Biochemical Characterization of the Copper Nitrite Reductase from Neisseria gonorrhoeae

Abstract

The copper-containing nitrite reductase from Neisseria gonorrhoeae has been shown to play a critical role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with a type 1 copper center per subunit and a type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized for the first time by EPR and CD spectroscopy, showing that the type 1 copper center has a high rhombicity due to its lower symmetry and more tetragonal structure, while the type 2 copper center has the usual properties, but with a smaller hyperfine coupling constant (A// = 10.5 mT). The thermostability of the enzyme was analyzed by differential scanning calorimetry, which shows a single endothermic transition in the thermogram, with a maximum at 94 °C, while the CD spectra in the visible region indicate the presence of the type 1 copper center up to 80 °C. The reoxidation of the N. gonorrhoeae copper-containing nitrite reductase in the presence of nitrite were analyzed by visible spectroscopy and showed a pH dependence, being higher at pH 5.5–6.0. The high thermostability of this enzyme may be important to maintaining a high activity in the extracellular space and to making it less susceptible to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.

Effect of pH on the thermostability and redox properties of cytochrome c552 from Wolinella succinogenes

“…The unfolding temperature observed at pH 7.0 is relatively higher than that observed for other proteins isolated from mesophilic bacteria (Nobrega et al, 2017), although some exceptions have been reported (Barreiro et al, 2023b). However, the T M is relatively low (90 °C, at pH 5.0) compared to that of cytochrome c 552 from thermophilic organisms: Hydrogenophilus thermoluteolus and H. thermophilus, which have a T M of 108 °C and 121 °C, respectively, at pH 5.0 (Oikawa et al, 2005;Nakamura et al, 2006).…”

Effect of pH on the thermostability and redox properties of cytochromec552fromWolinella succinogenes

“…The unfolding temperature observed at pH 7.0 is relatively higher than that observed for other proteins isolated from mesophilic bacteria (Nobrega et al, 2017), although some exceptions have been reported (Barreiro et al, 2023b). However, the TM is relatively low (90 ºC, at pH 5.0) compared to that of cytochromes c552 from thermophilic organisms: Hydrogenophilus thermoluteolus and H. thermophilus, which have a TM of 108 º C and 121 ºC, respectively, at pH 5.0 (Oikawa et al, 2005;Nakamura et al, 2006).…”

Elogio da Desnitrificação

“…Estes domínios adicionais ligam hemos do tipo-c ou centros CuT1 e medeiam a transferência de eletrões para o domínio conservado [23]. O domínio conservado nas várias famílias de CuNiR tem uma estrutura homotrimérica (Figura 3C), com cada subunidade contendo os dois centros mononucleares de cobre [17,24]. O centro CuT1 é coordenado por dois resíduos de histidina, uma metionina e uma cisteína, da mesma subunidade.…”

“…Por sua vez, o centro CuT2 está localizado na interface entre dois monómeros e é coordenado por três cadeias laterais de histidina, uma das quais é do monómero adjacente (Figura 3D) [25]. Estes dois centros estão ligados entre si por uma ponte de cisteína-histidina conservada que consiste na cisteína que coordena o centro CuT1 e na histidina que coordena o centro CuT2 [17,23,24].…”