Superoxide dismutases (SODs) are vital components in the resistance of aerobic organisms to the toxicity of oxygen. Escherichia coli contains two highly homologous cytoplasmic SODs, a manganese- and an iron-containing enzyme (MnSOD, FeSOD). We previously demonstrated that MnSOD and FeSOD have different physiological functions and that MnSOD is more effective in preventing oxidative damage to DNA. In this report, purified E. coli MnSOD was shown to bind nonspecifically to DNA by electrophoretic mobility shift assay and nitrocellulose-filter binding methodologies. From electrophoretic mobility shift assay, the equilibrium dissociation constants for interaction with a variety of double-stranded and single-stranded oligonucleotides ranged from 1.5 ± 0.2 to 8.4 ± 1.3 μM at 20 °C. This range of concentrations corresponds to MnSOD concentrations in aerobically grown E. coli. In vivo binding of MnSOD to DNA was supported by colocalization of MnSOD and the E. coli nucleoid in immunoelectron microscopy. Both MnSOD and DNA were inhomogeneously distributed in the cytosol, the concentration of each being higher in the center of the cell and relatively low near the inner membrane. In contrast, there was no evidence for physiologically relevant interaction of FeSOD with DNA. Binding to DNA in vitro was weak, K(d) > 40-220 μM, concentrations 7-40 times higher than found in vivo. In addition, the cytoplasmic distribution of FeSOD did not correlate with DNA. FeSOD concentration was higher near the inner membrane and lower in the center of the cytosol. These results demonstrate that E. coli MnSOD associates with DNA in vitro and in vivo. Combined with prior data demonstrating that MnSOD preferentially protects DNA in vivo while an equal enzymatic activity of FeSOD does not (Hopkin, K. A., Papazian, M. A., and Steinman, H. M. (1992) J. Biol. Chem. 267, 24253-24258), our data suggest that E. coli MnSOD acts as a 'tethered antioxidant'; association of MnSOD with DNA localizes dismutase activity near a target of oxidative stress and increases protection of DNA from oxidative damage. This model has implications for the therapeutic use of SODs as antioxidants.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Nov 18 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology