Biochemical characterization of the bacterial peroxidase from the human pathogen Neisseria gonorrhoeae

Interaction between Neisseria gonorrhoeae bacterial peroxidase and its electron donor, the lipid‐modified azurin

“…These results indicate that LAz donates electrons to E heme, activating Ng BCCP, and maintaining its catalytic activity. This can be attributed to a specific interaction between the two proteins and also to the reduction potential of LAz (277 ± 5 mV, at pH 7.0 ) that is close to the one of Ng BCCP E heme (+310 ± 10 mV, at pH 7.5 ).…”

“…Therefore, calcium ions are needed for maximum activity, as previously shown for the Ng BCCP/ABTS 2− pair. This could also be an indication that formation of a productive ET complex is dependent on calcium ions, probably due to a simplification of the Ng BCCP solution states .…”

“…Previously, it was shown that Ng BCCP has peroxidase activity using a synthetic electron donor, ABTS 2− as electron source, and that it needs reductive activation in the presence of calcium ions to reach maximum activity. Here, it was evaluated whether LAz was able to activate Ng BCCP and how fast this process is.…”

“…The two proteins were heterologously produced in Escherichia coli BL21 (DE3) and purified, in a soluble form without the N‐terminal H.8 epitope region as previously described . Proteins’ concentration was determined using the extinction coefficient established before .…”

“…The assay was performed at 25 °C in the same buffer, containing 10 μ m reduced LAz, 100 μ m H 2 O 2 (for the Michaelis–Menten curve H 2 O 2 ranged between 0.01 and 1 m m ), and initiated with 10 n m pre‐activated Ng BCCP . At the end of the assay, a small aliquot of potassium ferricyanide concentrated solution was added to fully oxidize LAz and confirm that all electron donor was oxidized.…”