Pauleta, S. R. | Search bioRxiv

bioRxiv2024

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

Cytochrome c552 from Wolinella succinogenes is one of the few examples of a low reduction potential class I c-type cytochrome with a mixture of high/low spin state populations observed in its visible spectrum. Analysis of its structural model suggests that the heme is Met/His coordinated and highly solvent-exposed. This supports the hypothesis that it is the solvent accessibility of the propionate groups that controls the reduction potential of small c-type cytochromes. The visible spectra obtained at different pH values reveal the presence of a protonable group with a pKa of 7.3, which also influences the reduction potential of this small cytochrome c552 (Em0 of 97 {+/-} 5 mV, pH 7.0) and can be either an H2O/OH- group distantly coordinating the heme iron, or one of the propionate groups. The thermostability of cytochrome c552 has been studied by circular dichroism and differential scanning calorimetry, indicating a highly stable protein at pH 5-7 (90 {degrees}C to 77 {degrees}C).

biochemistry↗

bioRxiv2022

Biochemical characterization of AniA from Neisseria gonorrhoeae

AniA, the nitrite reductase from Neisseria gonorrhoeae, has been shown to play a crucial role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with one type-1 copper center per subunit and one type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized by visible, EPR and CD spectroscopy for the first time, indicating that AniAs type 1 copper center has a high rhombicity, which is attributed to its tetrahedral geometry, and shorter Met-Cu bond, while type 2 copper center has the usual properties, though with a shorter hyperfine coupling constant (A//= 9.1 mT). The thermostability of AniA was analyzed by differential scanning calorimetry showing a single endothermic transition in the thermogram, with a maximum at 95 {degrees}C, while the CD spectra in the visible region indicates the presence of copper centers at 85-90 {degrees}C. The reoxidation rates of AniA in the presence of nitrite were analyzed by visible spectroscopy showing a pH dependence and being higher at pH 6.0. The high thermostability of this enzyme might be important for maintaining a high activity in the extracellular space and be less prone to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.

biochemistry↗