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Projeto de investigação
INSIGHTS INTO THE STRUCTURE AND REACTIVITY OF THE CATALYTIC SITE OF NITROUS OXIDE REDUCTASE
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The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase
Publication . Carreira, Cíntia; Nunes, Rute F.; Mestre, Olga; Moura, Isabel; Pauleta, Sofia R.; LAQV@REQUIMTE; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Springer
Abstract: Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state. Graphic abstract: [Figure not available: see fulltext.].
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.
The bacterial MrpORP is a novel Mrp/NBP35 protein involved in iron-sulfur biogenesis
Publication . Pardoux, Romain; Fiévet, Anouchka; Carreira, Cíntia; Brochier-Armanet, Céline; Valette, Odile; Dermoun, Zorah; Py, Béatrice; Dolla, Alain; Pauleta, Sofia R.; Aubert, Corinne; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Nature Publishing Group
Despite recent advances in understanding the biogenesis of iron-sulfur (Fe-S) proteins, most studies focused on aerobic bacteria as model organisms. Accordingly, multiple players have been proposed to participate in the Fe-S delivery step to apo-target proteins, but critical gaps exist in the knowledge of Fe-S proteins biogenesis in anaerobic organisms. Mrp/NBP35 ATP-binding proteins are a subclass of the soluble P-loop containing nucleoside triphosphate hydrolase superfamily (P-loop NTPase) known to bind and transfer Fe-S clusters in vitro. Here, we report investigations of a novel atypical two-domain Mrp/NBP35 ATP-binding protein named MrpORP associating a P-loop NTPase domain with a dinitrogenase iron-molybdenum cofactor biosynthesis domain (Di-Nase). Characterization of full length MrpORP, as well as of its two domains, showed that both domains bind Fe-S clusters. We provide in vitro evidence that the P-loop NTPase domain of the MrpORP can efficiently transfer its Fe-S cluster to apo-target proteins of the ORange Protein (ORP) complex, suggesting that this novel protein is involved in the maturation of these Fe-S proteins. Last, we showed for the first time, by fluorescence microscopy imaging a polar localization of a Mrp/NBP35 protein.
Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide reductase from Marinobacter hydrocarbonoclasticus – An electrochemical study
Publication . Carreira, Cíntia; dos Santos, Margarida M. C.; Pauleta, Sofia R.; Moura, Isabel; LAQV@REQUIMTE; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier Science B.V., Amsterdam.
Reduction of N2O to N2 is catalysed by nitrous oxide reductase in the last step of the denitrification pathway. This multicopper enzyme has an electron transferring centre, CuA, and a tetranuclear copper-sulfide catalytic centre, “CuZ”, which exists as CuZ*(4Cu1S) or CuZ(4Cu2S). The redox behaviour of these metal centres in Marinobacter hydrocarbonoclasticus nitrous oxide reductase was investigated by potentiometry and for the first time by direct electrochemistry. The reduction potential of CuA and CuZ(4Cu2S) was estimated by potentiometry to be +275 ± 5 mV and +65 ± 5 mV vs SHE, respectively, at pH 7.6. A proton-coupled electron transfer mechanism governs CuZ(4Cu2S) reduction potential, due to the protonation/deprotonation of Lys397 with a pKox of 6.0 ± 0.1 and a pKred of 9.2 ± 0.1. The reduction potential of CuA, in enzyme samples with CuZ*(4Cu1S), is controlled by protonation of the coordinating histidine residues in a two-proton coupled electron transfer process. In the cyclic voltammograms, two redox pairs were identified corresponding to CuA and CuZ(4Cu2S), with no additional signals being detected that could be attributed to CuZ*(4Cu1S). However, an enhanced cathodic signal for the activated enzyme was observed under turnover conditions, which is explained by the binding of nitrous oxide to CuZ0(4Cu1S), an intermediate species in the catalytic cycle.
Insights into the structure and reactivity of the catalytic site of nitrous oxide reductase
Publication . Carreira, Cíntia Catarina Sousa; Moura, Isabel; Pauleta, Sofia; Einsle, Oliver
Substantial part of the emissions of nitrous oxide (N2O), a powerful greenhouse gas, to the atmosphere comes from the incomplete denitrification in bacteria. N2O can only be detoxified by nitrous oxide reductase (N2OR), which catalyzes the last step of this pathway. This enzyme contains two distinct centers per monomer: CuA, the electron transfer center and “CuZ”, a tetranuclear copper-sulfide center, which can exists in two forms CuZ(4Cu2S) and CuZ*(4Cu1S).
Most of the studies on the denitrification pathway have used soil denitrifying bacteria as models, while marine bacteria are understudied. This thesis presents an analysis of denitrification pathway of Marinobacter hydrocarbonoclasticus a marine bacterium capable of respiring nitrate under oxygen-limiting conditions. Here, the effect of pH (6.5, 7.5 and 8.5) on the denitrification pathway of this organism, as well as on the N2OR isolated from each of those growths, was investigated. These enzymes were characterized through biochemical, spectroscopic and structural studies.
The expression profile of genes encoding the enzymes and accessory proteins involved in denitrification was analyzed, together with quantification of the by-products, nitrate and nitrite. These results showed lower levels of nirS expression at pH 6.5, which correlates with the accumulation of nitrite detected. In parallel, whole-cells reduction rates of NO and N2O demonstrated that denitrification is impaired at more acidic conditions, as the whole-cells are not able to reduce external N2O when grown at pH 6.5.
The N2OR isolated from each growth exhibits differences at the “CuZ center”. At acidic growth conditions, N2OR has “CuZ center” mainly as CuZ*(4Cu1S), whereas when isolated from growths at 7.5 and 8.5, it is mainly as CuZ(4Cu2S). This was supported by spectroscopic data, sulfide quantification, and inspection of “CuZ center” X-ray structure, demonstrating the presence of an additional sulfur atom in the CuZ(4Cu2S) form. The effect of exogenous ligands on both forms of the “CuZ center” was re-visited and clarified.
Direct electrochemistry of N2OR is reported for the first time, with the two signals observed, assigned to CuA and CuZ(4Cu2S) centers, with reduction potentials being in line with the ones determined by potentiometry (272 ± 10 mV and 65 ± 10 mV vs SHE at pH 7.6, respectively). This form of N2OR has lower specific activity (0.004 ± 0.001 U/mg) in the presence of physiological electron donor, cytochrome c552, compared to a N2OR with CuZ*(4Cu1S) (1.25 ± 0.07 U/mg).
Fully reduced CuZ*(4Cu1S) is catalytically competent and in the presence of a stoichiometric amount of N2O originates CuZº intermediate. CuZº species can be reduced through intramolecular electron transference (IET) from CuA center, in a reaction 104 faster than IET in the CuZ*(4Cu1S). In the absence of substrate or electrons a novel “CuZ center” intermediate species is formed with a maximum absorption band at 617 nm, and having a [1Cu2+-3Cu1+] oxidation state. These studies shed new lights on the catalytic cycle, which was reassessed and discussed here.
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SFRH/BD/87898/2012