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Walter G. Zumft - One of the best experts on this subject based on the ideXlab platform.
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Nature’s way of handling a greenhouse gas: the copper-sulfur cluster of purple nitrous oxide Reductase
Biological Chemistry, 2012Co-Authors: Anja Wüst, Peter M. H. Kroneck, Walter G. Zumft, Lisa Schneider, Anja Pomowski, Oliver EinsleAbstract:: The tetranuclear Cu(Z) cluster is the unique active site of nitrous oxide Reductase, the enzyme that catalyzes the reduction of nitrous oxide to dinitrogen as the final reaction in bacterial denitrification. Three-dimensional structures of orthologs of the enzyme from a variety of different bacterial species were essential steps in the elucidation of the properties of this center. However, while structural data first revealed and later confirmed the presence of four copper ions in spectroscopically distinct forms of Cu(Z), the exact structure and stoichiometry of the cluster showed significant variations. A ligand bridging ions Cu(Z1) and Cu(Z2) was initially assigned as a water or hydroxo species in the structures from Pseudomonas nautica (now Marinobacter hydrocarbonoclasticus) and Paracoccus denitrificans. This ligand was absent in a structure from 'Achromobacter cycloclastes', and could be reconstituted by iodide that acted as an inhibitor of catalysis. A recent structure of anoxically isolated nitrous oxide Reductase from Pseudomonas stutzeri revealed the bridging ligand to be sulfide, S2-, and showed an unprecedented side-on mode of nitrous oxide binding to this form of Cu(Z).
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N2O binding at a [4Cu:2S] copper-sulphur cluster in nitrous oxide Reductase.
Nature, 2011Co-Authors: Anja Pomowski, Walter G. Zumft, Peter M. H. Kroneck, Oliver EinsleAbstract:Nitrous oxide (N2O) gas, generated by both natural and anthropogenic processes, is a potentially important greenhouse gas because of its heat-trapping effects and its persistence in the atmosphere. Some bacteria use nitrous oxide Reductase (N2OR) to convert N2O to dinitrogen gas. The X-ray crystal structure of N2OR from Pseudomonas stutzeri has now been determined in the presence of N2O and, to generate the active purple form of the enzyme, in the absence of O2. The results reveal the nature of the copper–sulphur cluster at the substrate-binding site and show how N2O interacts with this complex metal centre. Nitrous oxide (N2O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO2 300-fold1,2. In bacterial denitrification, N2O is reduced to N2 by the copper-dependent nitrous oxide Reductase (N2OR)3. This enzyme carries the mixed-valent CuA centre and the unique, tetranuclear CuZ site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its CuZ site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N2OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N2O-pressurized crystals. The active CuZ cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N2O bound side-on at CuZ, in close proximity to CuA. With the substrate located between the two clusters, electrons are transferred directly from CuA to N2O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N2OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N2O bound to its Reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase4.
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Crystallization of purple nitrous oxide Reductase from Pseudomonas stutzeri.
Acta crystallographica. Section F Structural biology and crystallization communications, 2010Co-Authors: Anja Pomowski, Walter G. Zumft, Peter M. H. Kroneck, Oliver EinsleAbstract:Nitrous oxide Reductase (N(2)OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N(2)OR crystallized in either space group P1 or P6(5). Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6(5) contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å.
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Biogenesis of the bacterial respiratory CuA, Cu-S enzyme nitrous oxide Reductase.
Journal of Molecular Microbiology and Biotechnology, 2006Co-Authors: Walter G. ZumftAbstract:Nitrous oxide Reductase (NosZ, EC 1.7.99.6) is the terminal oxidoReductase of a respiratory electron transfer chain that transforms nitrous oxide to dinitrogen. The enzyme carries six Cu atoms. Two ar
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Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome cd1 Biosynthesis in Pseudomonas stutzeri
Journal of Bacteriology, 2001Co-Authors: Mari P. Heikkilä, Ulrike Honisch, Patrick Wunsch, Walter G. ZumftAbstract:By transforming N2O to N2, the multicopper enzyme nitrous oxide Reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O Reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N2O Reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a Reductase-overproducing strain, we show a function for TatE in N2O Reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite Reductase system. The cytochrome cd1 nitrite Reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.
Peter M. H. Kroneck - One of the best experts on this subject based on the ideXlab platform.
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Walking the seven lines: binuclear copper A in cytochrome c oxidase and nitrous oxide Reductase
JBIC Journal of Biological Inorganic Chemistry, 2018Co-Authors: Peter M. H. KroneckAbstract:The enzymes nitrous oxide Reductase (N2OR) and cytochrome c oxidase (COX) are constituents of important biological processes. N2OR is the terminal Reductase in a respiratory chain converting N_2O to N_2 in denitrifying bacteria; COX is the terminal oxidase of the aerobic respiratory chain of certain bacteria and eukaryotic organisms transforming O_2 to H_2O accompanied by proton pumping. Different spectroscopies including magnetic resonance techniques, were applied to show that N2OR has a mixed-valent Cys-bridged [Cu^1.5+(CyS)_2Cu^1.5+] copper site, and that such a binuclear center, called CuA, does also exist in COX. A sequence motif shared between the CuA center of N2OR and the subunit II of COX raises the issue of a putative evolutionary relationship of the two enzymes. The suggestion of a binuclear CuA in COX, with one unpaired electron delocalized between two equivalent Cu nuclei, was difficult to accept originally, even though regarded as a clever solution to many experimental observations. This minireview in honor of Helmut Sigel traces several of the critical steps forward in understanding the nature of CuA in N2OR and COX, and discusses its unique electronic features to some extent including the contributions made by the development of methodology and the discovery of a novel multi-copper enzyme. Graphical Abstract Left: X-band (9.130 GHz) and C-band (4.530 GHz, 1st harmonic display of experimental spectrum) EPR spectra of bovine heart cytochrome c oxidase, recorded at 20K. Right: Ribbon presentation of the CuA domain in cytochrome c oxidase and nitrous oxide Reductase.
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Nature’s way of handling a greenhouse gas: the copper-sulfur cluster of purple nitrous oxide Reductase
Biological Chemistry, 2012Co-Authors: Anja Wüst, Peter M. H. Kroneck, Walter G. Zumft, Lisa Schneider, Anja Pomowski, Oliver EinsleAbstract:: The tetranuclear Cu(Z) cluster is the unique active site of nitrous oxide Reductase, the enzyme that catalyzes the reduction of nitrous oxide to dinitrogen as the final reaction in bacterial denitrification. Three-dimensional structures of orthologs of the enzyme from a variety of different bacterial species were essential steps in the elucidation of the properties of this center. However, while structural data first revealed and later confirmed the presence of four copper ions in spectroscopically distinct forms of Cu(Z), the exact structure and stoichiometry of the cluster showed significant variations. A ligand bridging ions Cu(Z1) and Cu(Z2) was initially assigned as a water or hydroxo species in the structures from Pseudomonas nautica (now Marinobacter hydrocarbonoclasticus) and Paracoccus denitrificans. This ligand was absent in a structure from 'Achromobacter cycloclastes', and could be reconstituted by iodide that acted as an inhibitor of catalysis. A recent structure of anoxically isolated nitrous oxide Reductase from Pseudomonas stutzeri revealed the bridging ligand to be sulfide, S2-, and showed an unprecedented side-on mode of nitrous oxide binding to this form of Cu(Z).
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N2O binding at a [4Cu:2S] copper-sulphur cluster in nitrous oxide Reductase.
Nature, 2011Co-Authors: Anja Pomowski, Walter G. Zumft, Peter M. H. Kroneck, Oliver EinsleAbstract:Nitrous oxide (N2O) gas, generated by both natural and anthropogenic processes, is a potentially important greenhouse gas because of its heat-trapping effects and its persistence in the atmosphere. Some bacteria use nitrous oxide Reductase (N2OR) to convert N2O to dinitrogen gas. The X-ray crystal structure of N2OR from Pseudomonas stutzeri has now been determined in the presence of N2O and, to generate the active purple form of the enzyme, in the absence of O2. The results reveal the nature of the copper–sulphur cluster at the substrate-binding site and show how N2O interacts with this complex metal centre. Nitrous oxide (N2O) is generated by natural and anthropogenic processes and has a critical role in environmental chemistry. It has an ozone-depleting potential similar to that of hydrochlorofluorocarbons as well as a global warming potential exceeding that of CO2 300-fold1,2. In bacterial denitrification, N2O is reduced to N2 by the copper-dependent nitrous oxide Reductase (N2OR)3. This enzyme carries the mixed-valent CuA centre and the unique, tetranuclear CuZ site. Previous structural data were obtained with enzyme isolated in the presence of air that is catalytically inactive without prior reduction. Its CuZ site was described as a [4Cu:S] centre, and the substrate-binding mode and reduction mechanism remained elusive. Here we report the structure of purple N2OR from Pseudomonas stutzeri, handled under the exclusion of dioxygen, and locate the substrate in N2O-pressurized crystals. The active CuZ cluster contains two sulphur atoms, yielding a [4Cu:2S] stoichiometry; and N2O bound side-on at CuZ, in close proximity to CuA. With the substrate located between the two clusters, electrons are transferred directly from CuA to N2O, which is activated by side-on binding in a specific binding pocket on the face of the [4Cu:2S] centre. These results reconcile a multitude of available biochemical data on N2OR that could not be explained by earlier structures, and outline a mechanistic pathway in which both metal centres and the intervening protein act in concert to achieve catalysis. This structure represents the first direct observation, to our knowledge, of N2O bound to its Reductase, and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates. The principle of using distinct clusters for substrate activation and for reduction may be relevant for similar systems, in particular nitrogen-fixing nitrogenase4.
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Crystallization of purple nitrous oxide Reductase from Pseudomonas stutzeri.
Acta crystallographica. Section F Structural biology and crystallization communications, 2010Co-Authors: Anja Pomowski, Walter G. Zumft, Peter M. H. Kroneck, Oliver EinsleAbstract:Nitrous oxide Reductase (N(2)OR) from Pseudomonas stutzeri catalyzes the final step in denitrification: the two-electron reduction of nitrous oxide to molecular dinitrogen. Crystals of the enzyme were grown under strict exclusion of dioxygen by sitting-drop vapour diffusion using 2R,3R-butanediol as a cryoprotectant. N(2)OR crystallized in either space group P1 or P6(5). Interestingly, the key determinant for the resulting space group was the crystallization temperature. Crystals belonging to space group P1 contained four 130 kDa dimers in the asymmetric unit, while crystals belonging to space group P6(5) contained a single dimer in the asymmetric unit. Diffraction data were collected to resolutions better than 2 Å.
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ELECTRON PARAMAGNETIC RESONANCE POWER STUDIES ON THE MIXED-VALENCE ELECTRON TRANSFER CENTRE OF NITROUS OXIDE Reductase : PRESENCE OF A MODIFIED CUA SI TE IN THE ENZYME FROM PSEUDOMONAS STUTZERI
Molecular Physics, 1998Co-Authors: William E. Antholine, Peter M. H. Kroneck, Walter G. ZumftAbstract:The difference spectrum obtained by subtracting a low power EPR signal from a high power EPR signal from the copper enzyme nitrous oxide Reductase (N2OR) is assigned to a mixed-valence [(Cu(1.5+) … Cu(1.5+)] signal. The relaxation of the signal is much faster than the relaxation of the binuclear mixed-valence [CuA(1.5+) … CuA(1.5+)] signal from the electron transfer site in N2OR. This fast relaxing signal is denoted by A∗ and referred to as the underlying signal. It is suggested that this signal is a modified form of the mixed-valence [CuA(1.5+) … CuA(1.5+)] electron transfer site.
William B. Tolman - One of the best experts on this subject based on the ideXlab platform.
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Copper-sulfur complexes supported by N-donor ligands : Towards models of the Cuz site in nitrous oxide Reductase
Inorganica Chimica Acta, 2008Co-Authors: John T. York, Itsik Bar-nahum, William B. TolmanAbstract:Abstract The distinctive structure of the [(his) 7 Cu 4 (μ-S)] n + cluster in the “Cu Z ” active site of nitrous oxide Reductase and the intriguing mechanistic hypotheses for its catalytic reactivity provide inspiration for synthetic model studies aimed at characterizing relevant copper–sulfur compounds and obtaining fundamental insights into structure and bonding. In this brief review, we summarize such studies that have focused on the synthesis and characterization of a range of copper–sulfur complexes supported by N-donor ligands. Compounds with variable nuclearities and sulfur redox levels have been isolated, with the nature of the species obtained being dependent on the supporting ligand, sulfur source, and the reaction conditions. Spectroscopic data and theoretical calculations, often performed with a view toward drawing comparisons to oxygen analogs, have provided insight into the nature of the copper–sulfur bonding interactions in the complexes.
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Copper–sulfur complexes supported by N-donor ligands: Towards models of the CuZ site in nitrous oxide Reductase
Inorganica Chimica Acta, 2008Co-Authors: John T. York, Itsik Bar-nahum, William B. TolmanAbstract:Abstract The distinctive structure of the [(his) 7 Cu 4 (μ-S)] n + cluster in the “Cu Z ” active site of nitrous oxide Reductase and the intriguing mechanistic hypotheses for its catalytic reactivity provide inspiration for synthetic model studies aimed at characterizing relevant copper–sulfur compounds and obtaining fundamental insights into structure and bonding. In this brief review, we summarize such studies that have focused on the synthesis and characterization of a range of copper–sulfur complexes supported by N-donor ligands. Compounds with variable nuclearities and sulfur redox levels have been isolated, with the nature of the species obtained being dependent on the supporting ligand, sulfur source, and the reaction conditions. Spectroscopic data and theoretical calculations, often performed with a view toward drawing comparisons to oxygen analogs, have provided insight into the nature of the copper–sulfur bonding interactions in the complexes.
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Using synthetic chemistry to understand copper protein active sites: a personal perspective
JBIC Journal of Biological Inorganic Chemistry, 2006Co-Authors: William B. TolmanAbstract:The results of studies performed in the author’s laboratory are surveyed, with particular emphasis on demonstrating the value of a multidisciplinary synthetic modeling approach for discovering new and unusual chemistry helpful for understanding the properties of the active sites of copper proteins or assessing the feasibility of mechanistic pathways they might follow during catalysis. The discussion focuses on the progress made to date toward comprehending the nitrite Reductase catalytic site and mechanism, the electronic structures of copper thiolate electron transfer centers, the sulfido-bridged “Cu_Z” site in nitrous oxide Reductase, and the processes of dioxygen binding and activation by mono- and dicopper centers in oxidases and oxygenases.
Thomas C Hollocher - One of the best experts on this subject based on the ideXlab platform.
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Denitrification enzymes of Bacillus stearothermophilus
Fems Microbiology Letters, 1993Co-Authors: Tay P. Ho, Alison M Jones, Thomas C HollocherAbstract:The Reductases of the denitrification pathway of Bacillus stearothermophilus, a thermophilic denitrifier, were surveyed in vitro. Nitrate, nitrite and nitric oxide Reductases were found to be membrane-bound, and nitrite Reductase may be a copper-containing enzyme by virtue of its rapid inactivation by the chelator, diethyldithiocarbamate. Nitric oxide Reductase exhibited an optimal rate at about pH 7 rather than 5.0–5.5, as expected from the enzyme utilized by several mesophiles. Nitrous oxide Reductase was not detected in assays employing reduced benzyl or methyl viologen, in spite of the appreciable N2O-uptake activity of intact cells.
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A Diffusion-Controlled Step in the Catalytic Cycle of Nitrous Oxide Reductase from Wolinella succinogenes
Archives of Biochemistry and Biophysics, 1993Co-Authors: Chengeto Mukonoweshuro, Thomas C HollocherAbstract:Abstract The viscosity test was applied to the highly active nitrous oxide Reductase purified from Wolinella succinogenes to determine whether the catalytic cycle may contain a diffusion-controlled step. For this test, the benzyl viologen cation (BV + )-N 2 O oxidoReductase reaction, which exhibits a k catBV / K m BV ∼2 × 10 8 M −1 s −1 at 23°C and pH 7.1, was run in solutions of nominally nondenaturing viscogens that were used to increase microviscosity. The parameters k catN 2 O and k catBV were unaffected by viscosity in the range of viscogen concentrations that were not inhibitory and where the data were well behaved. K m BV , but not K m N 2 O , was observed to increase linearly with relative viscosity with a slope of 0.14 for all viscogens surveyed. The results, when considered in the context of a plausible kinetic model for the BV + -N 2 O oxidoReductase reaction, suggest that one of the two one-electron reactions between BV + and enzyme is diffusion-controlled but only partially rate determining. The ratio of the second-order-rate constants for these two one-electron steps is estimated to be about 6.1, and the larger rate constant to be about 1.1 × 10 9 M −1 s −1 . There would appear to be no diffusion-controlled step associated with the half-reaction which results in reduction of N 2 O to N 2 .
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Interaction of dichloromethane (methylene chloride) with the nitrous oxide Reductase from Wolinella succinogenes
World Journal of Microbiology & Biotechnology, 1993Co-Authors: Chunqing Zhang, Thomas C HollocherAbstract:Nitrous oxide Reductase from Wolinella succinogenes was tested for benzyl viologen cation (BV+)-chlorinated methane oxidoReductase activity, using di-, tri- and tetra-chloromethanes, and for the inhibition of BV+-N2O oxidoReductase activity by these chloromethanes. No BV+-chlorinated methane oxidoReductase activity was detected. Any such activity, if it exists, must be less than 0.1% of the BV+-N2O oxidoReductase activity of the enzyme. Inhibition of the BV+-N2O oxidoReductase activity by dichloromethane was detected and was apparently reversible and non-competitive, as is the case with the small metal-ligand type inhibitors of the enzyme (e.g. acettlene, azide, cyanide and carbon monoxide). Trichloromethane was a weaker inhibitor and inhibition was not detected with tetrachloromethane.
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the reaction of reduced cytochromes c with nitrous oxide Reductase of wolinella succinogenes
Biochimica et Biophysica Acta, 1993Co-Authors: Chunsheng Zhang, Thomas C HollocherAbstract:Abstract Kinetic studies were carried out on the oxidation of dithionite-reduced, monoheme cytochromes c by nitrous oxide Reductase from Wolinella succinogenes. These reduced cytochrome c-N2O oxidoReductase systems showed second-order kinetics, first-order each in reduced cytochrome c and enzyme, at concentrations of reduced cytochrome c between 1 and 10 μM. The second-order rate constant at 25°C and pH 6.8, k2, was 3.1 · 106, 9.3 · 104 and about 1 · 104 M−1 s−1 for cytochrome c from W. succinogenes, horse heart and Pseudomonas aeruginosa (cytochrome c-551), respectively, at enzyme concentrations ≦ 12, ≦3 and ≦ 11 nM, respectively. With horse-heart cytochrome c and cytochrome c-551, k2 diminished substantially at higher enzyme concentrations. Evidence for reaction via an E-S (Michaelis) complex was not obtained. Unlike systems for which the radical cation of benzyl viologen (BV·+) served as reducing agent, nitrous oxide Reductase failed to show turnover-dependent inactivation when a reduced cytochrome c was the reductant. The system thus mimicked the ability of nitrous oxide Reductase to turnover in vivo without inactivation. Cytochrome c oxidase activity of nitrous oxide Reductase was not observed when O2 replaced N2O. Similarly, BV·+-CO2 oxidoReductase activity was not detected with bicarbonate buffer (CO2 is isoelectronic with N2O). The values for Mr (9214), optical extinction coefficients and amino acid composition of the monoheme cytochrome c of W. succinogenes were found to be somewhat different from published values.
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An apparently allosteric effect involving N2O with the nitrous oxide Reductase from Wolinella succinogenes
Biochemical and Biophysical Research Communications, 1992Co-Authors: Chunqing Zhang, Alison M Jones, Thomas C HollocherAbstract:Summary It was shown that kcat for the benzyl viologen cation (BV+)-N2O oxidoReductase activity of nitrous oxide Reductase from Wolinella succinogenes was 2–3 times greater at high N2O concentrations than at low. This effect of N2O on kcat exhibited a titration curve implicating a single secondary binding site for N2O with a Kd of 130–200 μM (Km with respect to N2O is about 2.5 μM). This work represents the first evidence of an apparently allosteric kinetic effect among nitrous oxide Reductases. Its possible cause is discussed. BV+ was generated in these kinetic studies by addition of sub-stoichiometric amounts of dithionite. This means of reduction proved to be superior to the photochemical generation of BV+ that had been used previously with the enzyme. Mass spectrometric measurements suggested that the Mr of the subunit of the enzyme is about 95,500 rather than 88,000.
Isabel Moura - One of the best experts on this subject based on the ideXlab platform.
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Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide Reductase from Marinobacter hydrocarbonoclasticus - An electrochemical study.
Bioelectrochemistry, 2020Co-Authors: Cíntia Carreira, Sofia R. Pauleta, Margarida M. Correia Dos Santos, Isabel MouraAbstract:Abstract 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.
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The catalytic cycle of nitrous oxide Reductase - The enzyme that catalyzes the last step of denitrification.
Journal of Inorganic Biochemistry, 2017Co-Authors: Cíntia Carreira, Sofia R. Pauleta, Isabel MouraAbstract:Abstract The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide Reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide Reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper “CuZ center”. This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two “CuZ center” forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic “CuZ center” is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide Reductase and its intermediate species.
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Spectroscopic Definition of the CuZ° Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into the Catalytic Mechanism
Journal of the American Chemical Society, 2017Co-Authors: Esther M. Johnston, Sofia R. Pauleta, Simone Dell’acqua, Isabel Moura, Cíntia Carreira, Edward I. SolomonAbstract:Spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of CuZ°, an intermediate form of the Cu4S active site of nitrous oxide Reductase (N2OR) that is observed in single turnover of fully reduced N2OR with N2O. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that CuZ° is a 1-hole (i.e., 3CuICuII) state with spin density delocalized evenly over CuI and CuIV. Resonance Raman spectroscopy shows two Cu–S vibrations at 425 and 413 cm–1, the latter with a −3 cm–1 O18 solvent isotope shift. DFT calculations correlated to these spectral features show that CuZ° has a terminal hydroxide ligand coordinated to CuIV, stabilized by a hydrogen bond to a nearby lysine residue. CuZ° can be reduced via electron transfer from CuA using a physiologically relevant reductant. We obtain a lower limit on the rate of this intramolecular electron transfer (IET) that is >104 faster than the unobs...
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CHAPTER 7:Insights into Nitrous Oxide Reductase
Metalloenzymes in Denitrification, 2016Co-Authors: Sofia R. Pauleta, Cíntia Carreira, Isabel MouraAbstract:Nitrous oxide Reductase is the enzyme that catalyses the last step of the denitrification pathway, reducing nitrous oxide to dinitrogen gas. This enzyme is a functional homodimer with two copper centres, CuA and a “CuZ centre”, located in different domains. The CuA centre is the electron transferring centre, while the catalytic centre is the “CuZ centre”, a unique metal centre in biology—a tetranuclear copper centre with a µ4-bridging sulphide. The enzyme has been isolated with the “CuZ centre” in two different forms, CuZ(4Cu2S) and CuZ*(4Cu1S), with the first presenting an additional µ2-sulphur atom as a bridging ligand between CuI and CuIV of the “CuZ centre”, whereas the second form was identified as a water-derived molecule. Spectroscopic analysis of CuZ*(4Cu1S), together with computational studies, indicated that there is a hydroxide bound to CuI. Genomic analysis has identified the presence of two different types of nitrous oxide Reductase, the typical and “atypical”, with a single member of the last group having been isolated to date, from Wolinella succinogenes. Thus, here the structure of the “typical” nitrous oxide Reductase with either CuZ(4Cu2S) or CuZ*(4Cu1S), as well as its spectroscopic and catalytic properties, will be discussed.
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Protonation state of the Cu4S2 CuZ site in nitrous oxide Reductase: redox dependence and insight into reactivity.
Chemical Science, 2015Co-Authors: Esther M. Johnston, Sofia R. Pauleta, Simone Dell’acqua, Isabel Moura, Edward I. SolomonAbstract:Spectroscopic and computational methods have been used to determine the protonation state of the edge sulfur ligand in the Cu4S2 CuZ form of the active site of nitrous oxide Reductase (N2OR) in its 3CuICuII (1-hole) and 2CuI2CuII (2-hole) redox states. The EPR, absorption, and MCD spectra of 1-hole CuZ indicate that the unpaired spin in this site is evenly delocalized over CuI, CuII, and CuIV. 1-hole CuZ is shown to have a μ2-thiolate edge ligand from the observation of S–H bending modes in the resonance Raman spectrum at 450 and 492 cm−1 that have significant deuterium isotope shifts (−137 cm−1) and are not perturbed up to pH 10. 2-hole CuZ is characterized with absorption and resonance Raman spectroscopies as having two Cu–S stretching vibrations that profile differently. DFT models of the 1-hole and 2-hole CuZ sites are correlated to these spectroscopic features to determine that 2-hole CuZ has a μ2-sulfide edge ligand at neutral pH. The slow two electron (+1 proton) reduction of N2O by 1-hole CuZ is discussed and the possibility of a reaction between 2-hole CuZ and O2 is considered.