Interaction between Neisseria gonorrhoeae bacterial peroxidase and its electron donor, the lipid-modified azurin

cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728).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.publishersversionpublishe

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

Belgian Federal Science Policy Office (Belspo) (grant to BD, IAP7/44, iPROS project). co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728).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.publishersversionpublishe

Source and reduction of nitrous oxide

PTDC/BBB-BQB/0129/2014. UID/QUI/50006/2019. UlD/Multi/04378/2019. MSPC acknowledges FCT/MCTES for funding her "Research Position" (signed with FCT NOVA in accordance with DL.57/2016 and Lei 57/2017). Sem PDF conforme despacho.Nitrous oxide is a potent greenhouse gas with a global warming impact 300-fold higher than carbon dioxide. Due to its exponential increase in the atmosphere and its implications in climate change there is the need to develop strategies to mitigate its emissions and to reduce it to the inert dinitrogen gas. Only three enzymes have been reported to be able to reduce nitrous oxide, namely nitrogenase, one multicopper oxidase and nitrous oxide reductase, with the latter being the only one with a relevant physiological activity. In this enzyme, reduction of nitrous oxide occurs in a unique catalytic tetranuclear sulfide center, named “CuZ” center, a complex center required to overcome the high activation barrier of this reaction. Nitrous oxide reductase can be isolated with “CuZ” center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), that differ in their catalytic and spectroscopic properties. Recently, another step towards a better understanding of the catalytic and activation mechanism of this enzyme was taken by identifying and spectroscopically characterizing an intermediate species of its catalytic cycle, CuZ 0 . A different approach for N 2 O reduction can be attained using model compounds. The unique structural motif present in “CuZ” center, a Cu 4 (µ 4 -S), has been a challenge for inorganic synthesis but several synthetic clusters that mimic different forms of “CuZ” center have been reported. Model compounds for the oxidation states involved in N 2 O reduction are also available. The advances in this area will be discussed in light of the recent data, with structural and functional model compounds of N 2 OR active site.authorsversionpublishe

Multivalent enzymes that enable the use of hydrogen peroxide for microaerobic and anaerobic proliferation

This work was financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. FCT supported SRP through the projects PTDC/BIA-PRO/109796/2009 and PTDC/BIA-BQM/29442/2017, DSB and RNSO through the scholarships UI/BD/151168/2021, and “Verão com Ciência2020”, respectively.Bacterial peroxidases are responsible for the reduction of hydrogen peroxide to water. Found in the periplasm of gram-negative bacteria, they are one of the defense mechanisms against endogenous and exogenous peroxide stress under low oxygen tensions. Besides being involved in peroxide detoxification, bacterial peroxidases have been proposed to constitute an alternative pathway to the respiratory chain under anoxic conditions, as demonstrated in E. coli that can use hydrogen peroxide as an electron acceptor in the absence of oxygen. Bacterial peroxidases are c-type cytochromes with either two or three c-type hemes bound to the polypeptide chain, being divided into classical or non-classical, respectively. Orthologous to the classical bacterial peroxidases are the MauG enzymes that share some structural, spectroscopic and sequence similarities but have distinct physiological roles (though for most their function remains unknown). The spectroscopic and kinetic data on bacterial peroxidases are reviewed for both classes. Most classical bacterial peroxidases require reductive activation that consists in structural changes so that the catalytic heme becomes accessible to the substrate. However, non-classical enzymes are ready to bind the hydrogen peroxide as their catalytic center is penta-coordinated, which is also observed in their structural model. The few studies that report the involvement of bacterial peroxidases from pathogenic bacteria in biofilms, is an indication that these enzymes might contribute to their infection mechanism and thus can constitute alternative drug targetspublishersversionpublishe

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Biomol NMR Assign (2007) 1:81–83 DOI 10.1007/s12104-007-9022-3We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo–Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments

Electron transfer complex between nitrous oxide reductase and cytochrome c552 from Pseudomonas nautica: kinetic, nuclear magnetic resonance, and docking studies

Biochemistry. 2008 Oct 14;47(41):10852-62. doi: 10.1021/bi801375qThe multicopper enzyme nitrous oxide reductase (N 2OR) catalyzes the final step of denitrification, the two-electron reduction of N 2O to N 2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome c 552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c 552, the reaction rate is dependent on the ET reaction and independent of the N 2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c 552 concentration dependence, we estimate the following kinetic parameters: K m c 552 = 50.2 +/- 9.0 muM and V max c 552 = 1.8 +/- 0.6 units/mg. The N 2O concentration dependence indicates a K mN 2 O of 14.0 +/- 2.9 muM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c 552 is used as the electron donor (p K a = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by (1)H NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c 552 is placed near a hydrophobic patch located around the CuA center

Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed α3DIV-L21C three-helical bundle peptide

PB would thank the PTNMRPhD (PD/00065/2013). VLP thanks the NIH for support (GM141086).The rational design and functionalization of small, simple, and stable peptides scaffolds is an attractive avenue to mimic catalytic metal-centres of complex proteins, relevant for the design of metalloenzymes with environmental, biotechnological and health impacts. The de novo designed α3DIV-L21C framework has a rubredoxin-like metal binding site and was used in this work to incorporate a Mo-atom. Thermostability studies using differential scanning calorimetry showed an increase of 4 °C in the melting temperature of the Mo-α3DIV-L21C when compared to the apo-α3DIV-L21C. Circular dichroism in the visible and far-UV regions corroborated these results showing that Mo incorporation provides stability to the peptide even though there were almost no differences observed in the secondary structure. A formal reduction potential of ∼ −408 mV vs. NHE, pH 7.6 was determined. Combining electrochemical results, EPR and UV–visible data we discuss the oxidation state of the molybdenum centre in Mo-α3DIV-L21C and propose that is mainly in a Mo (VI) oxidation state.publishersversionpublishe

Biochemical Characterization of the Copper Nitrite Reductase from Neisseria gonorrhoeae

Funding Information: This research was funded by Fundação para a Ciência e a Tecnologia, I.P. (FCT), through project grants to SRP (PTDC/BIA-BQM/29442/2017). This work was also supported by national funds from FCT in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. DSB and RNSO were supported by FCT through the scholarships UI/BD/151168/2021 and “Verão com Ciência2020”, respectively. Publisher Copyright: © 2023 by the authors.The copper-containing nitrite reductase from Neisseria gonorrhoeae has been shown to play a critical role in the infection mechanism of this microorganism by producing NO and abolishing epithelial exfoliation. This enzyme is a trimer with a type 1 copper center per subunit and a type 2 copper center in the subunits interface, with the latter being the catalytic site. The two centers were characterized for the first time by EPR and CD spectroscopy, showing that the type 1 copper center has a high rhombicity due to its lower symmetry and more tetragonal structure, while the type 2 copper center has the usual properties, but with a smaller hyperfine coupling constant (A// = 10.5 mT). The thermostability of the enzyme was analyzed by differential scanning calorimetry, which shows a single endothermic transition in the thermogram, with a maximum at 94 °C, while the CD spectra in the visible region indicate the presence of the type 1 copper center up to 80 °C. The reoxidation of the N. gonorrhoeae copper-containing nitrite reductase in the presence of nitrite were analyzed by visible spectroscopy and showed a pH dependence, being higher at pH 5.5–6.0. The high thermostability of this enzyme may be important to maintaining a high activity in the extracellular space and to making it less susceptible to denaturation and proteolysis, contributing to the proliferation of N. gonorrhoeae.publishersversionpublishe

Structural Characterization of <i>Neisseria gonorrhoeae</i> Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases

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

The Tetranuclear Copper-Sulfide Center of Nitrous Oxide Reductase

Nitrous oxide reductase catalyzes the reduction of nitrous oxide (N2O) to dinitrogen (N2) and water at a catalytic tetranuclear copper sulfide center, named CuZ, overcoming the high activation energy of this reaction. In this center each Cu atom is coordinated by two imidazole rings of histidine side-chains, with the exception of one named CuIV. This enzyme has been isolated with CuZ in two forms CuZ(4Cu1S) and CuZ(4Cu2S), which differ in the CuI-CuIV bridging ligand, leading to considerable differences in their spectroscopic and catalytic properties. The Cu atoms in CuZ(4Cu1S) can be reduced to the [4Cu1+] oxidation state, and its catalytic properties are compatible with the nitrous oxide reduction rates of whole cells, while in CuZ(4Cu2S) they can only be reduced to the [1Cu2C-3Cu1C] oxidation state, which has a very low turnover number. The catalytic cycle of this enzyme has been explored and one of the intermediates, CuZ0, has recently been identified and shown to be in the [1Cu2+-3Cu1+] oxidation state. Contrary to CuZ(4Cu2S), CuZ0 is rapidly reduced intramolecularly by the electron transferring center of the enzyme, CuA, to [4Cu1+] by a physiologically relevant redox partner. The three-dimensional structure of nitrous oxide reductase with the CuZ center either as CuZ(4Cu1S) or as CuZ(4Cu2S) shows that it is a unique functional dimer, with the CuZ of one subunit receiving electrons from CuA of the other subunit. The complex nature of this center has posed some questions relative to its assembly, which are only partially answered, as well as to which is the active form of CuZ in vivo. The structural, spectroscopic, and catalytic features of the two forms of CuZ will be addressed here, as well as its assembly. The understanding of its catalytic features, activation, and assembly is essential to develop strategies to decrease the release of nitrous oxide to the atmosphere and to reduce its concentration in the stratosphere, as well as to serve as inspiration to synthetic inorganic chemists to develop new models of this peculiar and challenging copper sulfide center.publishersversionpublishe