Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects

Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders. In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view. The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity. This effect is strongly influenced by the HCO3-/CO2 buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues. Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite. The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry. The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25 kcal/mol at 25 °C, with respect to the non-oxidized protein. Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions.