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

Neisseria gonorrhoeae is an obligate human pathogen that expresses an array of molecular systems to detoxify reactive oxygen species as defense mechanisms during colonization and infection. One of these is the bacterial peroxidase that reduces H₂O₂ to water in its periplasm. The soluble form of this enzyme was heterologously expressed in E. coli in the holo-form binding two c-types hemes, a high-potential E heme and a low-potential P heme, with redox potentials of (+ 310 mV) and (− 190 mV/− 300 mV), respectively in the presence of calcium ions, at pH 7.5. Visible and EPR spectroscopic analysis together with activity assays indicate the presence of a calcium dependent reductive activation mechanism in thgonorrhoeaeNeisseria gonorrhoeae bacterial peroxidase, in which P heme is bis-His coordinated low-spin in the fully oxidized state of the enzyme, and becomes penta-coordinated high-spin upon reduction of E heme in the presence of calcium ions. The activated enzyme has a high affinity for H₂O₂ (K_M of 4 ± 1 μM), with maximum activity being attained at pH 7.0 and 37 °C, with the rate-limiting step in the catalytic cycle being the electron transfer between the two hemes. In this enzyme, dimer formation is not promoted at high ionic strength, thus differing from the classical bacterial peroxidases. These results contribute to the understanding of the involvement of Neisseria gonorrhoeae bacterial peroxidase has a first line defense mechanism against exogenously produced hydrogen peroxide in the host environment.