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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.
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.
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.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/87878/2012