Nitric Oxide Reductase Research Articles

Identification and examination of nitrogen metabolic genes in Lelliottia amnigena PTJIIT1005 for their ability to perform nitrate remediation

Lelliottia amnigena PTJIIT1005 is a bacterium that utilizes nitrate as the sole nitrogen source and can remediate nitrate from media. The annotation was done related to nitrogen metabolic genes using the PATRIC, RAST tools, and PGAP from the genome sequence of this bacterium. Multiple sequence alignments and phylogenetic analysis of respiratory nitrate reductase, assimilatory nitrate reductase, nitrite reductase, glutamine synthetase, hydroxylamine reductase, nitric oxide reductase genes from PTJIIT1005 were done to find out sequence identities with the most similar species. The identification of operon arrangement in bacteria was also identified. The PATRIC KEGG feature mapped the N-metabolic pathway to identify the chemical process, and the 3D structure of representative enzymes was also elucidated. The putative protein 3D structure was analyzed using I-TASSER software. It gave good quality protein models of all nitrogen metabolism genes and showed good sequence identity with reference templates, approximately 81–99%, except for two genes; assimilatory nitrate reductase and nitrite reductase. This study suggested that PTJIIT1005 can remove N-nitrate from water because of having N-assimilation and denitrification genes.

Ensifer meliloti denitrification is involved in infection effectiveness and N2O emissions from alfalfa root nodules

PurposeAlfalfa is one of the most valuable forage crops in temperate climate zones. Ensifer meliloti, the endosymbiont of alfalfa, contains all the denitrification genes but the capacity of alfalfa root nodules to produce nitrous oxide (N2O) is not known. In this work, N2O emissions as well as the influence of bacteroidal denitrification on nodulation competitiveness and N2O release from alfalfa nodules has been investigated.MethodsMedicago sativa cv. Victoria plants were inoculated with E. meliloti 1021, a periplasmic nitrate reductase (Nap) defective mutant, a Nap overexpressing strain and a nitrous oxide reductase defective mutant. Plants were grown in the presence of different nitrate and copper treatments and subjected to flooding during one week before harvesting. N2O production by the nodules was analysed by using gas chromatography. Methyl viologen-dependent nitrate reductase (MV+-NR), nitrite reductase (MV+-NIR) and nitrous oxide reductase (N2OR) enzymatic activities were measured in isolated bacteroids.ResultsAlfalfa root nodules produce N2O in response to nitrate and flooding. Overexpression of Nap improved nodulation competitiveness and induced N2O emissions from nodules. Copper is required for an effective symbiosis as well as triggered a reduction of N2O production due to the induction of the N2OR and a reduction of NIR activities in the bacteroids.ConclusionAlfalfa root nodules emit N2O. Nap is involved in nodulation competitiveness and in N2O emissions by the nodules. Bacteroidal N2OR and NIR activities are modulated by Cu and may be considered as effective targets for the mitigation strategies of N2O emissions derived from alfalfa crops.

Hot moment of N2O emissions in seasonally frozen peatlands.

Since the start of the Anthropocene, northern seasonally frozen peatlands have been warming at a rate of 0.6 °C per decade, twice that of the Earth's average rate, thereby triggering increased nitrogen mineralization with subsequent potentially large losses of nitrous oxide (N2O) to the atmosphere. Here we provide evidence that seasonally frozen peatlands are important N2O emission sources in the Northern Hemisphere and the thawing periods are the hot moment of annual N2O emissions. The flux during the hot moment of thawing in spring was 1.20 ± 0.82 mg N2O m-2 d-1, significantly higher than that during the other periods (freezing, -0.12 ± 0.02 mg N2O m-2 d-1; frozen, 0.04 ± 0.04 mg N2O m-2 d-1; thawed, 0.09 ± 0.01 mg N2O m-2 d-1) or observed for other ecosystems at the same latitude in previous studies. The observed emission flux is even higher than those of tropical forests, the World's largest natural terrestrial N2O source. Furthermore, based on soil incubation with 15N and 18O isotope tracing and differential inhibitors, heterotrophic bacterial and fungal denitrification was revealed as the main source of N2O in peatland profiles (0-200 cm). Metagenomic, metatranscriptomic, and qPCR assays further revealed that seasonally frozen peatlands have high N2O emission potential, but thawing significantly stimulates expression of genes encoding N2O-producing protein complexes (hydroxylamine dehydrogenase (hao) and nitric oxide reductase (nor)), resulting in high N2O emissions during spring. This hot moment converts seasonally frozen peatlands into an important N2O emission source when it is otherwise a sink. Extrapolation of our data to all northern peatland areas reveals that the hot moment emissions could amount to approximately 0.17 Tg of N2O yr-1. However, these N2O emissions are still not routinely included in Earth system models and global IPCC assessments.

The reducing effect of aglime onN2OandCO2emissions balance from an acidic soil: A study on intact soil cores

AbstractThe functioning of the nitrous oxide (N2O) reductase enzyme involved in the last step of denitrification is pH sensitive, with an optimum of 6.8. A solution to mitigate N2O emissions would be to bring soil pH close to neutrality by adding agricultural liming products (aglime). Nevertheless, the influence of aglime on the soil greenhouse gas (GHG) balance (CO2–N2O) is a subject of debate, particularly when considering the fate of the carbon (C) derived from carbonates. Our objective was to investigate the results of the effect of calcium carbonate (CaCO3) aglime on the CO2–N2O balance. Sixteen cylinders of undisturbed acidic soil were taken from a sandy loam profile and incubated at 20°C for 107 days in anaerobic conditions (water‐filled pore space >60%). Eight limed treatment cylinders received 1.45 g of aglime on the soil surface (2 t NV ha−1) and 0.08 g of N (100 kg of N ha−1). Eight control treatment cylinders received only 0.08 g of N. N2O and CO2fluxes were measured and converted into CO2equivalents to perform a GHG balance calculation. Furthermore, soil and leachate properties were measured. Aglime application triggered a reduction of N2O emissions, probably due to an increase in soil pH at the beginning of the experiment, which would have led to the N2O reductase activation. High ‐N content in the soil may inhibit the high N2O reduction potential in the limed treatment. CO2emissions were unexpectedly lower in the limed treatment. Aglime addition did not enhance C mineralisation, which may be explained by the possible stabilisation of soil organic carbon. A significant 11.3% reduction of GHG emissions was observed in the limed treatment. Overall, our results show that a strategy of liming acidic agricultural soil could be implemented for its potential in GHG mitigation. Nevertheless, future in‐depth research is necessary to better understand the fate of the C brought about by aglime.

Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins

BackgroundNorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families.ResultsWe show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.ConclusionsBased on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

Effect of tillage state of paddy soils with heavy metal pollution on the nosZ gene of N2O reductase

Paddy soils are an important source of atmospheric nitrous oxide (N2O). However, numerous studies have focused on N2O production during the soil tillage period, neglecting the N2O production during the dry fallow period. In this study, we conducted an incubation experiment using the acetylene inhibition technique to investigate N2O emission and reduction rates of paddy soil profiles (0-1 m) from Guangdong Province and Jinlin Province in China, with different heavy-metal pollution levels. The abundance and community structures of denitrifying bacteria were determined via quantitative-PCR and Illumina MiSeq sequencing of nosZ, nirK, and nirS genes. Our results showed that the potential N2O emission rate, N2O production rate, and denitrification rate have decreased with increasing soil vertical depth and heavy-metal pollution. More importantly, we found that the functional gene type of N2O reductase switched with the tillage state of paddy soils, which clade Ⅱ nosZ genes were the dominant gene during the tillage period, while clade Ⅰ nosZ genes were the dominant gene during the dry fallow period. The heavy-metal pollution has less effect on the niche differentiation of the nosZ gene. The N2O emission rate was significantly regulated by the genus Bradyhizobium, which contains both N2O reductase and nitrite reductase genes. Our findings suggests that the nosZ gene of N2O reductase can significantly impact the N2O emission from paddy soils.

Network analysis of 16S rRNA sequences suggests microbial keystone taxa contribute to marine N2O cycling

The mechanisms by which large-scale microbial community function emerges from complex ecological interactions between individual taxa and functional groups remain obscure. We leveraged network analyses of 16S rRNA amplicon sequences obtained over a seven-month timeseries in seasonally anoxic Saanich Inlet (Vancouver Island, Canada) to investigate relationships between microbial community structure and water column N2O cycling. Taxa separately broadly into three discrete subnetworks with contrasting environmental distributions. Oxycline subnetworks were structured around keystone aerobic heterotrophs that correlated with nitrification rates and N2O supersaturations, linking N2O production and accumulation to taxa involved in organic matter remineralization. Keystone taxa implicated in anaerobic carbon, nitrogen, and sulfur cycling in anoxic environments clustered together in a low-oxygen subnetwork that correlated positively with nitrification N2O yields and N2O production from denitrification. Close coupling between N2O producers and consumers in the anoxic basin is indicated by strong correlations between the low-oxygen subnetwork, PICRUSt2-predicted nitrous oxide reductase (nosZ) gene abundances, and N2O undersaturation. This study implicates keystone taxa affiliated with common ODZ groups as a potential control on water column N2O cycling and provides a theoretical basis for further investigations into marine microbial interaction networks.

Comparative investigation on heterotrophic denitrification driven by different biodegradable polymers for nitrate removal in mariculture wastewater: Organic carbon release, denitrification performance, and microbial community.

Heterotrophic denitrification is widely studied to purify freshwater wastewater, but its application to seawater wastewater is rarely reported. In this study, two types of agricultural wastes and two types of synthetic polymers were selected as solid carbon sources in denitrification process to explore their effects on the purification capacity of low-C/N marine recirculating aquaculture wastewater (NO3 --N 30 mg/L, salinity 32‰). The surface properties of reed straw (RS), corn cob (CC), polycaprolactone (PCL) and poly3-hydroxybutyrate-hydroxypropionate (PHBV) were evaluated by Brunauer-Emmett-Teller, Scanning electron microscope and Fourier-transform infrared spectroscopy. Short-chain fatty acids, dissolved organic carbon (DOC), and chemical oxygen demand (COD) equivalents were used to analyze the carbon release capacity. Results showed that agricultural waste had higher carbon release capacity than PCL and PHBV. The cumulative DOC and COD of agricultural waste were 0.56-12.65 and 1.15-18.75 mg/g, respectively, while those for synthetic polymers were 0.07-1.473 and 0.045-1.425 mg/g, respectively. The removal efficiency of nitrate nitrogen (NO3 --N) was CC 70.80%, PCL 53.64%, RS 42.51%, and PHBV 41.35%. Microbial community analysis showed that Proteobacteria and Firmicutes were the most abundant phyla in agricultural wastes and biodegradable natural or synthetic polymers. Quantitative real-time PCR indicated the conversion from nitrate to nitrogen was achieved in all four carbon source systems, and all six genes had the highest copy number in CC. The contents of medium nitrate reductase, nitrite reductase and nitrous oxide reductase genes in agricultural wastes were higher than those in synthetic polymers. In summary, CC is an ideal carbon source for denitrification technology to purify low C/N recirculating mariculture wastewater.

Effects of nitrogen input on N2O production and enzyme activity in Luoshijiang Wetland sediments, China

We examined the effects of nitrate (NO3--N) and ammonium (NH4+-N) input at different concentrations (0, 1, 5 and 25 mg·kg-1) on N2O production rate from the surface sediment (0-5 cm) of Luoshijiang Wetland, located upstream from Lake Erhai. The contribution of nitrification, denitrification, nitrifier denitrification, and other factors to the N2O production rate in sediments was studied by the inhibitor method. The relationships between N2O production and the activities of hydroxylamine (HyR), nitrate (NAR), nitric oxide (NOR), and nitrous oxide (NOS) reductases in sediments were analyzed. We found that NO3--N input significantly increased total N2O production rate (1.51-11.35 nmol·kg-1·h-1), which led to N2O release, whereas NH4+-N input decreased that (-0.80 to -0.54 nmol·kg-1·h-1), causing N2O absorption. NO3--N input did not change the dominant roles of nitrification and nitrifier denitrification in N2O production in sediments, but increased the contributions of these two factors to 69.5% and 56.5%, respectively. The NH4+-N input significantly changed N2O gene-ration process, and the nitrification and nitrifier denitrification changed from N2O release to uptake. There was a positive correlation between total N2O production rate and NO3--N input. NO3--N input significantly increased NOR activity and decreased NOS activity, thereby promoting N2O production. The total N2O production rate in sediments was negatively correlated with NH4+-N input. NH4+-N input significantly increased the activities of HyR and NOR, decreased NAR activity, and inhibited N2O production. Nitrogen inputs with different forms and concentrations changed the degree of contribution and mode of N2O production by affecting enzyme activities in sediments. NO3--N input significantly promoted N2O production, acting as a source of N2O, while NH4+-N input inhibited N2O production, resulting in an N2O sink.

Biochar-derived persistent free radicals and reactive oxygen species reduce the potential of biochar to mitigate soil N2O emissions by inhibiting nosZ

Biochar amendment has been proven to generally reduce soil nitrous oxide (N2O) emissions. However, the effects and underlying mechanisms of biochar-derived reactive oxygen species (ROS) on soil N2O emissions are still unclear. Thus, we firstly weakened the intensities of persistent free radicals (PFRs) within biochar using triethanolamine (TEA) as a quencher, and then used soil incubation methods to compare the potentials of TEA-quenched and un-quenched biochar in mitigating soil N2O emissions. The TEA-quenched biochar generated less hydrogen peroxide (H2O2) and hydroxyl radical (•OH), while having a higher soil N2O emission mitigation potential, than the un-quenched biochar. The N2O emissions and the N2O/(N2O + N2) ratio were significantly, positively, correlated with the generated H2O2 and •OH contents. These results demonstrated that biochar-derived ROS weakened biochar's mitigation of soil N2O emissions. The specific mechanisms of biochar-derived ROS on soil N2O emissions were further explored by a ROS scavenging experiment. It was found that scavenging H2O2 by catalase efficiently hindered the generation of •OH, resulting in decreases in N2O emissions and the N2O/(N2O + N2) ratio. Meanwhile, biochar-derived ROS exhibited a more severe inhibition on N2O reductase gene (nosZ) expression than that on the expression of genes responsible for N2O production (nirK and nirS), indicating that biochar-derived ROS weakened biochar's mitigation of soil N2O emissions by inhibiting microbial N2O reduction. Our results imply that controlling the content of biochar-derived ROS is a promising strategy to maximize biochar's potential for mitigating soil N2O emission.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

Cell density dependent restriction of N2O reductase in denitrifying Pseudomonas spp., a potential driver of N2O emissions in natural systems.

Denitrification is a major biological source and sink for the ozone-depleting greenhouse gas N2. Thus, the respiratory physiology of denitrifiers and the mechanisms determining their propensity for accumulation of N-oxides are of fundamental interest. Here, we report a pervasive positive correlation between cell density and N2O accumulation in Pseudomonas aeruginosa and P. fluorescens F113. We show that this was a result of quorum sensing by comparing the P. aeruginosa PAO1-UW wild type to a rhlI/lasI knockout mutant able to sense, but not synthesize the N-acyl-homoserine lactones (AHL) of the Rhl and Las circuits. Neither the transcription of nosZ (encoding N2O reductase, N2OR) nor the abundance of peptides of known relevance to denitrification could explain the restriction of N2O reduction in AHL-affected cultures. However, a protein shown to be involved in synthesis and repair of iron-sulphur (Fe-S) centers under NO stress, CyaY, was significantly downregulated in the AHL producing wild type. This hints to a possible route of N2OR-suppression via compromised Fe-S centers in the ancillary protein NosR. While the exact mechanism remains obscure, it appears that quorum sensing driven restriction of N2OR activity is common. Thus, given its ubiquity among prokaryotes, and the potential for cross-species and -strain effects, quorum sensing is plausibly a driver of N2O emissions in a range of systems.

Cadmium effects on net N2O production by the deep-sea isolate Shewanella loihica PV-4.

Deep-sea mining may lead to the release of high concentrations of metals into the surrounding seabed, which can disturb important ecosystem functions provided by microbial communities. Among these, the production of N2O and its reduction to N2 is of great relevance since N2O is an important greenhouse gas. Metal impacts on net N2O production by deep-sea bacteria are, however, currently unexplored. Here, we evaluated the effects of cadmium (Cd) on net N2O production by a deep-sea isolate, Shewanella loihica PV-4. We performed a series of Cd exposure incubations in oxic conditions and determined N2O fluxes during induced anoxic conditions, as well as the relative expression of the nitrite reductase gene (nirK), preceding N2O production, and N2O reductase gene (nosZ), responsible for N2O reduction. Net N2O production by S. loihica PV-4 exposed to Cd was strongly inhibited when compared to the control treatment (no metal). Both nirK and nosZ gene expression were inhibited in reactors with Cd, but nirK inhibition was stronger, supporting the lower net N2O production observed with Cd. The Cd inhibition of net N2O production observed in this study poses the question whether other deep-sea bacteria would undergo the same effects. Future studies should address this question as well as its applicability to complex communities and other physicochemical conditions, which remain to be evaluated.

Meta-Omics Analysis of a Formate Sidestream-Mediated Partial Nitritation-Coupled Anammox Process

A stable partial nitritation process that was achieved by treating activated sludge in sidestream with formate as a selective inhibitor has been previously described. However, little is known about the effect of formate on the microbial communities in both the partial nitritation and the subsequent anammox process. To this end, a formate sidestream-mediated partial nitritation-coupled anammox process was established and studied. The effluent ratio of nitrite/ammonia in the partial nitritation remained at 1.24 ± 0.19, and the total nitrogen removal efficiency of the anammox process was maintained at 80.0 ± 1.6%. Metagenomic and metatranscriptomic analysis showed that the nitrite oxidizing bacteria (NOB) was almost eliminated from the partial nitritation process, while Nitrosomonas europaea was the dominant ammonia-oxidizing bacteria (AOB) with an abundance of 30.8% of total reads. The high expression of nitrous oxide reductase genes in the partial nitritation process suggested a great potential for mitigating nitrous oxide release. Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia sinica were identified as the main anammox bacteria, and their abundances were as high as 49.3 and 11.8%, respectively. There was a highly expressed gene encoding a formate tetrahydrofolate ligase in the Candidatus Kuenenia stuttgartiensis metagenome-assembled genomes (MAG), suggesting that formate-related metabolic activity may contribute to its enrichment in the anammox process.

The effect of biochar on nitrogen availability and bacterial community in farmland

PurposeNitrification and denitrification in soil are key components of the global nitrogen cycle. This study was conducted to investigate the effect of biochar application on soil nitrogen and bacterial diversity.MethodsPot experiments were conducted to investigate the effects of different biochar-based rates 0% (CK), 0.5% (BC1), 1.0% (BC2), 2.0% (BC3), and 4.0% (BC4) on soil nutrient and bacterial community diversity and composition.ResultsThe results indicate that the total nitrogen (TN) and ammonium nitrogen (AN) contents in the soil increased by 4.7–32.3% and 8.3–101.5%, respectively. The microbial biomass nitrogen (MBN) content increased with increased amounts of biochar rate. The application of biochar also significantly changed the soil bacterial community composition. The copy number of 16S marker gene of related enzymes to the nitrification process in BC2 was reduced by 20.1%. However, the gene expressions of nitric oxide reductase and nitrous oxide reductase in BC3 increased by 16.4% and 16.0%, respectively, compared to those in CK. AN, nitrate nitrogen (NN), and NN/TN were the main factors affecting the structure of the soil bacterial community. In addition, the expressions of nitrite reductase, hydroxylamine, and nitric oxide reductase (cytochrome c) were also significantly correlated.ConclusionTherefore, the applied biochar improved soil nitrogen availability and which ultimately resulted in an environmental risk decrease by soil nitrogen release inhibition.

Isolation and characteristics of two heterotrophic nitrifying and aerobic denitrifying bacteria, Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6

In order to solve nitrogen pollution in environmental water, two heterotrophic nitrifying and aerobic denitrifying strains isolated from acid paddy soil were identified as Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6 respectively. Strain HNDS-1 and strain HNDS-6 exhibited amazing ability to nitrogen removal. When (NH4)2SO4, KNO3, NaNO2 were used as nitrogen resource respectively, the NH4+-N, NO3−-N, NO2−-N removal efficiencies of strain HNDS-1 were 93.31%, 89.47%, and 100% respectively, while those of strain HNDS-6 were 82.39%, 96.92%, and 100%. And both of them could remove mixed nitrogen effectively in low C/N (C/N = 5). Strain HNDS-1 could remove 76.86% NH4+-N and 75.13% NO3−-N. And strain HNDS-6 can remove 65.07% NH4+-N and 78.21% NO3−-N. A putative ammonia monooxygenase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein and nitric oxide reductase of strain HNDS-1, while hydroxylamine reductase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein, and nitric oxide reductase of strain HNDS-6 were identified by genomic analysis. DNA-SIP analysis showed that genes Nxr, narG, nirK, norB, nosZ were involved in nitrogen removal pathway, which indicates that the denitrification pathway of strain HNDS-1 and strain HNDS-6 was NO3−→NO2−→NO→N2O→N2 during NH4+-N removal process. And the nitrification pathway of strain HNDS-1 and strain HNDS-6 was NO2−→NO3−, but the nitrification pathway of NH4+→ NO2− needs further studies.

Trapping of a Mononitrosyl Nonheme Intermediate of Nitric Oxide Reductase by Cryo-Photolysis of Caged Nitric Oxide.

Characterization of short-lived reaction intermediates is essential for elucidating the mechanism of the reaction catalyzed by metalloenzymes. Here, we demonstrated that the photolysis of a caged compound under cryogenic temperature followed by thermal annealing is an invaluable technique for trapping of short-lived reaction intermediates of metalloenzymes through the study of membrane-integrated nitric oxide reductase (NOR) that catalyzes reductive coupling of two NO molecules to N2O at its heme/nonheme FeB binuclear center. Although NO produced by the photolysis of caged NO did not react with NOR under cryogenic temperature, annealing to ∼160 K allowed NO to diffuse and react with NOR, which was evident from the appearance of EPR signals assignable to the S = 3/2 state. This indicates that the nonheme FeB-NO species can be trapped as the intermediate. Time-resolved IR spectroscopy with the use of the photolysis of caged NO as a reaction trigger showed that the intermediate formed at 10 μs gave the NO stretching frequency at 1683 cm-1 typical of nonheme Fe-NO, confirming that the combination of the cryo-photolysis of caged NO and annealing enabled us to trap the reaction intermediate. Thus, the cryo-photolysis of the caged compound has great potential for the characterization of short-lived reaction intermediates.

Preliminary X-ray Structure Analysis of the Hyphomicrobium Nitrous Oxide Reductase and Insights into the Inter-protein Electron Transfer

Preliminary X-ray Structure Analysis of the <i>Hyphomicrobium</i> Nitrous Oxide Reductase and Insights into the Inter-protein Electron Transfer

Exploring second coordination sphere effects in flavodiiron nitric oxide reductase model complexes.

Flavodiiron nitric oxide reductases (FNORs) equip pathogens with resistance to nitric oxide (NO), an important immune defense agent in mammals, allowing these pathogens to proliferate in the human body, potentially causing chronic infections. Understanding the mechanism of how FNORs mediate the reduction of NO contributes to the greater goal of developing new therapeutic approaches against drug-resistant strains. Recent density functional theory calculations suggest that a second coordination sphere (SCS) tyrosine residue provides a hydrogen bond that is critical for the reduction of NO to N2O at the active site of FNORs [J. Lu, B. Bi, W. Lai and H. Chen, Origin of Nitric Oxide Reduction Activity in Flavo-Diiron NO Reductase: Key Roles of the Second Coordination Sphere, Angew. Chem., Int. Ed., 2019, 58, 3795-3799]. Specifically, this H-bond stabilizes the hyponitrite intermediate and reduces the energetic barrier for the N-N coupling step. At the same time, the role of the Fe⋯Fe distance and its effect on the N-N coupling step has not been fully investigated. In this study, we equipped the H[BPMP] (= 2,6-bis[[bis(2-pyridylmethyl)amino]methyl]-4-methylphenol) ligand with SCS amide groups and investigated the corresponding diiron complexes with 0-2 bridging acetate ligands. These amide groups can form hydrogen bonds with the bridging acetate ligand(s) and potentially the coordinated NO groups in these model complexes. At the same time, by changing the number of bridging acetate ligands, we can systematically vary the Fe⋯Fe distance. The reactivity of these complexes with NO was then investigated, and the formation of stable iron(II)-NO complexes was observed. Upon one-electron reduction, these NO complexes form Dinitrosyl Iron Complexes (DNICs), which were further characterized using IR and EPR spectroscopy.

Nitric oxide and Nitrous oxide accumulation, oxygen production during nitrite denitrification in an anaerobic/anoxic sequencing batch reactor: exploring characteristics and mechanism.

Nitrite denitrification has received increasing attention due to its high efficiency, low energy consumption, and sludge yield. However, the nitric oxide (NO) and nitrous oxide (N2O) which are harmful to the environment, microorganisms, and humans are produced in this process. In order to mitigate NO and N2O production, the biological mechanisms of NO and N2O accumulation, as well as NO detoxification during nitrite denitrification in a sequencing batch reactor were studied. Results showed that the peak of NO accumulation increased from 0.29 [Formula: see text] 0.01 to 3.12 [Formula: see text] 0.34mg L-1 with the increase of carbon to nitrogen ratio (COD/N), which is caused by the sufficient electron donor supply for NO2--N reduction process at high COD/N. Furthermore, the result suggested that NO accumulation with no pH adjustment was 12 times higher than that with pH adjustment. It is related to the inhibition on NO reductase caused by the high free nitrous acid (FNA) and NO concentration with no pH adjustment. The pathways of NO detoxification included NO emission, reduction, and dismutation, and the more NO produced, the high proportion of NO dismutation pathway. Result showed that the maximum of oxygen production during NO dismutation reached to 1.39mg L-1. N2O accumulation was mainly associated with FNA and NO inhibition, COD/N. The peak of N2O accumulation presented a completely opposite trend at pH adjustment and no pH adjustment, it is because that the higher FNA and NO concentration at high COD/N without pH adjustment will inhibit the N2O reductase activity, resulting in the N2O reduction was hindered during nitrite denitrification.