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Projeto de investigação
Insights into novel bacterial cytochrome c peroxidases from pathogenic bacteria, Neisseria gonorrhoeae and Escherichia coli
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YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity
Publication . Nóbrega, Cláudia S.; Devreese, Bart; Pauleta, Sofia R.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier BV
The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be −170 mV, +133 mV and +210 mV, respectively, at pH 7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH 7.0 and millimolar range KM values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (KM = 0.6 ± 0.2 and 1.8 ± 0.5 mM H2O2, respectively), with similar turnover numbers (kcat = 19 ± 2 and 13 ± 2 s−1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV) = O species was observed upon incubation of fully oxidized YhjA with H2O2. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.
Reduction of hydrogen peroxide in gram-negative bacteria – bacterial peroxidases
Publication . Nóbrega, Cláudia S.; Pauleta, Sofia R.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Academic Press
Bacteria display an array of enzymes to detoxify reactive oxygen species that cause damage to DNA and to other biomolecules leading to cell death. Hydrogen peroxide is one of these species, with endogenous and exogenous sources, such as lactic acid bacteria, oxidative burst of the immune system or chemical reactions at oxic-anoxic interfaces. The enzymes that detoxify hydrogen peroxide will be the focus of this review, with special emphasis on bacterial peroxidases that reduce hydrogen peroxide to water. Bacterial peroxidases are periplasmic cytochromes with either two or three c-type haems, which have been classified as classical and non-classical bacterial peroxidases, respectively. Most of the studies have been focus on the classical bacterial peroxidases, showing the presence of a reductive activation in the presence of calcium ions. Mutagenesis studies have clarified the catalytic mechanism of this enzyme and were used to propose an intramolecular electron transfer pathway, with far less being known about the intermolecular electron transfer that occurs between reduced electron donors and the enzyme. The physiological function of these enzymes was not very clear until it was shown, for the non-classical bacterial peroxidase, that this enzyme is required for the bacteria to use hydrogen peroxide as terminal electron acceptor under anoxic conditions. These non-classical bacterial peroxidases are quinol peroxidases that do not require reductive activation but need calcium ions to attain maximum activity and share similar catalytic intermediates with the classical bacterial peroxidases.
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.
Interaction between Neisseria gonorrhoeae bacterial peroxidase and its electron donor, the lipid-modified azurin
Publication . Nóbrega, Cláudia S.; Pauleta, Sofia R.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Federation of European Biochemical Societies | Wiley
The Neisseria gonorrhoeae bacterial cytochrome c peroxidase plays a key role in detoxifying the cells from H2O2 by reducing it to water using the lipid-modified azurin, LAz, a small type 1 copper protein, as electron donor. Here, the interaction between these two proteins was characterized by steady-state kinetics, two-dimensional NMR and molecular docking simulations. LAz is an efficient electron donor capable of activating this enzyme. This electron transfer complex is weak with a hydrophobic character, with LAz binding close to the electron transferring heme of the enzyme. The high catalytic rate (39 ± 0.03 s−1) is explained by the LAz pre-orientation, due to a positive dipole moment, and by the fast-dynamic ensemble of orientations, suggested by the small chemical shifts.
Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes
Publication . Carreira, Cíntia; Mestre, Olga; Nunes, Rute F.; Moura, Isabel; Pauleta, Sofia R.; LAQV@REQUIMTE; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; PeerJ Inc.
Background. Denitrification is one of the main pathways of the N-cycle, during which nitrate is converted to dinitrogen gas, in four consecutive reactions that are each catalyzed by a different metalloenzyme. One of the intermediate metabolites is nitrous oxide, which has a global warming impact greater then carbon dioxide and which atmospheric concentration has been increasing in the last years. The four denitrification enzymes have been isolated and biochemically characterized from Marinobacter hydrocarbonoclasticus in our lab. Methods. Bioinformatic analysis of the M. hydrocarbonoclasticus genome to identify the genes involved in the denitrification pathway. The relative gene expression of the gene encoding the catalytic subunits of those enzymes was analyzed during the growth under microoxic conditions. The consumption of nitrate and nitrite, and the reduction of nitric oxide and nitrous oxide by whole-cells was monitored during anoxic and microoxic growth in the presence of 10 mM sodium nitrate at pH 7.5. Results. The bioinformatic analysis shows that genes encoding the enzymes and accessory factors required for each step of the denitrification pathway are clustered together. An unusual feature is the co-existence of genes encoding a q- and a c-type nitric oxide reductase, with only the latter being transcribed at similar levels as the ones encoding the catalytic subunits of the other denitrifying enzymes, when cells are grown in the presence of nitrate under microoxic conditions. Using either a batch- or a closed system, nitrate is completely consumed in the beginning of the growth, with transient formation of nitrite, and whole-cells can reduce nitric oxide and nitrous oxide from mid-exponential phase until being collected (time-point 50 h). Discussion. M. hydrocarbonoclasticus cells can reduce nitric and nitrous oxide in vivo, indicating that the four denitrification steps are active. Gene expression profile together with promoter regions analysis indicates the involvement of a cascade regulatory mechanism triggered by FNR-type in response to low oxygen tension, with nitric oxide and nitrate as secondary effectors, through DNR and NarXL, respectively. This global characterization of the denitrification pathway of a strict marine bacterium, contributes to the understanding of the N-cycle and nitrous oxide release in marine environments.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-PRO/109796/2009