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Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Biobased production of 5-aminovalerate (5AVA) from biomass can support a sustainable and economic biorefinery process to produce bio-based nylon 5 for food packaging materials. Cost-competitive production of 5AVA from biomass is a key factor in the successful commercialization of nylon 5. Bioproduction of 5AVA is a promising candidate for the industrial process to the current petrochemical route. In this study, we developed an artificial 2-keto-6-aminocaproate-mediated pathway for cost-competitive and high efficiency production of 5AVA in engineered Escherichia coli. Firstly, the combination of native l-lysine α-oxidase (RaiP) from Scomber japonicas, α-ketoacid decarboxylase (KivD) from Lactococcus lactis and aldehyde dehydrogenase (PadA) from Escherichia coli could efficiently convert l-lysine into 5AVA. Moreover, the engineered strains ML03-P nirB -RKP, ML03-P PL-PR -RKP, ML03-P M1-93 -RKP induced by anaerobic condition, temperature-induced, constitutive expression instead of expensive isopropyl β-D-thiogalactoside were constructed, respectively. The use of nirB promoter induced by anaerobic condition not only could attain a higher titer of 5AVA than PL-PR and M1-93 promoters, but omit cost of expensive exogenous inducers. After the replacement of industrial materials, 5AVA titer successfully reached 33.68 g/L in engineered strain ML03-P nirB -RKP via biotransformation. This biotransformation process conduces to the cosmically industrial 5AVA bioproduction., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, financial or otherwise., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The ability of denitrifying microorganisms to respond to different ecological pressures remains unknown, especially in marine sediments rich in various heavy metals. Here, gene abundance and transcriptional abundance of five functional denitrification genes (narG, nirK, nirS, norB, and nosZ) in Bohai Sea sediments were examined, and high-throughput Illumina sequencing was used to analyze the community structure of nirK and nirS denitrifying bacteria. The nirS- and nirK-type denitrifying bacteria were classified into different genera. The heavy metal content in sediments was negatively correlated with transcriptional abundance of denitrifying genes, and RNA: DNA ratio for each gene was highest in central Bohai Sea. These results indicated the distribution of nitrite reductase denitrifying bacterial communities was affected by depth, total nitrogen, total phosphorus and sediment grain size. Heavy metal contamination in sediment environment may negatively regulate the transcriptional abundance of denitrifying genes and cause geographical differences in the denitrifying bacterial community structure., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Reduction of nitrite to nitric oxide in denitrification is catalysed by two different nitrite reductases, encoded by nirS or nirK. Long considered mutually exclusive and functionally redundant in denitrifying bacteria, we show expression of both genes co-occurring in Pseudomonas stutzeri. The differential expression patterns between strain AN10 and JM300 in relation to oxygen and nitrate and their different denitrification phenotypes, with AN10 reducing nitrate more rapidly and accumulating nitrite, suggest that nirS and nirK can have different roles. Dissimilar gene arrangements and transcription factors in the nir gene neighbourhoods could explain the observed differences in gene expression and denitrification activity., (Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k cat =3.9(4) s -1 ) lower than that of wt SmNir (k cat =240(50) s -1 ). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N δ1 H…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the N δ1 H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacterium's growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Large accumulation of nitrate in soil has resulted in "salt stress" and soil secondary salinization. Bacillus megaterium NCT-2 which was isolated from secondary salinization soil showed high capability of nitrate reduction. The genes encoding assimilatory nitrate and nitrite reductase from NCT-2 were cloned and over-expressed in Escherichia coli. The optimum co-expression condition was obtained with E. coli BL21 (DE3) and 0.1mM IPTG for 10h when expression was carried out at 20°C and 120rpm in Luria-Bertani (LB) medium. The molecular mass of nitrate reductase was 87.3kDa and 80.5kDa for electron transfer and catalytic subunit, respectively. The large and small subunit of nitrite reductase was 88kDa and 11.7kDa, respectively. The purified recombinant enzymes showed broad activity range of temperature and pH. The maximum activities were obtained at 35°C and 30°C, pH 6.2 and 6.5, which was similar to the condition of greenhouse soils. Maximum stimulation of the enzymes occurred with addition of Fe 3+ , while Cu 2+ caused the maximum inhibition. The optimum electron donor was MV+Na 2 S 2 O 4 +EDTA and MV+Na 2 S 2 O 4 , respectively. Kinetic parameters of K m and V max were determined to be 670μM and 58U/mg for nitrate reductase, and 3100μM and 5.2U/mg for nitrite reductase. Results of quantitative real-time PCR showed that the maximum expression levels of nitrate and nitrite reductase were obtained at 50mM nitrate for 8h and 12h, respectively. These results provided information on novel assimilatory nitrate and nitrite reductase and their properties presumably revealed adaption of B. megaterium NCT-2 to secondary salinization condition. This study also shed light on the role played by the nitrate assimilatory pathway in B. megaterium NCT-2., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory., (Copyright © 2016. Published by Elsevier Ltd.)

Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64A Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The microbial community and the nirS- and nirK-encoding denitrifiers in the intertidal sediments along Laizhou Bay in China were studied using pyrosequencing and real-time quantitative PCR (qPCR), respectively. There were three primary intertidal zones: Laizhou (La), Weifang Harbor (We), and Dongying (Do). Significant differences in composition and abundances at the different taxonomic levels were observed among the three bacterial communities. The qPCR results indicated that the nirS gene abundance varied from 8.67 × 10(5) to 5.68 × 10(6)copies/gwet weight (ww), whereas the nirK gene abundance varied from 1.26 × 10(5) to 1.89 × 10(6)copies/gww. The canonical correlation analysis (CCA) indicated that the sand percentage was the most important factor in shaping the bacterial community followed by silt percentage, NO2(-), TOC, DO, pH, and clay percentage, whereas the clay percentage, pH, NO3(-), DO, NO2(-), TOC, silt percentage, and sand percentage were the most important factors associated with regulating the abundance of nirS- and nirK-encoding denitrifiers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 heme per monomer (cd1NiR), encoded by the nirS gene. For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd1NiRs. It is now established that, during catalysis, cd1NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d1 heme, which, in turn, displays a peculiar "low" affinity for NO (KD=0.11 μM at pH 7.0). It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd1NiR it was considered a side reaction, rather than a genuine process controlling catalysis. The spectroscopic study of the reaction of cd1NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria., (Copyright © 2014 Elsevier Inc. All rights reserved.)

AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in Neisseria gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The entire nirK gene coding for a putative copper-nitrite reductase (Nir) from Sinorhizobium meliloti 2011 (Sm) was cloned and overexpressed heterologously in Escherichia coli for the first time. The spectroscopic and molecular properties of the enzyme indicate that SmNir is a green Nir with homotrimeric structure (42.5 kDa/subunit) containing two copper atoms per monomer, one of type 1 and the other of type 2. SmNir follows a Michaelis-Menten mechanism and is inhibited by cyanide. EPR spectra of the as-purified enzyme exhibit two magnetically different components associated with type 1 and type 2 copper centers in a 1:1 ratio. EPR characterization of the copper species obtained upon interaction of SmNir with nitrite, and catalytically-generated and exogenous NO reveals the formation of a Cu-NO EPR active species not detected before in closely related Nirs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite extensive use of silver nanoparticles for antimicrobial applications, cellular mechanisms underlying microbial response to silver nanoparticles remain to be further elucidated at the systems level. Here, we report systems-level response of Escherichia coli to silver nanoparticles using transcriptome-based biochemical and phenotype assays. Notably, we provided the evidence that anaerobic respiration is induced upon exposure to silver nanoparticles. Further we showed that anaerobic respiration-related regulators and enzymes play an important role in E. coli resistance to silver nanoparticles. In particular, our results suggest that arcA is essential for resistance against silver NPs and the deletion of fnr, fdnH and narH significantly increases the resistance. We envision that this study offers novel insights into modes of antimicrobial action of silver nanoparticles, and cellular mechanisms contributing to the development of microbial resistance to silver nanoparticles., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The model actinobacterium Streptomyces coelicolor A3(2) uses nitrate and sulfate as nitrogen and sulfur sources, respectively. The final step prior to assimilation into amino acids is the 6-electron reduction of the nitrite and sulfite anions, catalyzed by siroheme-dependent nitrite (NirBD) and sulfite (SirA) reductases. There are two predicted nitrite/sulfite reductases annotated in the genome of S. coelicolor, but it is unclear which is responsible for nitrite and which for sulfite reduction. Here we demonstrate that a knock-out in the genes SCO2487 and SCO2488 encoding NirBD prevents use of nitrite as a nitrogen source, while a knock-out in SCO6102 encoding SirA prevents sulfate assimilation. Both mutations could be phenotypically complemented by supplementation of the growth medium with ammonium or casamino acids in the case of the nirBD mutants or sulfur-containing amino acids in the case of the sirA mutants. No functional redundancy between the genes was observed and we demonstrate that NirBD is exclusively required for assimilatory nitrite (it does not detoxify nitrite) and SirA exclusively for assimilatory sulfite reduction., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Nitrate is a major nitrogen source for land plants but also acts as a signaling molecule that regulates gene expression, metabolism and plant growth. The genes for nitrate reductase (NR) and nitrite reductase (NIR), which are enzymes in the nitrate assimilation pathway, are typical nitrate-inducible genes. We previously identified the first authentic nitrate-responsive cis-element (NRE) for nitrate-inducible transcription by the analysis of the NIR gene promoter from Arabidopsis. Here we further characterize NRE-mediated regulation using transgenic Arabidopsis plants. First, NRE-mediated regulation is shown to be a primary response to nitrate that is caused by pre-existing components, because the NRE-mediated nitrate-inducible expression of the GUS reporter gene is unaffected by treatment with the protein synthesis inhibitor cycloheximide. Second, we show that NRE-like sequences are present in various dicotyledonous and monocotyledonous NIR gene promoters at similar positions and that they also drive nitrate-inducible expression in Arabidopsis. This suggests that NRE-mediated regulation might be conserved in higher plants. Finally, the NRE-mediated expression of the GUS reporter gene is shown to produce diurnal expression in transgenic Arabidopsis plants. This expression peaked at the beginning of the day and decreased during the day which is very similar to the reported diurnal pattern of the nitrate content, suggesting a role of NRE-mediated regulation in controlling diurnal expression in response to oscillation of the nitrate content over a day/night cycle. These findings further clarify the roles of the NRE-mediated regulatory system in higher plants., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Soil-denitrifying bacteria in highly-managed turfgrass systems were examined to assess their response to land-use change and time under management. Denitrifier community composition and diversity in a turfgrass chronosequence of 1 to 95-years-old were compared with those in an adjacent pine-dominant forest via molecular investigations of nirK and nosZ gene fragments. Both denaturing gradient gel electrophoresis and sequenced clone libraries revealed that the denitrifier community became more diverse after turf establishment, and the diversity was then preserved. Furthermore, the composition of the turfgrass denitrifier community was slightly affected by time under management. Meta-analysis of sequenced nirK and nosZ gene fragments from a variety of ecosystems showed that denitrifier communities in pine and turf were more similar to those in other environments than to each other, suggesting that land-use change substantially modified the composition and increased the diversity of denitrifiers. This study provides a useful baseline of nirK- and nosZ-type soil denitrifier communities to aid in the evaluation of ecological and environmental impacts of turfgrass systems., (2010 Elsevier Masson SAS. All rights reserved.)

O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.

Nitrite reductase (cd1NIR) from Pseudomonas aeruginosa, which catalyses the reduction of nitrite to nitric oxide (NO), contains a c-heme as the electron acceptor and a d1-heme where catalysis occurs. Reduction involves binding of nitrite to the reduced d1-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. Since NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the release of NO. We recently discovered that NO dissociation from the ferrous d1-heme is fast, showing that cd1NIR behaves differently from other hemeproteins. Here we demonstrate for the first time that the physiological substrate nitrite displaces NO from the ferrous enzyme, which enters a new catalytic cycle; this reaction depends on the conserved His369 whose role in substrate stabilization is crucial for catalysis. Thus we suggest that also in vivo the activity of cd1NIR is controlled by nitrite.

Macrophages produce nitric oxide (NO) via the inducible nitric oxide synthase as part of a successful response to infection. The gene norB of Neisseria meningitidis encodes a NO reductase which enables utilization and consumption of NO during microaerobic respiration and confers resistance to nitrosative stress-related killing by human monocyte-derived macrophages (MDM). In this study we confirmed that NO regulates cytokine and chemokine release by resting MDM: accumulation of TNF-alpha, IL-12, IL-10, CCL5 (RANTES) and CXCL8 (IL-8) in MDM supernatants was significantly modified by the NO-donor S-nitroso-N-penicillamine (SNAP). Using a protein array, infection of MDM with N. meningitidis was shown to be associated with secretion of a wide range of cytokines and chemokines. To test whether NO metabolism by N. meningitidis modifies release of NO-regulated cytokines, we infected MDM with wild-type organisms and an isogenic norB strain. Resulting expression of the cytokines TNF-alpha and IL-12, and the chemokine CXCL8 was increased and production of the cytokine IL-10 and the chemokine CCL5 was decreased in norB-infected MDM, in comparison to wild-type. Addition of SNAP to cultures infected with wild-type mimicked the effect observed in cultures infected with the norB mutant. In conclusion, NorB-catalysed removal of NO modifies cellular release of NO-regulated cytokines and chemokines.

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.

We present high-resolution crystal structures and functional analysis of T1Cu centre mutants of nitrite reductase that perturb the redox potential and the Cys130-His129 "hard-wired" bridge through which electron transfer to the catalytic T2Cu centre occurs. These data provide insight into how activity can be altered through mutational manipulation of the electron delivery centre (T1Cu). The alteration of Cys to Ala results in loss of T1Cu and enzyme inactivation with azurin as electron donor despite the mutant enzyme retaining full nitrite-binding capacity. These data establish unequivocally that no direct transfer of electrons occurs from azurin to the catalytic type 2 Cu centre. The mutation of the axial ligand Met144 to Leu increases both the redox potential and catalytic activity, establishing that the rate-determining step of catalysis is the intermolecular electron transfer from azurin to nitrite reductase.

Crystal structures of C-terminal despentapeptide nitrite reductase (NiRc-5) from Achromobacter cycloclastes were determined from 1.9 to 2.3A at pH 5.0, 5.4, and 6.2. NiRc-5, that has lost about 30% activity, is found to possess quite similar trimeric structures as the native enzyme. Electron density and copper content measurements indicate that the activity loss is not caused by the release of type 2 copper (T2Cu). pH-profile structural comparisons with native enzyme reveal that the T2Cu active center in NiRc-5 is perturbed, accounting for the partial loss of enzyme activity. This perturbation likely results from the less constrained conformations of two catalytic residues, Asp98 and His255. Hydrogen bonding analysis shows that the deletion of five residues causes a loss of more than half the intersubunit hydrogen bonds mediated by C-terminal tail. This study shows that the C-terminal tail plays an important role in controlling the conformations around the T2Cu site at the subunit interface, and helps keep the optimum microenvironment of active center for the full enzyme activity of AcNiR.

We provide the first atomic resolution (<1.20 A) structure of a copper protein, nitrite reductase, and of a mutant of the catalytically important Asp92 residue (D92E). The atomic resolution where carbon-carbon bonds of the peptide become clearly resolved, remains a key goal of structural analysis. Despite much effort and technological progress, still very few structures are known at such resolution. For example, in the Protein Data Bank (PDB) there are some 200 structures of copper proteins but the highest resolution structure is that of amicyanin, a small (12 kDa) protein, which has been resolved to 1.30 A. Here, we present the structures of wild-type copper nitrite reductase (wtNiR) from Alcaligenes xylosoxidans (36.5 kDa monomer), the "half-apo" recombinant native protein and the D92E mutant at 1.04, 1.15 and 1.12A resolutions, respectively. These structures provide the basis from which to build a detailed mechanism of this important enzyme., (Copyright 2003 Elsevier Science Ltd.)

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes. The mutant has no enzyme activity. Its crystal structure determined at 1.65A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the N epsilon atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.

The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin. The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site. The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR. The redox potentials of the 14- and 35-kDa fragments are +345 and +353mV vs. NHE at pH 7.0, respectively. Moreover, the intermolecular electron transfer rate constant of the 35-kDa fragment from an electron donor, cognate cytochrome c(550), is nearly the same as that of the native enzyme.

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.

Cyanide binding to fully reduced Pseudomonas aeruginosa cd(1) nitrite reductase (Pa cd(1) NiR) has been investigated for the wild-type enzyme and a site-directed mutant in which the active-site His369 was replaced by Ala. This mutation reduces the affinity toward cyanide (by approximately 13-fold) and especially decreases the rate of binding of cyanide to the reduced d(1) heme (by approximately 100-fold). The crystal structure of wild-type reduced Pa cd(1) NiR saturated with cyanide was determined to a resolution of 2.7 A. Cyanide binds to the iron of the d(1) heme, with an Fe-C-N angle of 168 degrees for both subunits of the dimer and only His369 is within hydrogen bonding distance of the nitrogen atom of the ligand. These results suggest that in Pa cd(1) NiR the invariant distal residue His369 plays a dominant role in controlling the binding of anionic ligands and allow the discussion of the mechanism of cyanide binding to the wild-type enzyme., (©2002 Elsevier Science (USA).)

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a soluble enzyme catalysing the reduction of nitrite (NO2(-)) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme domain and a d(1)-heme domain. The structures of the enzyme in both the oxidised and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues. The kinetic characterisation of site-directed mutants has shown that the substitution of either one of these two His with Ala dramatically reduces the physiologically relevant reactivity towards nitrite, leaving the reactivity towards oxygen unaffected. The three-dimensional structures of P. aeruginosa NIR mutant H327A, and H369A in complex with NO have been solved by multiple wavelength anomalous dispersion (MAD), using the iron anomalous signal, and molecular replacement techniques. In both refined crystal structures the c-heme domain, whilst preserving its classical c-type cytochrome fold, has undergone a 60 degrees rigid-body rotation around an axis parallel with the pseudo 8-fold axis of the beta-propeller, and passing through residue Gln115. Even though the distance between the Fe ions of the c and d(1)-heme remains 21 A, the edge-to-edge distance between the two hemes has increased by 5 A. Furthermore the distal side of the d(1)-heme pocket appears to have undergone structural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly different from that of the NO bound to the reduced wild-type structure. Our results provide insight into the flexibility of the enzyme and the distinction between nitrite and oxidase reduction mechanisms. Moreover they demonstrate that the two histidine residues play a crucial role in the physiological activity of nitrite reduction, ligand binding and in the structural organisation of nitrite reductase from P. aeruginosa., (Copyright 2001 Academic Press.)

Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre. The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69. Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here. The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1)., (Copyright 2001 Academic Press.)

Gene encoding of the blue copper-containing nitrite reductase (nir) from Alcaligenes xylosoxidans NCIMB 11015 has been cloned and characterized. The nir is translated into a polypeptide of 360 amino acid residues as a precursor, and the N-terminal 24 residues are subsequently removed upon transport into the periplasm as a mature protein. A specific transcription product of nir was detected only in the presence of nitrate. The aeration level of the culture medium did not show a significant effect on the transcriptional level. A varsigma54 binding sequence is identified upstream of the transcriptional initiation at 53 to 26 nucleotides. A putative fnr box has also been identified in the sequence of the upstream region. The mature polypeptide showed 70% sequence identity with those of the Achromobacter cycloclastes enzyme. The transcriptional start point has been determined at 92 nucleotides upstream of the initiation codon and is preceded by the binding sites for varsigma54 and the fnr box. These results suggest that gene expression depends on the presence of nitrate and is stimulated under an anaerobic environment., (Copyright 1999 Academic Press.)

The nitrite reductase (NIR) and blue copper protein (BCP) genes have been cloned from Achromobacter cycloclastes and characterized. NIR gene encodes a protein of 378 amino acid residues including a putative signal peptide of 37 residues. BCP gene encodes a protein of 148 residues with a 24-residue signal peptide. The DNA-derived amino acid sequence of NIR is in complete agreement with that from Edman degradation and the DNA coding sequence of BCP is also consistent with its partial N-terminal amino acid sequence. Both genes contain their own FNR box in the 5' upstream region and a TA-rich region that could be the transcription start site. These two genes are separated by at least 10 kb. Based on these observations it is very likely that these two genes, although functionally related, are regulated independently. Both proteins could be expressed in E. coli, and both of the expressed proteins could be recognized by their respective antisera. The expressed NIR demonstrates full enzymatic activity. The similarity of both proteins to the counterparts from Alcaligenes faecalis S-6 is discussed.

Spinach ferredoxin-nitrite reductase is a chloroplast enzyme that contains a coupled [Fe4S4]-siroheme-active site and catalyzes the six-electron reduction of nitrite to ammonia. An expression system which produced enzymatically active spinach nitrite reductase (NiR) in Escherichia coli was developed in order to study the structure-function relationships of the coupled active site using site-directed mutagenesis. The spinach NiR cDNA, without the sequences encoding the chloroplast transit peptide, was expressed as a beta-galactosidase fusion containing five additional amino acids at the N-terminus. The expressed NiR in aerobic cultures was mostly insoluble and inactive. After optimizing growth conditions, active NiR represented 0.5-1.0% of the total protein. E. coli-expressed NiR was purified approximately 200-fold to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis. The expressed NiR enzyme was recognized by rabbit anti-spinach NiR antibody as visualized by Western blot analysis. The absorption spectrum of the E. coli-expressed NiR was identical to authentic spinach NiR with a Soret and alpha band at 386 and 573 nm, respectively, and a A278/A386 = 1.9. The addition of nitrite to the oxidized enzyme preparation produced the characteristic shifts in the spectrum. The specific activity for the methyl viologen-dependent reduction of nitrite of E. coli-expressed NiR was 100 U/mg and the Km determined for nitrite was 0.3 mM, which are in agreement with reported values for this enzyme. These results indicate that the E. coli-expressed NiR is fully comparable to spinach NiR in purity, catalytic activity, and physical state.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive procedure for the determination of nitrate within the 1- to 50-microM range is described. The method is based on the photoreduction of nitrate to nitrite by whole cells of a nitrite reductase-less mutant strain of the unicellular cyanobacterium Synechococcus R2. Cell suspensions of this cyanobacterium retain high levels of nitrate photoreduction activity for at least 2 months when maintained in the presence of dimethyl sulfoxide at -70 degrees C.

Nitrate transport and its regulation by oxygen was studied in denitrifying halophilic Pseudomonas stutzeri, strain Zobell, and a Tn-5 transposon nitrite reductase mutant of this organism. The rate of nitrate transport was found to be 130 nanomoles nitrate min-1 mg protein-1 and 150 nanomoles nitrate min-1 mg protein-1 in the wildtype and the nitrite reductase mutant respectively as compared to 26.4 nanomoles nitrate min-1 mg protein-1 in a non-halophilic Pseudomonas stutzeri. Asparagine was found to be the best energy source for nitrate uptake. The ratio of nitrate import to nitrite export was established by measuring extracellular nitrate and nitrite concentrations using HPLC/UV analysis. There was a 1.3:1 (NO3-/NO2-) exchange. High concentrations of nitrate during growth was found to have a negative effect on nitrite metabolism. Oxygen exerted an inhibitory effect on nitrate uptake which was reversible and more pronounced in cells grown on low concentrations of nitrate compared to cells grown at high concentrations of nitrate.

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.