Studies on the Reconstitution of Bovine Erythrocyte Superoxide Dismutase

Two metal derivatives of superoxide dismutase have been prepared and partially characterized by optical, circular dichroic (CD), and EPR spectroscopies. Only 2 Co(II) ions will bind to apo superoxide dismutase and evidence is presented that these occupy the zinc-binding sites of the protein molecule. This derivative, referred to as 2Co protein, has an absorption maximum at 583 nm with each ion having a molar absorbance of ∼370. The CD spectrum possesses at least two weak negative bands near 585 nm and 530 nm. The EPR spectrum below 100°K shows g⊥ = 4 and g|| = 2.16 resonances. Addition of 2 meq of Cu(II) ions to the 2Co protein over a period of 1 to 24 hours results in the formation of a complex in which Cu(II) appears to be bound in its native position. This derivative, referred to as 2Co2Cu protein, has an absorption maximum near 680 nm with each Cu(II) having an absorbance index of ∼150 liters per mole cm. The Co(II) in this derivative has absorption maxima at 568 nm and 598 nm, the latter having an absorbance index per mole of Co(II) of 370. Evidence is presented that the optical and CD spectra of the 2Co2Cu protein are not the sum of the appropriate spectra of 2Co and native proteins; thus, the spectral properties of each ion depend on the presence of the other. Only 20 to 30% of the Cu(II) in the 2Co2Cu protein can be detected by EPR spectroscopy. Similarly, only a portion of the Co(II) is observed by EPR at helium temperatures. However, on reduction of the Cu(II) an EPR signal appears which has an intensity and parameters similar to that of the 2Co protein. Thus, the Co(II) and Cu(II) ions must be quite close to one another in this derivative. By analogy, this supports our previous contention that the zinc and copper ions are bound as two copper-zinc pairs in the native protein (Fee, J. A., and Gaber, B. P. (1972) J. Biol. Chem. 247, 60).