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N2O is a powerful greenhouse gas contributing both to global warming and ozone depletion. While fungi have been identified as a putative source of N2O, little is known about their production of this greenhouse gas. Here we investigated the N2O-producing ability of a collection of 207 fungal isolates. Seventy strains producing N2O in pure culture were identified. They were mostly species from the order Hypocreales order—particularly Fusarium oxysporum and Trichoderma spp.—and to a lesser extent species from the orders Eurotiales, Sordariales, and Chaetosphaeriales. The N2O 15N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰, and we observed a significant taxa effect, with Penicillium strains displaying lower SP values than the other fungal genera. Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect. The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains, and its genetic variability showed a strong congruence with the ITS phylogeny, indicating vertical inheritance of this trait. Taken together, this comprehensive set of findings should enhance our knowledge of fungi as a source of N2O in the environment.

Nitrite (NO₂ ⁻-N) accumulation in denitrification can provide the substrate for anammox, an efficient and cost-saving process for nitrogen removal from wastewater. This batch-mode study aimed at achieving high NO₂ ⁻-N accumulation over long-term operation with the acetate as sole organic carbon source and elucidating the mechanisms of NO₂ ⁻-N accumulation. The results showed that the specific nitrate (NO₃ ⁻-N) reduction rate (59.61 mg N VSS⁻¹ h⁻¹ at NO₃ ⁻-N of 20 mg/L) was much higher than specific NO₂ ⁻-N reduction rate (7.30 mg N VSS⁻¹ h⁻¹ at NO₃ ⁻-N of 20 mg/L), and the NO₂ ⁻-N accumulation proceeded well at the NO₃ ⁻-N to NO₂ ⁻-N transformation ratio (NTR) as high as 90 %. NO₂ ⁻-N accumulation was barely affected by the ratio of chemical oxygen demand (COD) to NO₃ ⁻-N concentration (C/N). With the addition of NO₃ ⁻-N, NO₂ ⁻-N accumulation occurred and the specific NO₂ ⁻-N reduction rate declined to a much lower level compared with the value in the absence of NO₃ ⁻-N. This indicated that the denitrifying bacteria in the system preferred to use NO₃ ⁻-N as electron acceptor rather than use NO₂ ⁻-N. In addition, the Illumina high-throughput sequencing analysis revealed that the genus of Thauera bacteria was dominant in the denitrifying community with high NO₂ ⁻-N accumulation and account for 67.25 % of total microorganism. This bacterium might be functional for high NO₂ ⁻-N accumulation in the presence of NO₃ ⁻-N.

Vascular stiffness is a major cause of cardiovascular disease during normal aging and in Hutchinson–Gilford progeria syndrome (HGPS), a rare genetic disorder caused by ubiquitous progerin expression. This mutant form of lamin A causes premature aging associated with cardiovascular alterations that lead to death at an average age of 14.6 years. We investigated the mechanisms underlying vessel stiffness in LmnaG609G/G609G mice with ubiquitous progerin expression, and tested the effect of treatment with nitrites. We also bred LmnaLCS/LCSTie2Cre+/tgand LmnaLCS/LCSSM22αCre+/tg mice, which express progerin specifically in endothelial cells (ECs) and in vascular smooth muscle cells (VSMCs), respectively, to determine the specific contribution of each cell type to vascular pathology. We found vessel stiffness and inward remodeling in arteries of LmnaG609G/G609G and LmnaLCS/LCSSM22αCre+/tg, but not in those from LmnaLCS/LCSTie2Cre+/tgmice. Structural alterations in aortas of progeroid mice were associated with decreased smooth muscle tissue content, increased collagen deposition, and decreased transverse waving of elastin layers in the media. Functional studies identified collagen (unlike elastin and the cytoskeleton) as an underlying cause of aortic stiffness in progeroid mice. Consistent with this, we found increased deposition of collagens III, IV, V, and XII in the media of progeroid aortas. Vessel stiffness and inward remodeling in progeroid mice were prevented by adding sodium nitrite in drinking water. In conclusion, LmnaG609G/G609G arteries exhibit stiffness and inward remodeling, mainly due to progerin‐induced damage to VSMCs, which causes increased deposition of medial collagen and a secondary alteration in elastin structure. Treatment with nitrites prevents vascular stiffness in progeria. Ubiquitous progerin expression causes decreased smooth muscle tissue content and increased collagen deposition in the media associated with inward remodeling and vessel stiffness. These alterations are reproduced in mice expressing progerin in vascular smooth muscle cells but not in endothelial cells. Treatment with nitrites prevents progerin‐induced inward remodeling and vessel stiffness.

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.

The development of electrocatalysts capable of efficient reduction of nitrate (NO 3 − ) to ammonia (NH 3 ) is drawing increasing interest for the sake of low carbon emission and environmental protection. Herein, we present a CuCo bimetallic catalyst able to imitate the bifunctional nature of copper-type nitrite reductase, which could easily remove NO 2 − via the collaboration of two active centers. Indeed, Co acts as an electron/proton donating center, while Cu facilitates NO x − adsorption/association. The bio-inspired CuCo nanosheet electrocatalyst delivers a 100 ± 1% Faradaic efficiency at an ampere-level current density of 1035 mA cm −2 at −0.2 V vs . Reversible Hydrogen Electrode. The NH 3 production rate reaches a high activity of 4.8 mmol cm −2 h −1 (960 mmol g cat −1 h −1 ). A mechanistic study, using electrochemical in situ Fourier transform infrared spectroscopy and shell-isolated nanoparticle enhanced Raman spectroscopy, reveals a strong synergy between Cu and Co, with Co sites promoting the hydrogenation of NO 3 − to NH 3 via adsorbed *H species. The well-modulated coverage of adsorbed *H and *NO 3 led simultaneously to high NH 3 selectivity and yield. Electroreduction of NO 3 − to NH 3 is drawing increasing interest. Here, the authors designed a CuCo catalyst imitating the bifunctional nature of Cu-type nitrite reductase to deliver an ampere-level current density for NH 3 formation.

Introduction The efficacy of nebulized sodium nitrite (AIR001) has been demonstrated in animal models of pulmonary arterial hypertension (PAH), but it was not known if inhaled nitrite would be well tolerated in human subjects at exposure levels associated with efficacy in these models. Methods Inhaled nebulized sodium nitrite was assessed in three independent studies in a total of 82 healthy male and female subjects. Study objectives included determination of the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) under normal and mildly hypoxic conditions, and following co-administration with steady-state sildenafil, assessment of nitrite pharmacokinetics, and evaluation of the fraction exhaled nitric oxide (FE NO ) and concentrations of iron-nitrosyl hemoglobin (Hb(Fe)-NO) and S -nitrosothiols ( R -SNO) as biomarkers of local and systemic NO exposure, respectively. Results Nebulized sodium nitrite was well tolerated following 6 days of every 8 h administration up to 90 mg, producing significant increases in circulating Hb(Fe)-NO, R -SNO, and FE NO . Pulmonary absorption of nitrite was rapid and complete, and plasma exposure dose was proportional through the MTD dosage level of 90 mg, without accumulation following repeated inhalation. At higher dosage levels, DLTs were orthostasis (observed at 120 mg) and hypotension with tachycardia (at 176 mg), but venous methemoglobin did not exceed 3.0 % at any time in any subject. Neither the tolerability nor pharmacokinetics of nitrite was impacted by conditions of mild hypoxia, or co-administration with sildenafil, supporting the safe use of inhaled nitrite in the clinical setting of PAH. Conclusion On the basis of these results, nebulized sodium nitrite (AIR001) has been advanced into randomized trials in PAH patients.

Nitrite reductases play a crucial role in the nitrogen cycle, demonstrating significant potential for applications in the food industry and environmental remediation, particularly for nitrite degradation and detection. In this study, we identified a novel nitrite reductase (AhNiR) from a newly isolated denitrifying bacterium, Acinetobacter haemolyticus YD01. We constructed a heterologous expression system using E. coli BL21/pET28a-AhNir, which exhibited remarkable nitrite reductase enzyme activity of 29 U/mL in the culture broth, substantially higher than that reported for other strains. Structural analysis of AhNiR revealed the presence of [Fe-S] clusters, with molecular docking studies identifying Tyr-282 and Ala-289 as key catalytic sites. The enzymatic properties of AhNiR demonstrated an optimal pH of 7.5 and an optimal catalytic temperature of 30 °C. Its kinetic parameters, Km and vmax, were 1.53 mmol/L and 10.18 mmol/min, respectively, fitting with the Michaelis–Menten equation. This study represents the first report of a nitrite reductase from a denitrifying bacterium, providing a new enzyme source for nitrite degradation applications in the food industry and environmental remediation, as well as for biosensing technologies aimed at nitrite detection.

Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45 °C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120 minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

Nitric oxide (NO) is a signalling molecule involved in several physiological processes, in both prokaryotes and eukaryotes, and nitrite is being recognised as an NO source particularly relevant to cell signalling and survival under challenging conditions. The “non-respiratory” nitrite reduction to NO is carried out by “non-dedicated” nitrite reductases, making use of metalloproteins present in cells to carry out other functions, such as several molybdoenzymes (a new class of nitric oxide-forming nitrite reductases). This minireview will highlight the physiological relevance of molybdenum-dependent nitrite-derived NO formation in mammalian, plant and bacterial signalling (and other) pathways. The mammalian xanthine oxidase/xanthine dehydrogenase, aldehyde oxidase, mitochondrial amidoxime-reducing component, plant nitrate reductase and bacterial aldehyde oxidoreductase and nitrate reductases will be considered. The nitrite reductase activity of each molybdoenzyme will be described and the review will be oriented to discuss the feasibility of the reactions from a (bio)chemical point of view. In addition, the molecular mechanism proposed for the molybdenum-dependent nitrite reduction will be discussed in detail.

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of \"Candidatus Nitrospira defluvii\" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO₂ fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.