Nitric Oxide Reductase Activity Research Articles

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

Investigating nitrous oxide emissions and mechanisms in kitchen waste composting with leachate reuse

Solid-liquid mixed composting is a novel approach for handling kitchen waste (KW). In this study, KW was employed as composting materials to explore the nitrous oxide (N2O) emissions and corresponding mechanisms associated with the reuse of three supplementary liquids (CK: deionized water; T1: kitchen waste composting leachate (KWCL); T2: kitchen waste leachate (KWL)) during the composting process. The analysis included N2O emissions, microbial community dynamics, and nitrogen metabolism pathways. The findings demonstrated that the addition of KWCL (T1) and KWL (T2) significantly increased the nutrient content in the compost. Notably, T1 exhibited significantly higher cumulative N2O emissions than CK and T2 (P < 0.01). Both CK and T2 demonstrated more robust microbial safety, while the addition of KWCL (T1) led to more significant alterations in the structure of bacterial and fungal communities. Denitrification was identified as the primary pathway for N2O emissions during the thermophilic period. Nitrous oxide reductase activity was significantly lower in T1 than in T2 (P < 0.05), and the abundance of the nosZ gene in T1 (3.39 %) was lower than that in CK (4.18 %) and T2 (4.51 %), contributing to higher N2O emissions. Correlation analysis revealed the vital role of bacteria in N2O production, while bacterial and fungal communities exhibited positive correlations with variables such as carbon-to-nitrogen ratio and total carbon. Environmental factors were identified as key drivers of N2O emissions (r = 0.837, P < 0.05). These findings provided valuable insights for the sustainable management and recycling of KW.

Structural and biochemical elucidation of class I hybrid cluster protein natively extracted from a marine methanogenic archaeon.

Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from Desulfovibrio species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from Methanococcales and many Bacteria corroborates this similarity. Indeed, Methanococcales HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in M. thermolithotrophicus, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.

Particle Swarm Fitting of Spin Hamiltonians: Magnetic Circular Dichroism of Reduced and NO-Bound Flavodiiron Protein.

A particle swarm optimization (PSO) algorithm is described for the fitting of ground-state spin Hamiltonian parameters from variable-temperature/variable-field (VTVH) magnetic circular dichroism (MCD) data. This PSO algorithm is employed to define the ground state of two catalytic intermediates from a flavodiiron protein (FDP), a class of enzymes with nitric oxide reductase activity. The bimetallic iron active site of this enzyme proceeds through a biferrous intermediate and a mixed ferrous-{FeNO}7 intermediate during the catalytic cycle, and the MCD spectra of these intermediates are presented and analyzed. The fits of the spin Hamiltonians are shown to provide important geometric and electronic insight into these species that is compared and contrasted with previous reports.

Rhizobium inoculation and exogenous melatonin synergistically increased thermotolerance by improving antioxidant defense, photosynthetic efficiency, and nitro-oxidative homeostasis in Medicago truncatula

Global warming negatively affects plant growth due to the detrimental effects of high temperature-induced heat stress. Rhizobium inoculation (RI) and exogenous melatonin (MT) have shown a positive role in resisting abiotic stress. However, their synergistic effect on avoiding heat-induced damages in Medicago truncatula has not been studied yet. Hence, the objective of the present study was to evaluate the impact of these amendments (RI and MT) to ameliorate the heat damages in Medicago truncatula. The study was comprised of two factors: (1) heat-induced stress: (i) optimum temperature (26 ± 1°C): (23 ± 1°C) (day: night), (ii) moderate heat (35 ± 1°C): (28 ± 1°C), and (iii) severe heat (41 ± 1°C): (35 ± 1°C) for 72 h, and (2) amendments: (i) no RI + no MT (NRI + NMT), (ii) Rhizobium inoculation (RI), (iii) 60 μM melatonin (MT), and (iii) RI + MT. Results showed that the combined application of RI and MT was better than their individual applications, as it prevented heat-induced membrane damages by declining the hydrogen peroxide (34.22% and 29.78%), superoxide anion radical (29.49% and 26.71%), malondialdehyde contents (26.43% and 21.96%), and lipoxygenase activity (44.75% and 25.51%) at both heat stress levels as compared to NRI + NMT. Moreover, RI + MT treated plants showed higher antioxidative and methylglyoxal detoxification enzymes (Gly I and Gly II) activities under heat stress. While, NRI + NMT treated plants showed a higher level of methylglyoxal contents (47.99% and 46.71%) under both levels of heat stress. Relative to NRI + NMT plants, RI + MT pretreated plants exhibited improved heat tolerance as indicated by higher chlorophyll (37.42% and 43.52%), carotenoid contents (32.41% and 47.08%), and photosynthetic rate (42.62% and 64.63%), under moderate and severe heat stress, respectively. Furthermore, RI + MT pretreated plants had considerably higher indole-3 acetic acid and abscisic acid concentrations under moderate (54.02% and 53.92%) and severe (68.36% and 64.61%) heat stress conditions. Similarly, plant dry biomass, NPK uptake, nitric oxide, and nitrate reductase activity were high in RI + MT treated plants, under both levels of stress. Therefore, this study advocates the positive synergistic effect of RI and MT pretreatment against moderate and severe heat-induced stress and for possible maintenance of plant growth under changing scenarios of global warming.

Ammonium and nitric oxide condition the establishment of Arabidopsis Ler/Kas-2 immune-related hybrid incompatibility

Main ConclusionHigh ammonium suppresses hybrid incompatibility between Ler and Kas-2 accessions through lowering nitric oxide levels and nitrate reductase activity required for autoimmunity.The immune-related hybrid incompatibility (HI) between Landsberg erecta (Ler) and Kashmir-2 (Kas-2) accessions is due to a deleterious genetic interaction between the RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like Ler locus and Kas-2 alleles of the receptor-like kinase SRF3 (STRUBBELIG RECEPTOR FAMILY 3). The genetic incompatibility is temperature-dependent and leads to constitutive activation of the salicylic acid (SA) pathway, dwarfism and cell death at 14–16 °C. Here we investigated the effect of nutrition on the occurrence of Ler/Kas-2 HI and found that high ammonium suppresses Ler/Kas-2 incompatible phenotypes independently of the ammonium/nitrate ratio. Ammonium feeding leads to compromised disease resistance to Pseudomonas syringae pv. tomato DC3000, lower total SA, nitric oxide and nitrate reductase activity in Ler/Kas-2 incompatible hybrids. In addition, we find that Ler/Kas-2 incompatibility is dependent on NPR1 (NONEXPRESSER OF PR GENES 1) and nitric oxide production. Overall, this work highlights the effect of nutrition on the expression of incompatible phenotypes independently of temperature.

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions.

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins.

Synergistically mitigating nitric oxide emission by co-applications of biochar and nitrification inhibitor in a tropical agricultural soil

Agricultural soils are the hotspots of nitric oxide (NO) emissions, which are related to atmospheric pollution and greenhouse effect. Biochar application has been recommended as an important countermeasure, however, its mitigation efficiency is limited as biochar, under certain conditions, can stimulate soil nitrification. Therefore, biochar co-applied with nitrification inhibitor could optimize the mitigation potential of biochar. Herein, a laboratory-scale experiment was conducted to investigate the effects of co-application of biochar and nitrification inhibitor on NO emission, nitrogen cycling function and bacterial community in a tropical vegetable soil. Results showed that a single application of biochar or nitrification inhibitor significantly decreased NO emissions, and this mitigation effectiveness was amplified by their co-applications. Soil NO2−−N intensity, along with abundances of AOB-amoA and nirK were significantly and positively correlated with cumulative NO emissions. The stimulated activity of ammonia monooxygenase and growths of AOB and total comammox Nitrospira by biochar were weakened by nitrification inhibitor, implying decreased nitrification-driven NO production. The nitric oxide reductase activity and related qnorB abundance in nitrification inhibitor-added soils were increased by biochar, indicating promoted NO consumption during denitrification. The nirK abundance and NO2−−N intensity were decreased more by co-applications of biochar or nitrification inhibitor. Moreover, both biochar and nitrification inhibitor changed bacterial β-diversity, and their co-application synergistically enriched Armatimonadetes and Verrucomicrobia abundances and decreased WPS-2 abundance. This study highlights that co-applications of biochar and nitrification inhibitor can make their respective advantages complementary to each other, thereby achieving a larger mitigation of NO emissions from agricultural soils in tropical regions.

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron–sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 μM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

Micro-analysis of nitrous oxide accumulation in denitrification under acidic conditions: The role of pH and free nitrous acid

Nitrous oxide (N2O), as an energy gas, is of increasing interest for its recovery from wastewater treatment. Acidic pH is one of the important factors affecting N2O accumulation during denitrification; however, its generation mechanism still needs further clarification. In this study, the production efficiency of N2O increased from 1.0 to 65.0% with the decrease in pH from 6.5 to 4.5 during nitrate-based denitrification performed in batch tests, which was accompanied by an increase in the nitrite accumulation. To further explore the mechanism, the net volumetric reaction rates of N2O within denitrified sludge aggregates were quantified by application of a microelectrode. When using N2O as a nitrogen source, the consumption rate of N2O was negatively correlated with both the hydrogen ion and free nitrous acid (FNA) concentrations. The inhibition of nitrous oxide reductase (NOS) activity in the absence of FNA demonstrates that hydrogen ion is the true inhibitor. When using NO as nitrogen source, the net volumetric production rates of N2O increased with decreasing pH, and they were higher in the presence of FNA compared to those in the absence of FNA, revealing that the increase in nitrite substrate promotes N2O production. Overall, the inhibition of NOS activity by hydrogen ions and FNA and the increase of substrate from nitrite accumulation are the main reasons for N2O enrichment under a low-pH condition.

Enhanced nitrate removal by biochar supported nano zero-valent iron (nZVI) at biocathode in bioelectrochemical system (BES)

In this study, activated biochar (BC), nano zero-valent iron (nZVI) and biochar supported nano zero-valent iron (BC-nZVI) were used to remove cathodic nitrate in bioelectrochemical system (BES). With an initial NO3–-N concentration of 100 mg/L, it was found that the complete nitrate removal could be observed within 28 h for the group of BC-nZVI, whereas the accumulated ammonia reached 63.33 mg/L. Meanwhile, nitrate removal reaction rate constants reached 0.171, 0.110, 0.023 and only 0.010 h−1 for BC-nZVI, nZVI, BC and the Control groups, respectively. Besides, enzymatic activity of nicotinamide adenine dinucleotide (NADH) and nitrous oxide reductase (NOS) of the BC-nZVI group reached the peak faster than three other groups. As a carrier for nZVI, loading of nZVI onto BC could increase the surface area of BC-nZVI other than nZVI for microbial aggregation, while embedded nZVI would contribute to the granulation of sludge and nitrate reduction. Moreover, Cyclic Voltammetry (CV) and chronoamperometry (CA) indicated that BC-nZVI group obtain better electrochemical performances. Importantly, it was found that not only abundances of microbial genus, but relative abundances of those iron mediated enzymes responsible for electro-consuming, hydrogen production and preferential ammonia accumulated nitrate removal could all be enriched. Therefore, BC-nZVI could be implemented to enhance the nitrate removal at biocathode in BES.

The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity

Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif, -G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.

Deepened snow enhances gross nitrogen cycling among Pan-Arctic tundra soils during both winter and summer

Many Arctic regions currently experience an increase in winter snowfall as a result of climate change. Deepened snow can enhance thermal insulation of the underlying soil during winter, resulting in warmer soil temperatures that promote soil microbial nitrogen (N)-cycle processes and the availability of N and other nutrients. We conducted an ex situ study comparing the effects of deepened snow (using snow fences that have been installed for 3–13 years) on microbial N-cycle processes in late summer (late growing season) and winter (late snow-covered season) among five tundra sites in three different geographic locations across the Arctic (Greenland (dry and wet tundra), Canada (mesic tundra), and Svalbard, Norway (heath and meadow tundra)). Soil gross N cycling rates (mineralization, nitrification, immobilization of ammonium (NH4+) and nitrate (NO3−), and denitrification) were determined using a15N pool dilution. Potential denitrification activity (PDA) and nitrous oxide reductase activity (N2OR) were measured to assess denitrifying enzyme activities.The deepened snow treatment across all sites had a significant effect of the potential soil capacity of accelerating N cycling rates in late winter, including quadrupled gross nitrification, tripled NO3−-N immobilization, and doubled denitrification as well as significantly enhanced late summer gross N mineralization, denitrification (two-fold) and NH4+-N availability. The increase in gross N mineralization and nitrification rates were primarily driven by the availability of dissolved organic carbon (DOC) and nitrogen (DON) across sites. The largest increases in winter DOC and DON concentrations due to deepened snow were observed at the two wetter sites (wet and mesic tundra), and N cycling rates were also more strongly affected by deepened snow at these two sites than at the three other drier sites. Together, these results suggest that the potential effects of deepened winter snow in stimulating microbial N-cycling activities will be most pronounced in relatively moist tundra ecosystems. Hence, this study provides support to prior observations that growing season biogeochemical cycles in the Arctic are sensitive to snow depth with altered nutrient availability for microorganisms and vegetation. It can be speculated that on the one hand growing season N availability will increase and promote plant growth, but on the other hand foster increased water- and gaseous (e.g. N2 and N2O) N-losses with implications for overall nutrient status.

Electrochemical insight into the activated algal biochar assisted hydrogenotrophic denitrification at biocathode using bioelectrochemical system (BES)

In this study, activated biochar prepared using Taihu blue algae was adopted to facilitate cathodic hydrogenotrophic denitrification in bioelectrochemical system (BES). It was found that addition of algal biochar (ABC)-800N (treated with nitric acid) at biocathode reached the highest nitrate removal constant (21.79), while the control reached only 6.94. Besides, activity of nitrous oxide reductase attained 5.16 U/L for the control in day 7, while group ABC-800N reached 16.06 U/L by then. Importantly, chronoamperometry (CA) detect indicated an electron exchange capacity for group ABC-800N as 872.68 μmol e−/gBC, compared to 162.81 μmol e−/gBC for the control. Through chronocoulomb (CC), better charge/discharge stability can be concluded in BES reactor using ABC-800N. Therefore, activated biochar could be implemented to enhance denitrification process at biocathode in BES.

Long-term effects of decabromodiphenyl ether on denitrification in eutrophic lake sediments: Different sensitivity of six-type denitrifying bacteria

The widespread use of polybrominated diphenyl ethers inevitably results in their increased release into natural waters and subsequent deposition in sediments. However, their long-term effects on the bacteria participating in each step of denitrification in eutrophic lake sediments are still unknown. Here, we conducted a one-year microcosm experiment to determine the long-term effects of decabromodiphenyl ether (BDE-209), at low (2 mg kg−1 dry weight) and high (20 mg kg−1 dry weight) contamination levels, on six-type denitrifying bacteria and their activities in sediments collected from Taihu Lake, a typical eutrophic lake in China. At the end of the experiment, sediment denitrifying reductase activities were inhibited by BDE-209 at both levels, with the greatest inhibition seen for nitric oxide reductase activity. The higher nitrate concentration in the contaminated sediments was attributed to the inhibition of nitrate reductase activities. The abundances of six-type denitrifying genes (narG, napA, nirK, nirS, norB, and nosZ) significantly decreased under high BDE-209 treatment, and narG and napA genes were more sensitive to the toxicity of BDE-209. The results from pyrosequencing showed that BDE-209, at either treatment concentration, decreased the six-type denitrifying bacterial diversities and altered their community composition. This shift of six-type denitrifying bacterial communities might also be driven by the debrominated products concentrations of BDE-209 and variations in sediment inorganic nitrogen concentrations. In particular, some genera from phylum Proteobacteria such as Pseudomonas, Cupriavidus, and Azoarcus were decreased significantly because of BDE-209 and its debrominated products.

Copper modulates nitrous oxide emissions from soybean root nodules

Agriculture is an important source of the greenhouse gas nitrous oxide (N2O) due to the over- or non-synchronised application of nitrogen to crops. Symbiotic nitrogen fixation (SNF) by legume-rhizobia symbiosis can be an effective strategy for N2O mitigation, but several environmental factors such as copper (Cu) availability might affect N2O emissions derived from legume crops. The aim of this research was to study how Cu can modulate N2O emissions by soybean root nodules. Soybean plants inoculated with Bradyrhizobium diazoefficiens USDA110 were grown in the presence of 4 mM KNO3 and a battery of Cu2+ concentrations added during growth (0, 5, 10, 20, 40, 60 or 100 μM). N2O emissions were measured by gas chromatography in both, nodulated roots and detached nodules, after root flooding. Also, 15N isotope dilution was assayed for SNF and N2O determinations. Results showed that an excess of added Cu during growth significantly affected plant physiology, nodulation and SNF of the soybean-B.diazoefficiens symbiosis, being 20 μM the threshold that soybean plants can tolerate without suffering Cu stress. Meanwhile, Cu addition reduced statistically N2O emissions by soybean nodules. This reduction was correlated with Cu accumulation in nodules, which affected the denitrifying enzymes activities. Cu excess produced a simultaneous decrease of nitrate and nitrite reductase activities, but an increase of nitrous oxide reductase activity. Finally, the modulation of bacteroidal nitrate reductase activity is proposed as an effective target for the strategies for mitigation of N2O emissions derived from soybean crops, probably more effective than nitrous oxide reductase activity.

The Catalytic Role of a Conserved Tyrosine in Nitric Oxide-Reducing Non-heme Diiron Enzymes

Recent studies have identified several key intermediates of the nitric oxide reductase (NOR) cycle of flavodiiron proteins (FDPs). These intermediates include, sequentially, a μ-hydroxo diferrous species, a mono-NO intermediate, a di-NO intermediate (FDPdiNO), and a bis-μ-hydroxo diferric species. This paper focuses on the reaction path connecting the last two intermediates on which the nitrous oxide product is released. On the basis of density functional theory calculations, a reaction sequence is proposed in which the enzyme passes through an intermediate with a bridging hyponitrite ligand, which accepts a proton, initiating a rate-determining ligand rotation from an N/N- to an N/O-coordinated conformation. This rotation is facilitated by a second-sphere tyrosine residue, which provides a transient hydrogen bond to one of the nitrogen atoms of the substrate near the transition state. The role of the tyrosine residue in the NOR activity has been tested by steady-state kinetics and rapid freeze-quench (RFQ) studies of the Y197F variant of the FDP from T. maritima in which the hydrogen bonding interaction is absent. The Y197F variant displayed little or no steady-state NOR activity in support of the importance of Y197. The RFQ samples, monitored by Mössbauer spectroscopy, showed that Y197F follows the same reaction path as a wild-type FDP up to and including the formation of FDPdiNO but diverges subsequently with the variant forming an inactive mono-NO species. The RFQ results demonstrate that Y197 enables the postFDPdiNO section of the reaction cycle in the wild-type FDP to proceed to N2O. The proposed refinement of the reaction mechanism provides an explanation for the lack of NOR activity of the variant Y197F of T. maritima and of other di-NO binding diiron enzymes and model compounds with active site structures like those of FDPs. The reported reductions of NO to N2O catalyzed by synthetic diiron complexes proceed only with the support of radiation, additional electrons, or an electron-rich ligand environment. However, higher potential diiron sites like those of FDPs require hydrogen bonding to second-sphere residues to turn over NO to N2O.

Heterologous expression and functional study of nitric oxide reductase catalytic reduction peptide from Achromobacter denitrificans strain TB

Biological denitrification is a promising and green technology for air pollution control. To investigate the nitric oxide reductase (NOR) that dominates NO reduction efficiency in biological purification, the heterologous prokaryotic expression system of the norB gene, which encodes the core peptide of the catalytic reduction structure in the NOR from Achromobacter denitrificans strain TB, was constructed in Escherichia coli BL21 (DE3). Results showed that the 1218 bp-long norB gene was expressed at the highest level under 1.0 mM IPTG for 5 h at 30 °C, and the relative expression abundance of norB in recombinant E. coli was increased by 16.6 times compared with that of the wild-type TB. However, the NO reduction efficiency and NOR activity of strain TB was 2.7 and 1.83 times higher than those of recombinant E. coli, respectively. On the basis of genomic reassembly and protein structure modeling, the core peptide of the NOR catalytic reduction structure from Achromobacter sp. TB can independently exert NO reduction. The low NO degradation efficiency of recombinant E. coli may be due to the lack of a NorC-like structure that increases the enzyme activity of the NorB protein. The results of this study can be used as basis for further research on the structure and function of NOR.

Influence of Copper Coordination Spheres on Nitrous Oxide Reductase (N2Or) Activity of a Mixed-Valent Copper Complex Containing a {Cu2S} Core.

A new mixed-valent dicopper complex [5] was generated from ligand exchange by dissolving a bis(CH3CN) precursor [3] in acetone. Introduction of a water molecule in place of an acetonitrile ligand was evidenced by base titration and the presence of a remaining coordinated CH3CN by IR, 19F NMR, and theoretical methods. The proposed structure (CH3CN-Cu-(SR)-Cu-OH2) was successfully DFT-optimized and the calculated parameters are in agreement with the experimental data. [5] has a unique temperature-dependence EPR behavior, with a localized valence from 10 to 120 K that undergoes delocalized at room temperature. The electrochemical signatures are in the line of the other aquo parent [2] and sensibly different from the rest of the series. Similar to the case of [2], [5] was finally capable of single turnover N2O reduction at room temperature. N2 was detected by GC-MS, and the redox character was confirmed by EPR and ESI-MS. Kinetic data indicate a reaction rate order close to 1 and a rate 10 times faster compared to [2]. [5] is thus the second example of that kind and highlights not only the main role of the Cu-OH2 motif, but also that the adjacent Cu-X partner (X = OTf- in [2] and CH3CN in [5]) is a new actor in the casting to establish structure/activity correlations.

Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway.

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK2-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK2 and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC, nirK, norCBQD, and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens, we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c550 soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc1 complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA, nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK2 as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK2 and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c-staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation.