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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.

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.

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.

Here, the draft genome of simultaneous nitrification-denitrification strain (SND) Klebsiella sp. KSND revealed possible existence of genes involved in N-assimilation and -dissimilation pathways. The change levels of genes under defined N-sources were analyzed by Quantitative Real-Time PCR. It suggested that NH4+-assimilation via NADP-glutamate dehydrogenase pathway would occur preferentially. NirBD genes were tightly regulated in a lower level, so that nitrite was rapidly consumed for detoxication by denitrification. Three types of nitrate reductase homologues are surprisingly present in KSND, whereas the dominant nitrate reduction for assimilation and denitrification processes mediates by NapA-type nitrate reductase. Nitric oxide reductase homologues FlRd and FlRd-red provide an adequate capacity for NO detoxification. The recombinant hydroxylamine reductase showed high activity in hydroxylamine to generate ammonium, which might contribute to detoxification mechanism in nitrogen cycling. Overall, this study firstly provides valuable insights into the genes expression and enzyme action, which helps understanding the mechanism of SND processes.

Abstract Nitrogen migration and transformation in the stormwater bioretention system were studied in laboratory experiments, in which the effects of drying-rewetting were particularly investigated. The occurrence and distribution of nitrogen in the plants, the soil, and the pore water were explored under different drying-rewetting cycles. The results clearly showed that bioretention system could remove nitrogen efficiently in all drying-rewetting cycles. The incoming nitrogen could be retained in the topsoil (0–10 cm) and accumulated in the planted layer. However, the overlong dry periods (12 and 22 days) cause an increase in nitrate in the pore water. In addition, nitrogen is mostly stored in the plants’ stem tissues. Up to 23.26% of the inflowing nitrogen can be immobilized in plant organ after a dry period of 22 days. In addition, the relationships between nitrogen reductase activity in the soil and soil nitrogen content were explored. The increase of soil TN content could enhance the activity of nitrate reductase. Meanwhile, the activity of hydroxylamine reductase (HyR) could be enhanced with the increase of soil NO3− content. These results provide a reference for the future development of nitrogen transformation mechanism and the construction of stormwater bioretention systems.

Tropospheric O3 can impact soil systems mediated by changing root biomass and exudates. This study investigated how elevated O3 alters soil CO2 and N2O emissions from a winter wheat field. Winter wheat (Triticum aestivum L.) were fumigated using elevated O3 concentration (EO3) and using ambient air within open-top chambers during 2015 and 2016. Soil CO2 and N2O emissions were measured using a static closed-chamber technique. Rhizosphere soils were collected to determine dissolved organic carbon (DOC) content, ammonium-nitrogen (NH4+-N) and nitrate-nitrogen (NO3−-N) concentrations, microbial biomass carbon (MBC) and nitrogen (MBN), and activities of nitrate reductase (NR), nitrite reductase (NiR) and hydroxylamine reductase (HR). Moreover, volumetric soil water content (θv) was monitored and plant dry biomass was quantified. The EO3 treatment exhibited decreased CO2 and increased N2O emissions from winter wheat soil. Soil NH4+-N, NO3−-N, NR and NiR increased under EO3, whereas DOC, MBC, MBN and HR decreased. Soil θv was higher under EO3 before irrigation, due to reduced transpiration. Elevated O3 decreased soil CO2 flux potentially due to reduced root biomass and associated carbon input. It increased N2O fluxes apparently through enhanced denitrification due to greater substrate availability and soil θv, despite the inhibition of nitrification-related routes.

Information on the biochemical pathways of carbon and energy metabolism in representatives of the deep lineage bacterial phylum Deferribacteres are scarce. Here, we report the results of the sequencing and analysis of the high-quality draft genome of the thermophilic chemolithoautotrophic anaerobe Deferribacter autotrophicus. Genomic data suggest that CO2 assimilation is carried out by recently proposed reversible tricarboxylic acid cycle (&ldquo, roTCA cycle&rdquo, ). The predicted genomic ability of D. autotrophicus to grow due to the oxidation of carbon monoxide was experimentally proven. CO oxidation was coupled with the reduction of nitrate to ammonium. Utilization of CO most likely involves anaerobic [Ni, Fe]-containing CO dehydrogenase. This is the first evidence of CO oxidation in the phylum Deferribacteres. The genome of D. autotrophicus encodes a Nap-type complex of nitrate reduction. However, the conversion of produced nitrite to ammonium proceeds via a non-canonical pathway with the participation of hydroxylamine oxidoreductase (Hao) and hydroxylamine reductase. The genome contains 17 genes of putative multiheme c-type cytochromes and &ldquo, e-pilin&rdquo, genes, some of which are probably involved in Fe(III) reduction. Genomic analysis indicates that the roTCA cycle of CO2 fixation and putative Hao-enabled ammonification may occur in several members of the phylum Deferribacteres.

To investigate the emission mechanism of ammonia (NH3) and nitrous oxide (N2O) during aerobic composting and the influence of powder bamboo biochar (PBB) on this process, this paper conducted a systematic study on the nitrogen-transforming functional microbial community, including functional genes, microbial structure and metabolism pathways. PBB reduced N2O and NH3 emissions by 1.25%-8.72% and 10.4%-11.8%, respectively. The quantitative PCR results indicated that the reduced N2O emission by PBB were mainly related to denitrifying genes (nirS, nirK, nosZ, and narG). The metagenome results demonstrated that Nitrosococcus was the main genus that could oxidize ammonia to nitrite decreased by PBB. The PBB significantly affected the nitrogen metabolism pathway, reduced the activity of glutamate dehydrogenase to inhibit the formation of NH4+ to reduce NH3 emission. The higher N2O emission in the control group was also related to the higher relative contents of hydroxylamine reductase and nitrite reductase.

Treponema denticola is a major pathogen in periodontal disease and is frequently isolated from the lesions of patients with chronic periodontitis. Treponema denticola utilizes serum components as nutrient sources so as to colonize and proliferate in the gingival crevice. However, the mechanisms of serum utilization remain unclear. Therefore, the aim of the present study was to identify T. denticola serum utilization genes. Precultured T. denticola cells were suspended in a tryptone-yeast extract-gelatin-volatile fatty acids medium containing 0, 1% and 10% serum, respectively, and incubated anaerobically for 17 h. Total RNA was isolated, and T. denticola gene expression was compared by microarray and reverse transcription-polymerase chain reaction. In serum-depleted conditions, the expression levels of a potential hydroxylamine reductase, several ABC transporters, and phosphoenolpyruvate synthase were increased, while those of genes encoding methyl-accepting chemotaxis proteins and a transcriptional regulator were decreased. These results suggest that T. denticola may uptake serum components mainly through the action of ABC transporters. In particular, the decrease in the dmcA expression level with decreasing serum concentration suggests its involvement in chemotaxis toward serum-rich environments.

Microbes capable of performing heterotrophic nitrification and aerobic denitrification simultaneously have application in nitrogen level management in effluent treatment plants. Klebsiella pneumoniae EGD-HP19-C is a metabolically versatile bacterium capable of utilising NH3-N, NO2-N and NO3-N as sole sources of nitrogen. The annotation was done for the genes involved in N-assimilation and N-dissimilation pathways from the draft genome sequences of this bacterium (NCBI GenBank accession no. AUTW02000000.1). The sequence data also suggested possible existence of plasmid associated with this bacterium. Multiple gene sequence alignments of glutamine synthetase (gln), hydroxylamine reductase (har), nitrite reductase (nir), nitric oxide reductase (nor), assimilatory nitrate reductase (nas) and respiratory nitrate reductase (nar) genes from EGD-HP19-C genome were performed to compare sequence identities with that of closely related bacterial species. The metabolic pathways were mapped using KAAS and 3D structures for representative enzyme sub-units were also elucidated. The study suggested that the organism, though it has incomplete nitrification and denitrification pathways still removes the inorganic nitrogen content from the system via ammonification reaction.

Porphyromonas gingivalis , the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 ( PG0893 ∷ ermF ) and FLL456 ( PG2213 ∷ ermF ) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181 , predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.

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 K(m) value of 0.40 mM and a k(cat) value of 3.8 s(-1) at 70 degrees 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-X(7)-C-X(5)-C motif close to the N-terminus, which is similar to the CXXC-X(8)-C-X(5)-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.

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 His6-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 Km values for NH2OH and methyl viologen were 1 mm and 7 μm, respectively, at the pH and temperature optima (9.3 and 40 °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 NH2OH as nitrogen source, and up to 10 mm NH2OH 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 NH2OH. Also, E. coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH2OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.

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.

Paracoccus pantotrophus cytochrome cd(1) is a physiological nitrite reductase and an in vitro hydroxylamine reductase. The oxidised "as isolated" form of the enzyme has bis-histidinyl coordinated c-heme and upon reduction its coordination changes to histidine/methionine. Following treatment of reduced enzyme with hydroxylamine, a novel, oxidised, conformer of the enzyme is obtained. We have devised protocols for freeze-quench near-ir-MCD spectroscopy that have allowed us to establish unequivocally the c-heme coordination of this species as His/Met. Thus it is shown that the catalytically competent, hydroxylamine reoxidised, form of P. pantotrophus cytochrome cd(1) has different axial ligands to the c-heme than "as isolated" enzyme.

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. he 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 microM, Km2 = 404 microM in dog; Km1 = 69 microM, Km2 = 503 microM in human). Estimates of maximal velocity were significantly, but not dramatically, higher for dog NDHR (Vmax1 = 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 in humans, 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 CYP2D isoform in pig, there is no evidence for involvement of CYP450 in the reduction of sulphamethoxazole hydroxylamine in either dogs or humans.

With the development of methods to crystallize proteins to sizes suitable for X-ray analysis, structures of many proteins have been determined to elucidate the relationships between their structures and functions. However, crystals of biological macromolecules have been frequently found to be twinned by adherence of two crystal lattices. Some crystals show splitting of diffraction spots owing to the different tilts of the two lattices. Others pretend to be single crystals with no split spots, and their symmetries of intensity distribution vary with every data set. These latter have been called hemihedral, and in them the unique axes of the two crystals are exactly reversely parallel with each other. The observed intensities were found to be detwinned by the least-squares method after Britton's algorithm [Britton (1972). Acta Cryst. A28, 296–297], when several sets of intensity data were observed. Fortran programs have been successfully used to determine the scale factors and degrees of twinning from the data of hydroxylamine oxidoreductase. The analysis of data collected at various positions of one crystal of this enzyme suggested a heterogeneous distribution of twinning in the crystal.

1,3,5-トリアジンのハロメチル置換基が硝化細菌の硝酸化成作用に及ぼす影響について調べることを目的として, 26種のハロメチル-1,3,5-トリアジン系化合物を用いて試験を行なった. 1,3,5-トリアジン系化合物の硝酸化成抑制効果は二つの実験方法によって測定した. 畑土壌を用いたアンモニアから硝酸 (NO3--N) への酸化と, Nitrosomonas europaea ATCC 25978の細胞懸濁液を用いたアンモニアから亜硝酸 (NO2--N) への酸化を測定した. その結果, トリクロルメチル基を有する化合物は畑土壌と菌体の両方において, 部分的に塩素化されたメチル基 (CH2ClおよびCHCl2) をもつ化合物よりも強い硝酸化成抑制効果を示すことが判明した. トリブロモメチル基をもつ1,3,5-トリアジン系化合物は N. europaea ATCC 25978の細胞懸濁液では非常に強い抑制効果を示したが, 土壌試験においては弱い抑制効果しか示さなかった. また, N. europaea ATCC 25978を用いた試験では基質として (NH4)2SO4を用いた場合には抑制効果がみられたが, NH2OH・HClを用いたときにはみられなかった. したがって, 検討したすべてのハロメチル-1,3,5-トリアジン系化合物の作用点はアンモニアからヒドロキシルアミンへの酸化過程にあると推測された.

The process of nitrate reduction via nitrite controls the fate and bioavailability of mineral nitrogen within ecosystems; i.e. whether it is retained as ammonium (ammonification) or lost as nitrous oxide or dinitrogen (denitrification). Here, we present experimental evidence for a novel pathway of microbial nitrate reduction, the reverse hydroxylamine:ubiquinone reductase module (reverse-HURM) pathway. Instead of a classical ammonia-forming nitrite reductase that performs a 6 electron-transfer process, the pathway is thought to employ two catalytic redox modules operating in sequence: the reverse-HURM reducing nitrite to hydroxylamine followed by a hydroxylamine reductase that converts hydroxylamine to ammonium. Experiments were performed on Nautilia profundicola strain AmH, whose genome sequence led to the reverse-HURM pathway proposal. N. profundicola produced ammonium from nitrate, which was assimilated into biomass. Furthermore, genes encoding the catalysts of the reverse-HURM pathway were preferentially expressed during growth of N. profundicola on nitrate as an electron acceptor relative to cultures grown on polysulfide as an electron acceptor. Finally, nitrate-grown cells of N. profundicola were able to rapidly and stoichiometrically convert high concentrations of hydroxylamine to ammonium in resting cell assays. These experiments are consistent with the reverse-HURM pathway and a free hydroxylamine intermediate, but could not definitively exclude direct nitrite reduction to ammonium by the reverse-HURM with hydroxylamine as an off-pathway product. N. profundicola and related organisms are models for a new pathway of nitrate ammonification that may have global impact due to the wide distribution of these organisms in hypoxic environments and symbiotic or pathogenic associations with animal hosts.

Although the Gram-negative, anaerobic periodontopathogen Porphyromonas gingivalis must withstand nitrosative stress, which is particularly high in the oral cavity, the mechanisms allowing for protection against such stress are not known in this organism. In this study, microarray analysis of P. gingivalis transcriptional response to nitrite and nitric oxide showed drastic upregulation of the PG0893 gene coding for hybrid cluster protein (Hcp), which is a putative hydroxylamine reductase. Although regulation of hcp has been shown to be OxyR dependent in Escherichia coli , here we show that in P. gingivalis its expression is dependent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing an ermF-ermAM insertion within the hcpR (PG1053) gene, was significantly reduced in the presence of nitrite ( P < 0.002) and nitric oxide-generating nitrosoglutathione (GSNO) ( P < 0.001), compared to that of the wild-type W83 strain. Furthermore, the upregulation of PG0893 ( hcp ) was abrogated in V2807 exposed to nitrosative stress. In addition, recombinant HcpR bound DNA containing the hcp promoter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstrating that HcpR plays an important role in P. gingivalis virulence. This work gives insight into the molecular mechanisms of protection against nitrosative stress in P. gingivalis and shows that the regulatory mechanisms differ from those in E. coli .

Major questions concerning the sources and mechanisms of the reduction of nitric oxide by enteric bacteria remain unresolved. The membrane-associated nitrate reductase is the major source of NO generated from nitrite, but at least one other source remains to be identified. Nitrite reductases are primarily detoxification systems that decrease rather than increase the accumulation of NO in the cytoplasm. Whether they also catalyze NO formation is unresolved. The FNR protein that regulates transitions between aerobic and anaerobic growth is inactivated as a consequence of nitrosative damage, but we challenge the idea that FNR is a physiologically relevant sensor of NO, except under the most severe nitrosative stress. As none of the three enzymes that reduce NO account for the majority of the rate of NO reduction, additional mechanisms remain to be discovered. Little is known about the biochemistry of damage repair. Whatever the growth conditions and however severe the nitrosative stress, groups of proteins are synthesized to protect the bacterial cytoplasm against the side effects of nitrate and nitrite reduction. The enigmatic hybrid cluster protein is more likely to be part of a repair pathway than a hydroxylamine reductase, as annotated in many genome databases.

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-A-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).

The incidence of adverse reactions to sulfamethoxazole-trimethoprim (SMX-TMP) combination products is higher in patients with AIDS than in the general population. Idiosyncratic adverse reactions to SMX are believed to be dependent upon the formation of the reactive intermediate, sulfamethoxazole hydroxylamine (SMX-HA), and its further oxidation product, nitroso-SMX. Changes in the disposition of SMX have been proposed to contribute to the increased risk of SMX adverse reactions in patients with AIDS. Activation of host defense mechanisms is known to alter drug metabolism and could decrease the enzymatic reduction of SMX-HA to the parent SMX, causing an imbalance in bioactivation and detoxification. We tested this hypothesis in a rat model of lipopolysaccharide (LPS)-evoked host defense activation. Rats were treated i.p. with 1 mg/kg of LPS, and hepatic microsomes were isolated 24 hr after treatment. The bioactivation of SMX to SMX-HA was reduced 50% by pretreatment with LPS (113 ± 10 vs 65 ± 4 pmol/min/mg; P < 0.05). However, the NADH-dependent reduction of SMX-HA to SMX was reduced by over 80% (454 ± 90 vs 81 ± 48 pmol/min/mg; P < 0.05). A decreased ability to reduce SMX-HA to SMX could predispose patients with systemic activation of host defense mechanisms, such as those with AIDS, to the occurrence of SMX-associated adverse reactions.

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 K m value of 0.40 mM and a k cat 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.

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 K m value of 0.40 mM and a k cat 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.

DMPP (3,4-dimethylpyrazole phosphate) has been used to reduce nitrogen (N) loss from leaching or denitrification and to improve N supply in agricultural land. However, its impact on soil nitrifying organisms and enzyme activities involved in N cycling is largely unknown. Therefore, an on-farm experiment, for two years, has been conducted, to elucidate the effects of DMPP on mineral N (NH4(+)-N and NO3(-)-N) leaching, nitrifying organisms, and denitrifying enzymes in a rice-oilseed rape cropping system. Three treatments including urea alone (UA), urea + 1% DMPP (DP), and no fertilizer (CK), have been carried out. The results showed that DP enhanced the mean NH4(+)-N concentrations by 19.1%--24.3%, but reduced the mean NO3(-)-N concentrations by 44.9%--56.6% in the leachate, under a two-year rice-rape rotation, compared to the UA treatment. The population of ammonia oxidizing bacteria, the activity of nitrate reductase, and nitrite reductase in the DP treatment decreased about 24.5%--30.9%, 14.9%--43.5%, and 14.7%--31.6%, respectively, as compared to the UA treatment. However, nitrite oxidizing bacteria and hydroxylamine reductase remained almost unaffected by DMPP. It is proposed that DMPP has the potential to either reduce NO3(-)-N leaching by inhibiting ammonia oxidization or N losses from denitrification, which is in favor of the N conversations in the rice-oilseed rape cropping system.

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates nitrate under anaerobic phototrophic growth conditions. A 17 kb DNA region encoding the nitrate assimilation (nas) system of this bacterium has been cloned and sequenced. This region includes the genes coding for a putative ABC (ATP-binding cassette)-type nitrate transporter (nasFED) and the structural genes for the enzymes nitrate reductase (nasA), nitrite reductase (nasB) and hydroxylamine reductase (hcp). Three genes code for putative regulatory proteins: a nitrite-sensitive repressor (nsrR), a transcription antiterminator (nasT) and a nitrate sensor (nasS). Other genes probably involved in nitrate assimilation are also present in this region. The sequence analysis of these genes and the biochemical properties of the purified nitrate, nitrite and hydroxylamine reductases are reviewed.

The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH(2)OH to NO(2)(-). The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O(2) uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization.

The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autotrophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct phylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosospira groups. Hybridization patterns were used to assess the potential of functional probes in non-PCR-based molecular analysis of natural AAO populations and their activity. The objective of this study was to obtain an overview of functional gene homologies by hybridizing probes derived from N. europaea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with 32P to Southern blots containing genomic DNA from four Nitrosospira representatives. Probes were specific for genes encoding ammonia monooxygenase (amoA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy). These probes produced hybridization signals, at low stringency (30 degreesC), with DNA from each of the four representatives; signals at higher stringency (42 degreesC) were greatly reduced or absent. The hybridization signals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional genes in the ammonia oxidation pathway have diverged between the Nitrosomonas and Nitrosospira groups. The hao probe produced the most consistent hybridization intensities among the Nitrosospira representatives, suggesting that hao sequences would provide the best probes for non-PCR-based molecular analysis of terrestrial AAOs. Since N. europaea can also denitrify, an additional objective was to hybridize genomic DNA from AAOs with probes for Pseudomonas genes involved in denitrification. These probes were specific for genes encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and Nitrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in their mechanisms and capacities for denitrification.

Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochromeb 5, its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83–92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxicN-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason whyN-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

The inclusion of cysteine and Na-EDTA in the extracting buffer lowered the activity of sulphite reductase extracted from wheat leaves while nitrite and hydroxylamine reductases were not so affected. Maximum activity for the three enzymes was achieved with reduced methyl viologen as the electron donor. The three enzyme activities were found in the chloroplasts. Nitrite reductase was detected in the leaves of the seedlings only when grown with nitrate and exposed to light. Sulphite and hydroxylamine reductases were not, however, influenced by either of these treatments. These results suggest that nitrite reductase is a distinct enzyme and is not associated with sulphite reductase and hydroxylamine reductase in wheat leaves.

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 enzyme systems and the mechanisms involved in the biotransformation of amphetamine, methamphetamine and the related compounds are briefly reviewed. The major themes discussed in this review are : 1) cytochrome P-450 isozymes involved in the deamination, N-dealkylation and aromatic hydroxylation ; 2) the role of cytochrome P-450 and flavin-containing monooxygenase in the N-dealkylation ; 3) the enzymes functioning as hydroxylamine reductase and oxidase ; 4) molecular mechanisms of deamination and N-dealkylation ; 5) metabolic-intermediate complex.

NADPH-sulfite reductase (EC 1.8.1.2) from Escherichia coli is a complex hemoflavoprotein, molecular weight 670,000, containing 4 FMN, 4 FAD, 20 to 21 atoms of iron, 14 to 15 labile sulfides, and 3 to 4 molecules of a novel type of heme per enzyme molecule. This heme has been identified as an octacarboxylic iron-tetrahydroporphyrin (Murphy, M. J., Siegel, L. M., Kamin, H., and Rosenthal, D. (1973) J. Biol. Chem. 248, 2801). The enzyme catalyzes the stoichiometric conversion of sulfite to sulfide at the expense of 3 NADPH. The Km values for sulfite and NADPH are both 4 to 5 µm. Reduced methyl or benzyl viologens can serve as electron donors for sulfite reduction, but NADH cannot. In addition to sulfite reduction, the enzyme catalyzes the NADPH-dependent reduction of a variety of "diaphorase" acceptors (cytochrome c, ferricyanide, 2,6-dichlorophenolindophenol, menadione, FMN, FAD) as well as NADPH oxidase, NADPH-3-acetylpyridine adenine dinucleotide phosphate transhydrogenase, NADPH-nitrite and -hydroxylamine reductase and reduced methyl viologen-NADP+ reductase activities. All NADPH-dependent activities examined were competitively inhibited by NADP+. Agents which react with the heme prosthetic group, i.e. CO, cyanide, and arsenite, inhibit the reductions of sulfite, nitrite, and hydroxylamine (with either NADPH or reduced methyl viologen as electron donor), while all other activities are unaffected. Cyanide and CO binding to and CO dissociation from the enzyme (determined spectrophotometrically) parallel the respective development and relief of inhibition of NADPH-sulfite reductase activity. Development of inhibition requires the presence of reductant (NADPH) as well as inhibitor, in accord with the observation that CO, cyanide, or arsenite can react with reduced, but not oxidized enzyme. Treatment of enzyme with 1 µm p-chloromercuriphenylsulfonate causes the dissociation of virtually all of the FMN while permitting retention of FAD and heme. This treatment inhibits all pyridine nucleotide-dependent reactions of the enzyme except transhydrogenase and FMN reductase. The methyl viologen-sulfite reductase is unaffected. The development of fluorescence due to FMN release parallels the development of the observed inhibitions. The FAD of the FMN-free enzyme is reducible by NADPH, but the heme is not. If exogenous FMN is added, the heme becomes reducible and all NADPH-dependent activities are restored. We have concluded that electron flow from NADPH to sulfite follows the minimum linear sequence: NADPH → FAD → FMN → heme → sulfite In this scheme, FAD serves as the "entry port" for electrons from NADPH. It can transfer electrons directly to FMN (internal or external) or to pyridine nucleotides and their analogues. The heme is required for electron transfer to sulfite (and nitrite and hydroxylamine). The FMN is required for electron transfer from the reduced FAD to the heme (and hence to acceptors dependent on the heme) or, more directly, to diaphorase-type acceptors and O2. Reduced methyl viologen can donate electrons to both the FMN and heme. The patterns of inhibition by a variety of salts of the NADPH-cytochrome c and reduced methyl viologen-sulfite reductase reactions are consistent with the hypothesis that these two reactions involve independent portions of the enzyme molecule.

NADH-nitrite and -hydroxylamine reductases were co-purified from Derxia gummosa. The stoichiometries for the reduction of nitrite and hydroxylamine to ammonia were 3 NADH:1 NO−2:1 NH3 and 1 NADH:1 NO−2:1 NH3. The Km values for nitrite and hydroxylamine were 4.8 μM and 5.3 mM, respectively, and for NADH they were 6.3 μM for nitrite reductase and 150 μM for hydroxylamine reductase. The optimal pH value for both enzyme activities was 8.5. Both activities were inhibited by NADH in the absence of the appropriate substrate, namely nitrite or hydroxylamine. Studies with amino acid modifiers indicate that histidine, glutamate/aspartate, sulphydryl and tyrosine are essential components of the enzyme protein. Kinetic studies show that nitrite and hydroxylamine were competitive for the same binding site on the enzyme. The results indicate that although nitrite and hydroxylamine reductases are associated with the same enzyme, its main function is the reduction of nitrite to ammonia. Azaserine inhibited the induction of the enzyme.

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.

Nitrite, hyponitrite and hydroxylamine reductases in Neurospora are shown to be metallo-flavoprotein enzymes; the first two require Fe and Cu and the last one is Mn-dependent, based on metal deficiency and inhibitor studies. Uncoupling reagents depressed nitrite and hyponitrite reductases but were without effect on the hydroxylamine reductase. The aliphatic sequence for the reduction of nitrate is probably nitrate→nitrite→hyponitrite→hydroxylamine→ammonia.

Nitrite reductase was purified between 760- and 1300-fold from vegetable marrow (Cucurbita pepo L.) and residual hydroxylamine reductase activity was low or negligible by comparison. With ferredoxin as electron donor, nitrite loss and ammonia formation at pH7.5 were stoicheiometrically equivalent. Crude nitrite reductase preparations showed negligible activity with NADPH as electron donor maintained in the reduced state by glucose 6-phosphate, whereas by comparison, activity was high when either ferredoxin or benzyl viologen were also present and reduced by the NADPH–glucose 6-phosphate system, whereas FMNH2 produced variable and relatively low activity under the same conditions. At pH values below 7, non-enzymic reactions occurred between reduced benzyl viologen and nitrite, and intermediate reduction products were inferred to be produced instead of ammonia. Activity with ferredoxin (0.1mm), reduced by chloroplast grana in the light, was 25 times that produced with ferredoxin (40μm) reduced with NADPH and glucose 6-phosphate. For an approximate molecular weight 61000–63000 derived by chromatography on Sephadex G-100 and G-200, and a specific activity of 46μmol of nitrite reduced/min per mg of protein with light and chloroplast grana, a minimum turnover number of 3×103mol of nitrite reduced/min per mol of enzyme was found. Two hydroxylamine reductases were separated on Sephadex gels. One (HR1) was initially associated with nitrite reductase during gel filtration but disappeared during later fractionation. This HR1 fraction showed nearly comparable activity with reduced benzyl viologen, ferredoxin or FMNH2. The other (HR2), of molecular weight approx. 35000, reacted with reduced benzyl viologen but showed negligible activity with ferredoxin or NADPH. Activity with FMNH2 was associated with an irregular trailing boundary during gel filtration, with much diminished activity in the HR2 region. Activity with NADPH was about 30% of that with FMNH2, reduced benzyl viologen or ferredoxin and was considered to reside in fraction HR1. Hydroxylamine yielded ammonia under all assay conditions. No activity with hyponitrite or sulphite was observed with reduced benzyl viologen as electron donor in either the nitrite reductase or the hydroxylamine reductase systems, but pyruvic oxime produced about 4% of the activity of hydroxylamine.

A c-type cytochrome from Shewanella oneidensis MR-1, containing eight hemes, has been previously designated as an octaheme tetrathionate reductase (OTR). The structure of OTR revealed that the active site contains an unusual lysine-ligated heme, despite the presence of a CXXCH motif in the sequence that would predict histidine ligation. This lysine ligation has been previously observed only in the pentaheme nitrite reductases, suggesting that OTR may have a possible role in nitrite reduction. We have now shown that OTR is an efficient nitrite and hydroxylamine reductase and that ammonium ion is the product. These results indicate that OTR may have a role in the biological nitrogen cycle.

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)

The hydroxylamine reductase of rat liver mitochondria has been studied. This enzyme reduces hydroxylamine (HA) to ammonia, using NADH; the stoichiometry of the reaction is 1:1:1. Although extensive efforts have been made to solubilize the enzyme, it remains firmly attached to the mitochondrial membrane. The preparations used were mitochondria fragmented either by small amounts of lysolecithin, or by brief sonication. These treatments yield a very active enzyme, which is destroyed by acetone, n -butanol, and other reagents, including phospholipase B. The rate of the reaction increases with increasing concentrations of HA up to 1 × 10 −3 m . Greater amounts were toxic to the enzyme. Alteration of the amount of NADH (from 0.5 to 2.0 × 10 −4 m ) however, did not affect the rate of the reaction. The enzyme is less stable to acid (pH 5.4) than to alkali (pH 8.4). Heat damage could be prevented by the presence of NADH, but not of HA. The activation energy is about 11,500 cals/mole. Sulfhydryl reagents inhibit, and with PCMB, NADH protects the enzyme if added before the mercurial, and cysteine can reverse the inhibition. Neither effect is obtained when N -ethylmaleimide is used as an inhibitor. The possible identity of the hydroxylamine reductase and the enzyme which reduces anthranilic hydroxamate to the amide is discussed.

The enzymes considered in this chapter include nitrate reductase, nitrite reductase, hyponitrite reductase, hydroxylamine reductase, enzymes of denitrification, and some other enzymes such as aldehyde oxidase with related properties. The properties of the various enzymes are described in relation to the different plants and micro-organisms from which they have been obtained1.