Nitrite Reductase Enzyme Research Articles

Enhanced biological denitrification of high concentration of nitrite with supplementary carbon source

Nitrite accumulates during biological denitrification processes when carbon sources are insufficient. Acetate, methanol, and ethanol were investigated as supplementary carbon sources in the nitrite denitrification process using biogranules. Without supplementary external electron donors (control), the biogranules degraded 200 mg l(-1) nitrite at a rate of 0.27 mg NO(2)-N g(-1) VSS h(-1). Notably, 1,500 mg l(-1) acetate and 700 mg l(-1) methanol or ethanol enhanced denitrification rates for 200 mg l(-1) nitrite at 2.07, 1.20, and 1.60 mg NO(2)-N g(-1) VSS h(-1), respectively; these rates were significantly higher than that of the control. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nitrite reductase (NiR) enzyme identified three prominent bands with molecular weights of 37-41 kDa. A linear correlation existed between incremental denitrification rates and incremental activity of the NiR enzyme. The NiR enzyme activity was enhanced by the supplementary carbon sources, thereby increasing the nitrite denitrification rate. The capacity of supplementary carbon source on enhancing NiR enzyme activity follows: methanol > acetate > ethanol on molar basis or acetate > ethanol > methanol on an added weight basis.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (Delta narGH) and nitrite reductase (Delta nirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to Delta nirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.

Denitrifying Bacterial Community Composition Changes Associated with Stages of Denitrification in Oxygen Minimum Zones

Denitrification in the ocean is a major sink for fixed nitrogen in the global N budget, but the process is geographically restricted to a few oceanic regions, including three oceanic oxygen minimum zones (OMZ) and hemipelagic sediments worldwide. Here, we describe the diversity and community composition of microbes responsible for denitrification in the OMZ using polymerase chain reaction, sequence and fragment analysis of clone libraries of the signature genes (nirK and nirS) that encode the enzyme nitrite reductase, responsible for key denitrification transformation steps. We show that denitrifying assemblages vary in space and time and exhibit striking changes in diversity associated with the progression of denitrification from initial anoxia through nitrate depletion. The initial denitrifying assemblage is highly diverse, but succession on the scale of 3-12 days leads to a much less diverse assemblage and dominance by one or a few phylotypes. This progression occurs in the natural environment as well as in enclosed incubations. The emergence of dominants from a vast reservoir of rare types has implications for the maintenance of diversity of the microbial population and suggests that a small number of microbial dominants may be responsible for the greatest rates of transformations involving nitrous oxide and global fixed nitrogen loss. Denitrifying blooms, driven by a few types responding to episodic environmental changes and distributed unevenly in time and space, are consistent with the sampling effect model of diversity-function relationships. Canonical denitrification thus appears to have important parallels with both primary production and nitrogen fixation, which are typically dominated by regionally and temporally restricted blooms that account for a disproportionate share of these processes worldwide.

Ammonium, Nitrate and Nitrite Nitrogen and Nitrate Reductase Enzyme Activity in Groundnut (Arachis hypogaea L.) Fields After Diazinon, Imidacloprid and Lindane Treatments

Impacts of diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate), imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] and lindane (1,2,3,4,5.6-hexachlorocyclohexane) treatments on ammonium, nitrate, and nitrite nitrogen and nitrate reductase enzyme activities were determined in groundnut (Arachis hypogaea L.) field for three consecutive years (1997 to 1999). Diazinon was applied for both seed- and soil-treatments but imidacloprid and lindane were used for seed treatments only at recommended rates. Diazinon residues persisted for 60 days in both the cases. Average half-lives (t1/2) of diazinon were found 29.3 and 34.8 days respectively in seed and soil treatments. In diazinon seed treatment, NH4 +, NO3 −, and NO2 − nitrogen and nitrate reductase activity were not affected. Whereas, diazinon soil treatment indicated significant increase in NH4 +-N in a 1-day sample, which continued until 90 days. Some declines in NO3 −N were found from 15 to 60 days. Along with this decline, significant increases in NO2 −N and nitrate reductase activity were found between 1 and 30 days. Imidacloprid and lindane persisted for 90 and 120 days with average half-lives (t1/2) of 40.9 and 53.3 days, respectively. Within 90 days, imidacloprid residues lost by 73.17% to 82.49% while such losses for lindane residues were found 78.19% to 79.86 % within 120 days. In imidacloprid seed-treated field, stimulation of NO3 −N and the decline in NH4 +NO2 −-N and nitrate reductase enzyme activity were observed between 15 to 90 days. However, lindane seed treatment indicated significant increases in NH4 +-N, NO2 −-N and nitrate reductase activity and some adverse effects on NO3 −N between 15 and 90 days.

Crystal structures of the nitrite and nitric oxide complexes of horse heart myoglobin

Nitrite is an important species in the global nitrogen cycle, and the nitrite reductase enzymes convert nitrite to nitric oxide (NO). Recently, it has been shown that hemoglobin and myoglobin catalyze the reduction of nitrite to NO under hypoxic conditions. We have determined the 1.20 Å resolution crystal structure of the nitrite adduct of ferric horse heart myoglobin (hh Mb). The ligand is bound to iron in the nitrito form, and the complex is formulated as Mb III(ONO −). The Fe–ONO bond length is 1.94 Å, and the O–N–O angle is 113°. In addition, the nitrite ligand is stabilized by hydrogen bonding with the distal His64 residue. We have also determined the 1.30 Å resolution crystal structures of hh Mb IINO. When hh Mb IINO is prepared from the reaction of metMb III with nitrite/dithionite, the FeNO angle is 144° with a Fe–NO bond length of 1.87 Å. However, when prepared from the reaction of NO with reduced Mb II, the FeNO angle is 120° with a Fe–NO bond length of 2.13 Å. This difference in FeNO conformations as a function of preparative method is reproducible, and suggests a role of the distal pocket in hh Mb IINO in stabilizing local FeNO conformational minima.

The effects of salinity and other factors on nitrite reduction by Ochrobactrum anthropi 49187

The nitrite reductase (NIR) gene was cloned from Ochrobactrum anthropi 49187 and found to contain an open reading frame of 1131 nucleotides, encoding a polypeptide of 376 amino acids. The O. anthropi NIR gene encodes a copper-type dissimilatory reductase based on sequence homology with other genes. The polypeptide product is predicted to form a trimeric holoenzyme of 37 kDa subunits based on molecular weight estimates of extracts in activity gels. Expression of the enzyme is up-regulated by nitrate, presumably through the intermediate nitrite, and its activity is influenced by inhibitors. Salinity enhances the activity of existing NIR enzyme, but appears to decrease the expression of new enzyme.

Long-range interfacial electron transfer of metalloproteins based on molecular wiring assemblies

We address some physical features associated with long-range interfacial electron transfer (ET) of metalloproteins in both electrochemical and electrochemical scanning tunneling microscopy (ECSTM) configurations, which offer a brief foundation for understanding of the ET mechanisms. These features are illustrated experimentally by new developments of two systems with the blue copper protein azurin and enzyme nitrite reductase as model metalloproteins. Azurin and nitrite reductase were assembled on Au(111) surfaces by molecular wiring to establish effective electronic coupling between the redox centers in the proteins and the electrode surface for ET and biological electrocatalysis. With such assemblies, interfacial ET proceeds through chemically defined and well oriented sites and parallels biological ET. In the case of azurin, the ET properties can be characterized comprehensively and even down to the single-molecule level with direct observation of redox-gated electron tunnelling resonance. Molecular wiring using a pi-conjugated thiol is suitable for assembling monolayers of the enzyme with catalytic activity well-retained. The catalytic mechanism involves multiple-ET steps including both intramolecular and interfacial processes. Interestingly, ET appears to exhibit a substrate-gated pattern observed preliminarily in both voltammetry and ECSTM.

Sensing Nitrite through a Pseudoazurin–Nitrite Reductase Electron Transfer Relay

Nitrite is converted to nitric oxide by haem or copper-containing enzymes in denitrifying bacteria during the process of denitrification. In designing an efficient biosensor, this enzymic turnover must be quantitatively assessed. The enzyme nitrite reductase from Alcaligenes faecalis contains a redox-active blue copper centre and a nonblue enzyme-active copper centre. It can be covalently tethered to modified gold-electrode surfaces in configurations in which direct electron transfer is possible. A surface cysteine mutant of the enzyme can be similarly immobilised on bare electroactive gold substrates. Under such circumstances, however, electron transfer cannot be effectively coupled with substrate catalytic turnover. In using either the natural redox partner, pseudoazurin, or ruthenium hexammine as an "electron-shuttle" or "conduit" between enzyme and a peptide-modified electrode surface, the coupling of electron transfer to catalysis can be utilised in the development of an amperometric nitrite sensor.

Heterologous metalloprotein biosynthesis inEscherichia coli: conditions for the overproduction of functional copper-containing nitrite reductase and azurin fromAlcaligenes xylosoxidans

This paper reports on the optimization of conditions for the overproduction and isolation of two recombinant copper metalloproteins, originally encoded on the chromosome of the dentrifying soil bacterium Alcaligenes xylosoxidans, in the heterologous host Escherichia coli. The trimeric enzyme nitrite reductase (NiR) contains both type-1 and type-2 Cu centres, whilst its putative redox partner, azurin I, is monomeric and has only a type-1 Cu centre. Both proteins were processed and exported to the periplasm of E. coli, which is consistent with their periplasmic location in their native host A. xylosoxidans. NiR could be readily purified from the periplasmic fraction of E. coli but the enzyme as isolated possessed only type-1 Cu centres. The type-2 Cu centre could be fully reconstituted by incubation of the periplasmic fraction with copper sulfate prior to enzyme purification. Azurin I could only be isolated with a fully occupied type-1 centre when isolated from the crude cell extract but not after isolation from the periplasmic fraction, suggesting loss of the copper due to proteolysis. Based on a number of criteria, including spectroscopic, mass spectrometric, biochemical and structural analyses, both recombinant proteins were found to be indistinguishable from their native counterparts isolated from A. xylosoxidans. The findings of this work have important implications for the overproduction of recombinant metalloproteins in heterologous hosts.

Diversity of nitrite reductase genes (nirS) in the denitrifying water column of the coastal Arabian Sea

Denitrification often occurs in the water column, underlying zones of intense productiv- ity and decomposition in upwelling regions. In the denitrifying zone off the southwest coast of India, high concentrations of nitrite (>15 µM) and nitrous oxide (>500 nM) have been reported near the sediment-water interface (<80 m). We investigated the chemical and molecular indicators of denitri- fication along the southwest coast of India during the southwest monsoon season of October 2001. Nitrite reduction to nitric oxide is the key step in the denitrification pathway, and is catalyzed by the enzyme nitrite reductase, which is encoded by the genes nirS and nirK. Here we report the diversity and distribution of nirS genes in relation to nitrite and nitrate distribution in the Arabian Sea coastal denitrifying region. nirS gene fragments were PCR-amplified, cloned, and sequenced from DNA extracted from the water column. Clone libraries were also subjected to restriction fragment length polymorphism (RFLP) and rarefaction analyses. These are the first nitrite reductase sequences reported from a water column denitrifying regime. nirS was amplified from DNA extracted from all water samples in which nitrite was present at high concentrations within the low oxygen waters, but was only rarely amplified from waters containing hydrogen sulfide or from well-oxygenated waters. Phylogenetic analysis grouped 132 nirS Arabian Sea sequences into 12 major clusters. Most of the nirS sequences from the coastal water column did not show a high level of identity with other nirS sequences previously reported from marine and estuarine sediments. Identities of the Arabian Sea sequences to those in the public database ranged from 44 to 99% at the amino acid level. The domi- nant sequence type from 1 surface sample showed 99% identity to the nirS sequence of the cultivated denitrifier Pseudomonas aeruginosa. Rarefaction analysis, based on both sequence and RFLP data, indicated the highest diversity in a sample in which relatively high nitrite concentrations implied the presence of active denitrification, and the lowest diversity in a surface sample where nitrite was undetectable, suggesting a link between functional diversity and ecosystem chemistry.

Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine.

Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)

The chemistry and biochemistry of the copper–radical interaction

This Perspective describes the plausible oxidation state combinations of Cun/Lm systems where Lm is an O- or N-based redox system with a paramagnetic intermediate such as O2˙−, NO˙, phenoxyl, o-semiquinone or azo radical anion. The biochemical relevance is discussed using superoxide dismutase, nitrite reductase, galactose oxidase and amine oxidase enzyme examples. Particular emphasis will be given to the identification of oxidation states and to the manifestations of intramolecular electron transfer in enzymes and model compounds.

The electrophilic reactions of pentacyanonitrosylferrate(II) with hydrazine and substituted derivatives. Catalytic reduction of nitrite and theoretical prediction of eta(1)-, eta(2)-N(2)O bound intermediates.

The electrophilic reactivity of the pentacyanonitrosylferrate(II) ion, [Fe(CN)(5)NO](2)(-), toward hydrazine (Hz) and substituted hydrazines (MeHz, 1,1-Me(2)Hz, and 1,2-Me(2)Hz) has been studied by means of stoichiometric and kinetic experiments (pH 6-10). The reaction of Hz led to N(2)O and NH(3), with similar paths for MeHz and 1,1-Me(2)Hz, which form the corresponding amines. A parallel path has been found for MeHz, leading to N(2)O, N(2), and MeOH. The reaction of 1,2-Me(2)Hz follows a different route, characterized by azomethane formation (MeNNMe), full reduction of nitrosyl to NH(3), and intermediate detection of [Fe(CN)(5)NO](3)(-). In the above reactions, [Fe(CN)(5)H(2)O](3)(-) was always a product, allowing the system to proceed catalytically for nitrite reduction, an issue relevant in relation to the behavior of the nitrite and nitric oxide reductase enzymes. The mechanism comprises initial reversible adduct formation through the binding of the nucleophile to the N-atom of nitrosyl. The adducts decompose through OH(-) attack giving the final products, without intermediate detection. Rate constants for the adduct-formation steps (k = 0.43 M(-)(1) s(-)(1), 25 degrees C for Hz) decrease with methylation by about an order of magnitude. Among the different systems studied, one-, two-, and multielectron reductions of bound NO(+) are analyzed comparatively, with consideration of the role of NO, HNO (nitroxyl), and hydroxylamine as bound intermediates. A DFT study (B3LYP) of the reaction profile allows one to characterize intermediates in the potential hypersurface. These are the initial adducts, as well as their decomposition products, the eta(1)- and eta(2)-linkage isomers of N(2)O.

Copper complexes with N-donor ligands as models of the active centres of nitrite reductase and related enzymes

Denitrification, the enzymatic reduction of nitrate (NO3 ‐) and nitrite (NO2 ‐ ) to gaseous NO, N2O and/or N2, is part of the nitrogen cycle which is essential to life. Studies of denitrification have gained impetus in recent years because of environmental concerns over the widespread agricultural use of potentially polluting nitrate and nitrite, and the damage to the atmosphere from NO and N2O that can be produced by their degradation. 1 Denitrification is mediated by a number of metalloenzymes; in particular we concentrate in this article on the reduction of nitrite, which is catalysed by two distinct types of nitrite reductase enzyme. One type involves a multi-haeme system and the second, upon which we shall focus, is a multicopper enzyme (CuNiR). 2 X-ray studies of CuNiR from a number of sources have shown that the enzyme is a homotrimer in which each monomer contains a so-called Type 1 copper, with a (His)2(Cys)(Met) ligand set, linked by sequential protein residues (CysHys) to a Type 2 copper, bound at the interface between subunits of the protein by a facial array of three histidine residues. The two copper centres are around 12.6 A apart. A fourth ligand, water or hydroxide, completes the pseudo-tetrahedral coordination of the Type 2 copper which is situated in a hydrophobic pocket, some 12 A from the protein surface (Fig. 1).3‐6

The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite.

Escherichia coli possesses two distinct nitrite reductase enzymes encoded by the nrfA and nirB operons. The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment. To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using nrfA-lacZ and nirB-lacZ reporter fusions. Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimal nrfA-lacZ expression occurred only at low to intermediate levels of nitrate and where nirB-lacZ expression was induced only by high nitrate conditions. Under these conditions, the presence of high levels of nitrate suppressed nrfA gene expression. While either NarL or NarP was able to induce nrfA-lacZ expression in response to low levels of nitrate, only NarL could repress at high nitrate levels. The different expression profile for the alternative nitrite reductase operon encoded by nirBDC under high-nitrate conditions was due to transcriptional activation by either NarL or NarP. Neither response regulator could repress nirB expression. Nitrite was also an inducer of nirB and nrfA gene expression, but nitrate was always the more potent inducer by >100-fold. Lastly, since nrfA operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment. In contrast, the nirB nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it. Revised regulatory schemes are presented for NarL and NarP in control of the two operons.

The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of thenirAGene and Characterization of the Recombinant Enzyme

The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed in Escherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Enzymatic Spectrophotometric Determination of Nitrites in Beer

ABSTRACT A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10−8 – 10−2 M range with a detection limit of 10−8 M and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NiR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10−5 – 10−2 M) and a higher detection limit (10−5 M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Two nitrite reductase isoforms are present in tomato cotyledons and are regulated differently by UV-A or UV-B light and during plant development

The regulation by UV-A or UV-B light of the nuclear gene(s) encoding the plastidic enzyme nitrite reductase (NiR; EC 1.7.7.1) was examined in the cotyledons of tomato (Lycopersicon esculentum L.). Two NiR isoforms designated NiR1 and NiR2 with apparent molecular masses of 63 kDa and 62 kDa, respectively, were detected by immunoblot analysis in total soluble protein extracts derived from tomato seedling cotyledons. Genomic Southern blot analysis indicated the presence of two NiR genes per haploid tomato genome. In etiolated tomato cotyledons, the total NiR protein pool was almost exclusively constituted by NiR1. In contrast, NiR2 was the predominant NiR isoform in the cotyledons of tomato seedlings grown in white light. Illumination of etiolated tomato cotyledons with UV-A or UV-B light resulted in an increase in both the total NiR transcript level and the NiR2 protein abundance. Blue light stimulated the NiR2 protein pool above the level obtained with red light of equal photon fluence rate. These results show that NiR2 protein expression is light-inducible and that the light-stimulation of NiR2 protein accumulation involves the action of both phytochrome and a specific blue-light photoreceptor. The NiR1 protein level remained virtually unaffected by the light treatments. The change in the relative proportion of the NiR isoforms during greening of etiolated tomato cotyledons is, therefore, due to the different light-responsiveness of the genes corresponding to NiR1 or NiR2. The physiological significance of the presence of NiR isoforms that are regulated differently by light in tomato cotyledons is discussed.

The Covalent Structure of the Blue Copper-Containing Nitrite Reductase from Achromobacter xylosoxidans

The complete amino acid sequence of the blue copper-containing nitrite reductase enzyme (NiR) from Achromobacter xylosoxidanshas been determined by chemical analysis, supported by high precision mass analysis. The polypeptide chain contains 336 residues with an overall charge of 0, including the +2 state of each of the copper ions. The two NiR enzymes for which the three-dimensional structures have been solved are green in color and have different absorption spectra than those of the blue-colored protein from A. xylosoxidans.The ligands to the two copper atoms are conserved. Therefore, the difference between the blue and the green NiR must depend on subtle changes in the geometry of the type I copper-sulfur bond. Both overall protein charge and active site charge are different in A. xylosoxidansNiR which may reflect the use of azurin as electron donor as opposed to the other enzymes that use pseudoazurin.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis.

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the pathway, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.