Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content—Order restored

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234–240; 2005) the authors argued that the catalytic rate constant, kcat, for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sample that already contained excess copper did not remove spurious activity, which is incompatible with well-known stability constants of copper complexes and contradicts previous observations. In the present study we verified that the addition of EDTA eliminates completely the effect of excess copper on the decomposition rate of O2− in the presence of Cu,Zn-SOD. We determined that kcat = (2.82 ± 0.02) × 109 M−1 s−1 at low ionic strength (2 < I < 15 mM) and (1.30 ± 0.02) × 109 M−1 s−1 in the presence of 50 mM phosphate at pH 7.8 (I ≈ 150 mM), which are about twice higher than those reported by Michel et al. We also determined kcat by the cytochrome c assay and demonstrated the correlation between these direct and indirect assays. The phenotypic deficits imposed by deletion of SODs, and the oxygen dependence of these deficits, have repeatedly demonstrated that the several SODs do in fact, as well as is theory, provide an important protection against that facet of oxidative stress imposed by O2−.