Role of the Dimeric Structure in Cu,Zn Superoxide Dismutase

Abstract

To investigate the structural/functional role of the dimeric structure in Cu,Zn superoxide dismutases, we have studied the stability to a variety of agents of the Escherichia coli enzyme, the only monomeric variant of this class so far isolated. Differential scanning calorimetry of the native enzyme showed the presence of two well defined peaks identified as the metal free and holoprotein. Unlike dimeric Cu,Zn superoxide dismutases, the unfolding of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly polar nature of its molecular surface. The melting temperature of the E. coli enzyme was found to be pH-dependent with the holoenzyme transition centered at 66°C at pH 7.8 and at 79.3°C at pH 6.0. The active-site metals, which were easily displaced from the active site by EDTA, were found to enhance the thermal stability of the monomeric apoprotein but to a lower extent than in the dimeric enzymes from eukaryotic sources. Apo-superoxide dismutase from E. coli was shown to be nearly as stable as the bovine apoenzyme, whose holo form is much more stable and less sensitive to pH variations. The remarkable pH susceptibility of the E. coli enzyme structure was paralleled by the slow decrease in activity of the enzyme incubated at alkaline pH and by modification of the EPR spectrum at lower pH values than in the case of dimeric enzymes. Unlike eukaryotic Cu,Zn superoxide dismutases, the active-site structure of the E. coli enzyme was shown to be reversibly perturbed by urea. These observations suggest that the conformational stability of Cu,Zn superoxide dismutases is largely due to the intrinsic stability of the ␤-barrel fold rather than to the dimeric structure and that pH sensitivity and weak metal binding of the E. coli enzyme are due to higher flexibility and accessibility to the solvent of its activesite region.

Monomeric Cu,Zn-superoxide Dismutase Is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis

“…The monomer intermediate is common to the aggregation of both wild type and fALS-associated mutant SODs. Because the dimeric structure is necessary for proper enzymatic action and stability (33,34), this may account for higher levels of oxidative stress in ALS patients (14) and reports of decreased enzymatic activity in some fALS SOD1 mutants (35). We found that monomerization and aggregation results from the oxidative modification of relatively few residues that are removed from the dimer interface of SOD1.…”

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

“…Bacterial Cu,Zn-SODs easily lose their metal cofactors and are therefore irreversibly inactivated in the presence of EDTA (30,49). The observed rates of EDTA-mediated inactivation vary for different enzymes (30), are affected by the stability of the quaternary structure (43,49), and are strongly influenced by pH, being faster at alkaline pH (29,30).…”

“…SodCI has a very high affinity for copper ions and cannot be completely demetallated by the procedures previously described to obtain apoSODs lacking both copper and zinc ions (30,43). Therefore, to remove the copper, the two StSOD enzymes were treated with potassium ferrocyanide to reduce the metal, then dialyzed for 12 h against 100 mM potassium phosphate buffer, 50 mM KCN, pH 6.0 (44).…”

“…The effect of EDTA on SodCI and SodCII activity was analyzed as described (30). Cu,Zn-SOD samples at a concentration of 0.04 mg/ml were incubated at 37°C in 100 mM phosphate buffer, 0.1 mM EDTA, pH 6.2 or 7.8.…”

“…More significantly, SodCI is a dimeric protein, whereas SodCII is a monomer (16). Extensive characterization of different bacterial Cu,Zn-SODs has established that monomeric and dimeric Cu,Zn-SODs have very different activity, thermal stability, metal affinity, and resistance to proteases (29,30). Slauch and co-workers have recently reported that SodCII is more susceptible than SodCI to protease digestion (25), but found other properties of the two enzymes to be identical (23).…”