In this paper, we show that amino acids E73 and D77 of staphylococcal nuclease cooperate unequally with E75 to stabilize its structure located between the C-terminal helix and b-barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24% and 16% and the thermal stability losses of 22% and 26%, respectively, in comparison with the WT protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD222nm. Furthermore, X-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between E75 and K9, Y93 and H121. In experiments using double-point mutations, E73G-D77G, E73G-E75G and E75G-D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47%, 59% and 58%, for E73G-D77G, E73G-E75G and E75G-D77G, respectively. The triple mutant, E(73,75)G-D77G, results in fluorescence intensity and CD222nm close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.