Hydroxylamine Reductase Activity Research Articles

Innovative fertilizer management system maintains higher maize productivity with lower environmental costs in the Loess Plateau region of China

ContextInnovative fertilizer management is the most effective strategy for increasing crop yields and reducing environmental pollution. ObjectivesDeep fertilization reduces NH3 emissions and increases crop yield, but its effects on N2O emissions are still unclear. Nitrification or urease inhibitors trigger cross-contamination of N2O and NH3. Little is known about whether innovative fertilizer management strategies can reduce nitrogen emissions while maintaining higher yields. MethodsWe conducted field experiments using maize in the semi-humid and drought-prone areas of the Loess Plateau in China from 2019 to 2021. To further verify the research results, experiments were conducted in semi-arid and drought-prone areas in 2022 and 2023 using surface fertilization (SF), surface fertilization with nitrification (SNI) or urease inhibitor (SUI), deep fertilization (DF), and deep fertilization with urease and nitrification inhibitor (DUN) as experimental treatments. ResultsThe trends were similar at the three experimental locations over five years, where the ability of DF to reduce emissions was equivalent to those of SNI for N2O and SUI for NH3. However, the increase in the crop productivity was significantly higher under DF than SUI and SNI. Compared with SF, DF significantly reduced the N2O and NH3 emissions by 39.85 % and 46.08 %, respectively, and significantly the increased maize yield, N uptake, and nitrogen use efficiency (NUE) by 16.86 %, 15.83 %, and 32.79 %. The innovative fertilizer management strategies combined the advantages of inhibitors and deep fertilization, and they obtained the highest maize yield and NUE, as well as the lowest N2O and NH3 emissions and gaseous nitrogen loss intensity (GNLI) among all treatments. Compared with SF, DUN reduced the urease, nitrate reductase, nitrite reductase, and hydroxylamine reductase activities by 22.03 %, 58.81 %, 52.72 %, and 50.27 %, respectively, the soil NO3–-N and NH4+-N contents by 82.94 % and 53.71 %, and N2O and NH3 emissions by 80.79 %, and 79.33 %. DUN increased the maize yield and NUE by 23.30 % and 46.91 %, respectively, and decreased the GNLI by 84.69 %. ConclusionsDUN reduced the concentration of substrates for producing gaseous nitrogen by regulating the activities of soil nitrogen circulating enzymes to significantly reduce the nitrogen loss, and it achieved the highest maize yield (11,818.10 kg ha–1) and NUE (46.62 %) with the lowest GNLI (0.71 g N kg–1 grain). Innovative fertilizer management strategies can help to balance economic and environmental benefits in the Loess Plateau region of China. ImplicationsOur findings provide a reference for obtaining higher maize productivity with low environmental pollution in the Loess Plateau region of China.

Class III hybrid cluster protein homodimeric architecture shows evolutionary relationship with Ni, Fe-carbon monoxide dehydrogenases

Hybrid cluster proteins (HCPs) are Fe-S-O cluster-containing metalloenzymes in three distinct classes (class I and II: monomer, III: homodimer), all of which structurally related to homodimeric Ni, Fe-carbon monoxide dehydrogenases (CODHs). Here we show X-ray crystal structure of class III HCP from Methanothermobacter marburgensis (Mm HCP), demonstrating its homodimeric architecture structurally resembles those of CODHs. Also, despite the different architectures of class III and I/II HCPs, [4Fe-4S] and hybrid clusters are found in equivalent positions in all HCPs. Structural comparison of Mm HCP and CODHs unveils some distinct features such as the environments of their homodimeric interfaces and the active site metalloclusters. Furthermore, structural analysis of Mm HCP C67Y and characterization of several Mm HCP variants with a Cys67 mutation reveal the significance of Cys67 in protein structure, metallocluster binding and hydroxylamine reductase activity. Structure-based bioinformatics analysis of HCPs and CODHs provides insights into the structural evolution of the HCP/CODH superfamily.

Effect of Litter Removal and Addition on Root Exudation and Associated Microbial N Transformation in a Pinus massoniana Plantation

In forest ecosystems, variations in aboveground litter input caused by global changes, substantially alter soil N cycling. In field-grown plants, few studies have directly measured root exudation rates and quantified their effects on N transformations under litter manipulation. We quantified soil N transformation rate responses to litter manipulation in a Pinus massoniana plantation, and unravelled the effect of root exudation on soil N transformations. We measured in situ P. massoniana root exudation rates as well as soil microbial biomass, soil C and N concentrations, the activities of four soil enzymes involved in soil N transformations, and net N mineralization and net nitrification rates after experimental litter removal and litter addition treatments. Litter removal and litter addition treatments had little impact on soil C and N concentrations, microbial biomass, soil enzyme (urease, hydroxylamine reductase, nitrate reductase, and nitrite reductase) activity, and net N mineralization rates. However, both litter removal and addition increased net N nitrification rates. Additionally, litter removal significantly decreased root C exudation rates (in April 2021 and annually), whereas litter addition had no significant effects on root C exudation rates across all seasons. Furthermore, root C exudation rates were positively associated with urease and nitrate reductase activities, but negatively associated with hydroxylamine reductase and nitrite reductase activities, as well as net N nitrification rate. Overall, we demonstrated that root exudates may be an important physiological adjustment by which trees respond to changes in litter input caused by global environmental changes, regulating underground N biochemical processes. Furthermore, we provide new evidence from root exudates for understanding the potential influence of litter inputs on soil N cycling. A strong correlation exists between root exudates and N transformation, shedding new light on the dynamics of rhizosphere nutrient cycling crucial for maintaining forest ecosystem stability and productivity under changing environmental conditions.

Nitrogen occurrence forms and bacterial community in sediment influenced by Bellamya purificata bioturbation

Endogenous water pollution and excessive nitrogen in ponds have always been prominent problems threatening aquaculture. To solve this problem, a 70-day indoor simulation experiment was conducted. The snail Bellamya purificata was stocked at four different densities: 0, 15, 30, and 60 individuals/tank, represented as CON (control), LD (low density), MD (medium density), and HD (high density), respectively, to investigate the effects of B. purificata on the nitrogen occurrence forms and bacterial communities in the sediment. At the end of the experiment, the nitrate concentration was significantly higher, while the total nitrogen content was significantly lower in the MD group than in the other three groups. Ammonia monooxygenase activity was significantly lower in the CON group than in the other three groups, and hydroxylamine oxidase activity was significantly higher in the HD group than in the other three groups. The CON and MD groups showed the highest and lowest values in nitrate reductase activity, respectively. The hydroxylamine reductase activity decreased significantly with increasing density. Through 16S ribosomal RNA (rRNA) high-throughput sequencing, significantly affected bacterial communities by B. purificata were found. Alpha diversity results showed that, a significantly lower Shannon index was observed in the MD group than in the other three groups. The LD and MD groups showed the highest and lowest Chao1 index values, respectively. Phyla Nitrospinae and family Nitrosomonadaceae were significantly enriched in the HD and MD groups, respectively. Redundancy analysis (RDA) indicated a significant correlation between differential bacterial taxa and TN content. Predicted functional analysis based on FAPROTAX (Functional annotation of prokaryotic taxa) database showed that functional groups aerobic ammonia oxidation and aerobic nitrite oxidation were significantly enriched in the MD group. Overall, B. purificata significantly altered the bacterial community composition, increased hydroxylamine oxidase and ammonia monooxygenase activities, enhanced the bacterial nitrification process, and promoted the transformation of total nitrogen to nitrate. Moreover, B. purificata stocked at 30 and 60 individuals/tank appeared to have a significant promotion effect on the bacterial community and nitrogen occurrence forms in the sediment. Hence, co-culturing B. purificata may be a feasible and effective ecological restoration method to alleviate excess nitrogen and reduce water eutrophication.

Successive corn stover and biochar applications mitigate N2O emissions by altering soil physicochemical properties and N-cycling-related enzyme activities: A five-year field study in Northeast China

As the most important source of commodity grain in China, Northeast China plays a crucial role in guaranteeing national food security. The application of biochar in agricultural ecosystems has shown immense potential in ensuring crop yields while mitigating N2O emissions. However, the long-term impacts and mechanisms of successive application of corn stover and its derived biochar on soil N2O emission in the corn monocropping region, such as Northeast China, remain unclear. A five-year field experiment was conducted in Northeast China to fill this research gap regarding the soil nitrogen (N) cycle. Three experimental treatments were established: a control with no corn stover or biochar application (CK), corn stover returned yearly at a rate of 7.5 t ha−1 (ST), and biochar added yearly at a rate of 2.6 t ha−1 (BC) corresponding to 35% of the stover addition rate in ST. Identical mineral fertilizers were applied in all three treatments. The total accumulated N2O emissions across five analyzed growing seasons within the CK, ST, and BC treatments were 260.2, 184.0, and 151.0 mg N2O m−2, respectively, with significant 29.3% and 42.0% decreases observed in ST and BC treatments, respectively, compared with the control. Based on the annual average from 2013 to 2017, ST and BC treatments significantly increased soil pH and total N (TN) content and significantly decreased the levels of NO3––N, NH4+-N, and soil bulk density (BD). Additionally, BC treatment remarkably lowered the average NH4+-N content than that under ST treatment by 24.1%. Both ST and BC showed a significant inhibitory effect on nitrate reductase (NR), nitrite reductase (NiR), and hydroxylamine reductase (HyR) activities related to denitrification. Soil N2O emission was significantly positively correlated with BD, NO3–-N, NH4+-N, NR, NiR, and HyR activities. Path analysis suggested that N2O emissions were primarily driven by NR and HyR activities and NO3–-N content. Overall, the applications of corn stover and its derived biochar are a feasible means of mitigating soil N2O emissions in the context of continuous corn cropping systems in mid-latitude regions like Northeast China, with biochar being more beneficial than stover application.

Impacts of silver nanoparticles on enzymatic activities, nitrifying bacteria, and nitrogen transformation in soil amended with ammonium and nitrate

Silver nanoparticles (AgNPs) are effective antimicrobial compounds that are used in a myriad of applications. Soil microorganisms play crucial roles in nitrogen cycling, but there is a lack of comprehensive understanding of the effects of AgNPs on enzymatic activity in the nitrogen cycle, nitrifying bacteria, and nitrogen transformation in soil. Herein, enzyme activities were determined following the addition of different forms of nitrogen, ammonium nitrogen ((NH4)2SO4), nitrate nitrogen (KNO3), and amide nitrogen (urea, CO(NH2)2) at 200 mg N kg–1, into the soil amended with AgNPs at 0, 10, 50, and 100 mg kg–1. After 7 d of incubation with 10 mg kg–1 AgNPs, the activities of urease, nitrite reductase (NiR), nitrate reductase (NaR), and hydroxylamine reductase (HyR) were reduced by 12.5%, 25.0%, 12.2%, and 24.2%, respectively. Of particular note, more than 53.5%, 61.7%, and 34.7% of NaR, NiR, and HyR activities, respectively, were inhibited at 100 mg kg–1 AgNPs. The abundance (most probable number) of ammonia- and nitrite-oxidizing bacteria (AOB and NOB, respectively) was measured using real-time quantitative polymerase chain reaction (qPCR) and the Cochran method. The abundance of AOB and NOB decreased when AgNPs were present in the soil. The NH4NO3 amendment increased copy numbers of bacterial and archaeal amoA nitrification functional genes, by 38.3% and 12.4%, respectively, but AgNPs at 50 mg kg–1 decreased these values by 70% and 56.4%, respectively. The results of 15N enrichment (atom% excess) of NH4+ and NO3– experiments illustrated the influence of AgNPs on soil nitrogen transformation. According to the 15N atom% excess detected, the conversion of 15N-labeled NH4+ to NO3– was significantly inhibited by the different levels of AgNPs in soil. The reduced gross nitrification rate further confirmed this finding. Overall, this study revealed considerable evidence that AgNPs inhibited nitrogen cycle enzyme activity, the number of nitrifying bacteria, the abundance of the amoA gene, and the gross nitrification rate. Silver nanoparticles inhibited nitrogen transformation, and the rate of nitrification was also negatively correlated with AgNP levels.

A Mechanistic Investigation of Cytochrome c Nitrite Reductase Catalyzed Reduction of Nitrite to Ammonia: The Search for Catalytic Intermediates

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were done with the wild type enzyme (wtccNiR) as well as with R103Q, Y206F, and H257Q variants. A UV/Vis stopped-flow investigation of the reaction between nitrite-loaded wtccNiR and TMPD revealed for the first time that the reaction proceeds via a transient 1-electron reduced intermediate. Generation of this species is pH-independent, whereas its decay to a previously characterized 2-electron reduced intermediate is fastest at pH 6.8 and significantly slower at higher pH. The pH dependence is ascribed to the rate-limiting cleavage of the nitrite N – O bond, which requires a prior di-protonation predicted to be slow in earlier computational studies. Under steady-state conditions, S. oneidensis ccNiR catalyzed the slow 1-electron reduction of nitrite to nitric oxide, which was monitored by tracking the concomitant appearance of the colored TMPD+ radical product. The rate of TMPD+ formation was found to be directly proportional to the concentration of TMPD+ at low TMPD concentrations, providing important insights about the mechanism of NO˙ release. The steady-state studies also showed that nitrite is a substrate inhibitor of NO˙ release when TMPD is the electron source, probably because it blocks exit by NO˙ through the nitrite entry channel. The H257Q variant of ccNiR was found to have 1/400th of the wild type enzyme's nitrite reductase activity in the standard assay in which methyl viologen monocation radical is the electron source, but nearly normal hydroxylamine reductase activity. This demonstrated that H257 is essential for nitrite reduction but not for reduction of hydroxylamine, a putative intermediate in the catalytic process. UV/Vis spectropotentiometry showed that the nitrite-loaded active site of H257Q still reduced at fairly high applied potential, but the reduction was by one electron, whereas the wild type is reduced in a concerted 2-electron step. The experiments with the variant, coupled with the pH-dependent stopped flow experiments with wtccNiR, confirm H257's importance in facilitating nitrite reduction, but suggest that its role is to modulate the pKa values of one or more of the waters that form a complex hydrogen bonding network in the active site. This view is more conservative than earlier theoretical predictions of direct histidine involvement in nitrite diprotonation leading to N – O bond cleavage.

Soil N2O and NOx emissions are directly linked with N-cycling enzymatic activities

Manure application and crop residue returning have been performed to enhance crop yield and reduce soil reactive nitrogen (Nr) gas (N2O and NOx) emissions. However, the dynamic characteristics of soil nitrogen-cycling (N-cycling) enzyme activities, and the relationship among N-cycling enzyme activities, Nr contents and Nr gas emissions are not clearly understood. Six fertilization regimes, including control (CK-no fertilizer), synthetic N fertilizer alone (N), synthetic nitrogen, phosphorus and potassium fertilizer (NPK), pig manure (OM), pig manure (30% of N rate) with synthetic NPK (70% of N rate) (OMNPK), and crop residues (20% of N rate) with synthetic NPK (80% of N rate) (CRNPK), were used for 11 years to form stable soil conditions. The total N application rate in each fertilization regime, except CK, is the same (130 kg N ha−1) in the winter wheat season. The results showed that the cumulative emissions of N2O plus NOx in OM increased significantly by 43.7%, while those in OMNPK and CRNPK decreased by 8.2 and 15.3% compared to conventional fertilization regimes of NPK. The long-term application of manure or returning crop residues to partly replace synthetic N fertilizer may significantly enhance soil hydrolase activities compared with NPK at most stages of wheat growth. In contrast to OM, OMNPK and CRNPK primarily inhibited the activities of nitrate reductase (NR) and nitrite reductase (NiR) but increased hydroxylamine reductase (HyR) activity. Redundancy analysis (RDA) and stepwise multiple linear regression showed positive NR and NiR activities, while PRO activity is negative for N2O emission. NOx emissions were mainly controlled by HyR activities. Thus, soil reactive nitrogen (Nr) gas emissions may be mitigated by different fertilization regimes through the regulation of soil enzyme activities. Fertilization regimes, such as manure application and returning crop residues with synthetic NPK, could be recommended as optimal N fertilization strategies in Regosols to mitigate Nr gas emissions.

Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system.

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).

A thermostable hybrid cluster protein from Pyrococcus furiosus: effects of the loss of a three helix bundle subdomain

Pyrococcus furiosus hybrid cluster protein (HCP) was expressed in Escherichia coli, purified, and characterized. This is the first archaeal and thermostable HCP to be isolated. Compared with the protein sequences of previously characterized HCPs from mesophiles, the protein sequence of P. furiosus HCP exhibits a deletion of approximately 13 kDa as a single amino acid stretch just after the N-terminal cysteine motif, characteristic for class-III HCPs from (hyper)thermophilic archaea and bacteria. The protein was expressed as a thermostable, soluble homodimeric protein. Hydroxylamine reductase activity of P. furiosus HCP showed a Km value of 0.40 mM and a kcat value of 3.8 s−1 at 70 °C and pH 9.0. Electron paramagnetic resonance spectroscopy showed evidence for the presence of a spin-admixed, S = 3/2 [4Fe–4S]+ cubane cluster and of the hybrid cluster. The cubane cluster of P. furiosus HCP is presumably coordinated by a CXXC–X7–C–X5–C motif close to the N-terminus, which is similar to the CXXC–X8–C–X5–C motif of the Desulfovibrio desulfuricans and Desulfovibrio vulgaris HCPs. Amino acid sequence alignment and homology modeling of P. furiosus HCP reveal that the deletion results in a loss of one of the two three-helix bundles of domain 1. Clearly the loss of one of the three-helix bundles of domain 1 does not diminish the hydroxylamine reduction activity and the incorporation of the iron–sulfur clusters.

The Nitric Oxide Reductase Activity of Cytochrome c Nitrite Reductase from Escherichia coli

Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO. than the parent strain led to the proposal that NrfA might also participate in NO. detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO. reductase activity. NO. reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO. reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO. reduction by NrfA is described by a K(m) of the order of 300 microm. The predicted turnover number of approximately 840 NO. s(-1) is much higher than that of the dedicated respiratory NO. reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO. detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO. encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO. levels such that any NO. reaching the cytoplasm is efficiently removed by flavorubredoxin (K(m) approximately 0.4 microm).

Hydroxylamine Assimilation by Rhodobacter capsulatus E1F1: REQUIREMENT OF THE hcp GENE (HYBRID CLUSTER PROTEIN) LOCATED IN THE NITRATE ASSIMILATION nas GENE REGION FOR HYDROXYLAMINE REDUCTION

Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP). Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene. A His(6)-HCP was overproduced in Escherichia coli and purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant. The apparent K(m) values for NH(2)OH and methyl viologen were 1 mM and 7 microM, respectively, at the pH and temperature optima (9.3 and 40 degrees C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents. R. capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH(2)OH as nitrogen source, and up to 10 mM NH(2)OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D,L-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However, R. capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH(2)OH. Also, E. coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH(2)OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.

Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli.

The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties. Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified. While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown. Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity. These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O. Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction.

Converting the NiFeS Carbon Monoxide Dehydrogenase to a Hydrogenase and a Hydroxylamine Reductase

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.

Hydroxylamine Reductase Activity of the Hybrid Cluster Protein from Escherichia coli

Hydroxylamine Reductase Activity of the Hybrid Cluster Protein from Escherichia coli

NADH-dependent reduction of sulphamethoxazole hydroxylamine in dog and human liver microsomes

1. Reduction of hydroxylamine drug metabolites by NADH-dependent hydroxylamine reductase (NDHR) has been suggested to be involved in the pathogenesis of idiosyncratic sulphonamide toxicity in humans. The dog represents a naturally occurring clinical model for sulphonamide toxicity in humans. The purpose of these studies, therefore, was to characterize the presence of hepatic NADH-dependent hydroxylamine reductase activity in the dog and to compare this activity with that found in humans. 2. NDHR activity was characterized by the presence of two enzymes in both dog and human liver microsomes, with comparable estimates of Km (Km1 = 75 μM, Km = 404 μM in dog; Km1 = 69 μM, Km = 503 μM in human). Estimates of maximal velocitywere significantly, but not dramatically, higher for dog NDHR (Vmax = 2.09 nmole mg−1 min−1, Vmax2 = 4.58 nmole mg−1 min−1) compared with human NDHR(Vmax1 = 0.42 nmole mg−1 min−1, Vmax2 = 1.56 nmole mg−1 min−1). NDHR in dog, as inhumans, preferred NADH to NADPH, was more active at pH 6.3 than at 7.4 and was not inhibited by carbon monoxide, azide, anaerobic conditions, the CYP substrate inhibitors tolbutamide, dextromethorphan, or erythromycin, or antibodies directed against CYP2C, CYP2D or CYP3A. 3. It is concluded that two forms of NDHR are present in dog and humans with similar biochemical characteristics. Although NDHR activity has been attributed to a CYP2Disoform in pig, there is no evidence for involvement of CYP450 in the reduction of sulphamethoxazole hydroxylamine in either dogs or humans.

Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase.

Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide.

The isolation of a hexaheme cytochrome from Desulfovibrio desulfuricans and its identification as a new type of nitrite reductase.

Desulfovibrio desulfuricans (ATCC 27774), a strictly anaerobic sulfate-reducing bacteria, is able to perform anaerobic nitrate respiration in which nitrate is first reduced to nitrite by the action of nitrate reductase, and nitrite reductase then catalyzes the six-electron reduction of nitrite to ammonia. The nitrite reductase was found to be a membrane-bound enzyme and has been purified to electrophoretic homogeneity. The purified enzyme has a minimal Mr = 66,000 as judged by sodium dodecyl sulfate gel electrophoresis and contains 6 c-type heme groups/molecule. Pure nitrite reductase exhibits a typical c-type cytochrome absorption spectrum with reduced alpha-band at 552.5 nm. NADH and NADPH do not function as direct electron donors for the nitrite reductase. Desulfovibrio vulgaris hydrogenase, however, is able to transfer electrons from H2 to the nitrite reductase using FAD as the electron transfer mediator. The dithionite-reduced nitrite reductase was demonstrated to be auto-oxidizable even in the presence of potassium cyanide. On addition of nitrite, the dithionite-reduced enzyme is re-oxidized immediately. Hydroxylamine, however, can only partially re-oxidize the reduced enzyme. Ascorbate reduces the enzyme to a limited extent and the partially reduced enzyme is neither auto-oxidizable nor re-oxidizable by nitrite or hydroxylamine. Purified nitrite reductase has a pH optimum in the range of 8.0-9.5 and optimal activity at 57 degrees C. Purified nitrite reductase also has hydroxylamine reductase activity, and the Km for nitrite was determined to be 1.14 mM and that for hydroxylamine is 113.5 mM. The difference in Km values seems to exclude the possibility of hydroxylamine being a free intermediate in the reduction of nitrite.

Nitrite reductase system involved in the terminal oxidation of the Streptomyces griseus respiratory particle

A nitrite reductase system which was associated with the electron transfer system of the respiratory particle in Streptomyces griseus was studied. The electron transfer pathway consisted of the cytochrome oxidase and the nitrite reductase systems under aerobic and anaerobic conditions respectively, and these systems showed the exact opposite response to 2- n-heptyl-4-hydroxyquinoline- N-oxide and azide. Azide inhibited specifically the nitrite reductase system. It seems that cytochrome d works as the nitrite reductase and the reduced cytochrome b works as an intermediate electron donor for cytochrome d respectively. The respiratory particle also had a hydroxylamine reductase activity and ammonia was identified as the product of hydroxylamine reduction by the respiratory particle. A terminal electron transfer pathway in Streptomyces griseus was proposed.

Hydroxylamine Reductase Enzymes from Maize Scutellum and Their Relationship to Nitrite Reductase

Three enzymes contribute to the total hydroxylamine reductase activity of corn (Zea mays L.) scutellum extracts. Two of these resemble enzymes previously prepared from leaves, while the third, which accounts for a major part of the activity, appears to have no counterpart in leaf tissue. One of the hydroxylamine reductases found only in small amounts is associated with nitrite reductase and is induced, together with nitrite reductase, by nitrite. The other two enzymes are noninducible by nitrite and can be totally separated from nitrite reductase, which subsequently remains capable of catalyzing the reduction of nitrite to ammonia. Possible causes of the decline of hydroxylamine reductase activity during the induction of nitrite reductase are discussed.