Copper-zinc superoxide dismutase reduced form

Superoxide dismutase will scavenge the Of formed and will therefore inhibit the reduction of the dianisidine radical by Of. Consequently the dianisidine radical will dismute to yield the divalently oxidized dianisidine. In the presence of superoxide dismutase this reaction is augmented (Fig. 6). The possibility that Of could reduce the final product of dianisidine oxidation and reverse the change in absorbance at 460 nm was tested and was excluded. The assay has been used to determine the rate constant for purified swordfish liver copper/zinc superoxide dismutase (Bannister et al., 1979) and could be applied to crude extracts. The assay was also found applicable to polyacryalmide gels (Misra and Fridovich, 1977c). Gels soaked in riboflavin plus dianisidine, and subsequently illuminated, developed stable brown bands. Peroxidases are also stained by this procedure due to the photochemical production of hydrogen peroxide. However, the development of bands due to peroxidase activity is much slower than the development of bands due to dismutase activity. 

Cu—Zn superoxide dismutases (SODs) [87,88] are abundant in eukaryotic cells and may serve to protect cells against the toxic effects of superoxide or deleterious oxy-products derived from 02 . The active site copper and zinc ions are 6.3 A apart and are bridged by a histidine imidazolate. In the oxidized form Cu(II) is roughly pentacoordinate, with four His N s and a water molecule. A highly conserved Arg residue is thought to stabilize Cu(II)-bound anions (e.g., Cu(II)—02 ) a redox reaction releases 02, generating Cu(I), which can reduce more 02 substrate to give peroxide and Cu(II). 

Cadmium-113 nuclear magnetic resonance studies of the cadmium substituted bovine superoxide dismutase were carried out Only a very small chemical-shift difference between the 2 Cd(Il) protein (Cd(II) is bound to the zinc site and the copper site is unoccupied) and the 2 Cd(ll)—2 Cu(I) enzyme (analogous to the reduced form of the native protein) was found. This was interpreted in that the imidazolate bridge is protonated at the Cu site after reduction. 

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