Thermal stability and kinetic unfolding of Neisseria gonorrhoeae copper nitrite reductase.
TL;DR
The results show that enzyme unfolding is irreversible and kinetically controlled and is best described by a three-state Lumry-Eyring model involving a reversible intermediate followed by an irreversible transition to an aggregated stated, likely contributing to enzyme robustness under the dynamic conditions encountered during host-pathogen interactions.
Abstract
Copper nitrite reductases catalyse the reduction of nitrite to nitric oxide in the periplasm of Gram-negative bacteria as the second step of the denitrification pathway. Although these enzymes have been extensively studied, limited data are available regarding their thermal stability. The unfolding mechanism and the role of the copper centres in the thermostability of the copper nitrite reductase from the pathogenic bacterium Neisseria gonorrhoeae were investigated using differential scanning calorimetry and spectroscopic techniques. The results show that enzyme unfolding is irreversible and kinetically controlled and is best described by a three-state Lumry-Eyring model involving a reversible intermediate followed by an irreversible transition to an aggregated stated. Visible spectroscopy revealed that type-1 copper centre exhibits remarkable local stability, remaining spectroscopically intact up to 80 °C and undergoing irreversible disruption only at higher temperatures. In contrast, removal of the copper centres does not significantly alter the secondary structure, but reduces thermal stability by 30 °C, highlighting their critical role in stabilizing the protein. Together, these findings provided new insight into the unfolding mechanism of a copper nitrite reductase from a pathogen and demonstrate the important contribution of the metal cofactors to the protein exceptional thermostability. These features likely contribute to enzyme robustness under the dynamic conditions encountered during host-pathogen interactions.