the Zn(I1)-free protein, "Ncyanide addition does not change the echo envelope spectrum appreciably, while "N-cyanide addition to the holoprotein does. Proton balance experiments suggest that the Zn(I1)-imid-Cu(I1) structure remains intact in the holoprotein when cyanide is added. These results demonstrate that cyanide binding alters the bonding of Cu(I1) to imidazole I4N in the holoprotein.