The catalytic activity of wild type Escherichia coli Cu,Zn superoxide dismutases and of two mutants in which two lysine residues conserved in most bacterial Cu,Zn superoxide dismutases have been replaced by serine was investigated by pulse radiolysis and Brownian dynamics simulations. Experimental and computational data show that neutralization of Lys60 strongly reduces the catalytic activity of the enzyme (approximately 50%), indicating that this residue has a primary role in the electrostatic attraction of the substrate towards the catalytic copper. Neutralization of Lys63 does not significantly influence the catalytic rate constant. The results suggest that prokaryotic Cu,Zn superoxide dismutases have evolved an electrostatic mechanism to facilitate the enzyme-substrate encounter that is functionally equivalent to that already found in the eukaryotic enzymes.