title: Biochemical characterization of the bacterial peroxidase from the human pathogen Neisseria gonorrhoeae.
abstract: 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 H2O2to 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 (+310mV) and (-190mV/-300mV), respectively in the presence of calcium ions, at pH7.5. 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(KMof 4±1μM), with maximum activity being attained at pH7.0 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.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal.
authors.forename: Cláudia S
authors.initials: CS
authors.lastname: Nóbrega
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal.
authors.forename: Mariana
authors.initials: M
authors.lastname: Raposo
authors.affiliation: Laboratory of Protein Biochemistry and Biomolecular Engineering, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
authors.forename: Gonzalez
authors.initials: G
authors.lastname: Van Driessche
authors.affiliation: Laboratory of Protein Biochemistry and Biomolecular Engineering, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal. Electronic address: srp@fct.unl.pt.
authors.forename: Sofia R
authors.initials: SR
authors.lastname: Pauleta
mesh_terms: D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D009195:Peroxidase
keywords: Bacterial peroxidase; Heme enzyme; Kinetics; Neisseria gonorrhoeae; Reactive oxygen species