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Projeto de investigação
BIOCHEMICAL AND PHYSIOLOGICAL INSIGHTS INTO THE BACTERIAL CYTOCHROME C PEROXIDASE FROM ESCHERICHIA COLI
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Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases
Publication . Nóbrega, Cláudia S.; Carvalho, Ana Luísa; Romão, Maria João; Pauleta, Sofia R.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.
Biochemical and physiological insights into bacterial cytochrome c peroxidases from Escherichia coli and Neisseria gonorrhoeae
Publication . Nóbrega, Cláudia Raquel da Silva; Pauleta, Sofia; Devreese, Bart
Bacteria display an array of enzymes to detoxify reactive oxygen species that cause cell damage and death, such as the bacterial cytochrome c peroxidase (BCCP) that reduces H2O2 to water in the periplasm. The BCCPs studied up-to-date are soluble dihemic enzymes from non-pathogenic bacteria. This thesis focus on the trihemic BCCP from Escherichia coli (YhjA), and the dihemic BCCP from the obligate human pathogen Neisseria gonorrhoeae (NgBCCP). These two enzymes are membrane anchored thus, soluble recombinant proteins of their conserved globular domains were produced, purified and characterized biochemically and spectroscopically.
Recombinant NgBCCP, a 38 kDa protein, forms a homodimer in the presence of calcium ions. It contains a high-potential E heme (+310 mV, pH 7.5) and a low-potential P heme (-190 mV/-300 mV, pH 7.5), the active site, with a unique high-spin EPR signal at low temperatures in the mixed-valence active form. NgBCCP has catalytic activity with ABTS2- (synthetic electron donor) and a Lipid-modified Azurin (LAz) as electron donors (low KM values 4.0 and 0.4 μM H2O2, respectively) which was dependent on reductive activation and calcium ions, and optimum at physiological pH (7.0) and temperature (37 ºC). LAz, identified as NgBCCP physiological electron donor, was capable of activating the enzyme. The NgBCCP/LAz electron transfer complex has a low binding affinity (micromolar range), and the interaction is dynamic and of a hydrophobic nature according to NMR, docking and preliminary calorimetry studies.
The peroxidase activity was inhibited by exogenous ligands bound at the active site, such as azide, cyanide and imidazole, as demonstrated by spectroscopic, kinetic and structural analysis. The structure of NgBCCP was determined for the mixed-valenced and azide-inhibited form, and a catalytic mechanism for BCCPs was proposed based on the structural analysis of NgBCCP active site.
The recombinant YhjA, a 50 kDa monomer, has a C-terminal domain homologous to dihemic BCCPs and a N-terminal (NT) domain. This domain was characterized for the first time, demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P, NT and E hemes were determined: –170 mV, +133 mV and +210 mV, at pH 7.5, respectively. YhjA has quinol peroxidase activity in vitro (millimolar range KM values) using hydroquinone and menadiol (menaquinol analogue), as electron donors. Calcium ions were needed for maximum activity but not reductive activation, as P heme is always high-spin penta-coordinated. This property allowed to detect the formation of an intermediate radical species upon incubation with H2O2. Real Time PCR data showed that YhjA was expressed under anaerobic conditions, which agrees with the use of menaquinol in those conditions. Hence it was suggested a role in H2O2 detoxification when transitioning from anaerobic to aerobic environments.
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Fundação para a Ciência e a Tecnologia
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OE
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SFRH/BD/87878/2012