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Identification of wheat (Triticum aestivum L.) chromosomes with genes controlling the level of nitrate reductase, nitrite reductase, and acid proteinase using the Chinese Spring-Hope substitution lines.

The levels of nitrate reductase, nitrite reductase, and acid proteinase were compared in the primary leaves of 8-day-old wheat seedlings of Chinese Spring, Hope, and the 21 disomic substitution lines of Hope in Chinese Spring. Two chromosomes, 7B and 7D, were considered to contain genes controlling the level of nitrate reductase. Substitution of Hope chromosome 7B caused a highly significant increase in the in vitro stability of nitrate reductase. Nitrite reductase appeared to be controlled by two major genes, located on chromosomes 4D and 7D, and two minor genes, located on chromosomes 3D and 5A. In the case of acid proteinase, substitution of chromosome 1D caused a significant reduction in enzyme activity.

Chromosome Mapping

Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings.

1. In rice seedlings synthesis of methyl viologen-nitrite reductase was stimulated by light, as was that of NADH-nitrate oxidoreductase (EC 1.6.6.1). A small residual effect of light on the synthesis of the enzymes persisted in the dark for a short time. 2. In etiolated seedlings exposed to light and nitrate, a lag period of 3h was necessary before enzyme synthesis commenced, whereas in green seedlings kept in the dark for 36h, synthesis of both the enzymes started as soon as light and nitrate were provided. 3. Experiments with cycloheximide suggested that fresh protein synthesis in light was necessary for formation of active enzymes. Mere activation by light of inactive enzymes or their precursors, was not involved. 4. In green seedlings synthesis of nitrite reductase was more sensitive to chloramphenicol than that of nitrate reductase. In chloramphenicol-treated etiolated seedlings, however, synthesis of both the enzymes was inhibited to the same extent on subsequent light-treatment. 5. A close correlation was observed between inhibition of the Hill reaction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and simazin [2-chloro-4,6-bis(ethylamino)-s-triazine] (at high concentration) and the inhibition of enzyme synthesis. At lower concentrations, however, simazin stimulated nitrate reductase. 6. In a single leaf synthesis of enzymes was observed only in portions exposed to light, whereas little activity was present in the dark covered part. 7. CO(2) deprivation severely inhibited the synthesis of enzymes in the light. Sucrose could not reverse this effect. 8. In excised embryos cultured in synthetic media containing sucrose, light was also essential for enzyme formation. 9. It is suggested that redox changes taking place in the green tissues as a result of the Hill reaction create conditions favourable for the induced synthesis of nitrate reductase and nitrite reductase.

Carbon Dioxide

Functional domains of assimilatory nitrate reductases and nitrite reductases.

Biochemical investigation of nitrate assimilation enzymes spans the past four decades. With the molecular cloning of genes for nitrate reductases and nitrite reductases, exciting new prospects are developing for the study of these enzymes. As large, complex enzymes with multiple redox centers, these two types of reductases should help us gain understanding of structural, functional and evolutionary relationships among the diverse group of multicenter redox enzymes.

Amino Acid Sequence

The reaction of Pseudomonas nitrite reductase and nitrite. A stopped-flow and EPR study.

The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy. The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2). [formula: see text] The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1. The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O. Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551. When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.

Electron Spin Resonance Spectroscopy

A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities.

The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples.

Candida

Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd1) among denitrifying pseudomonads.

The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques. Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization. The assignment of P. perfectomarina to the stutzerigroup (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase. Crossreactivity of nitrite reductase (cytochrome cd1) with a respective P. perfectomarina rabbit antiserum was limited to strain DSM 50227 of P. stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species.

Complement Fixation Tests

Nitrate reductase and nitrite reductase transcript levels in various mutants of Aspergillus nidulans: confirmation of autogenous regulation.

The regulation of the expression of the A. nidulans niiA and niaD genes (encoding nitrite reductase and nitrate reductase activities, respectively) was investigated by Northern blotting. It was demonstrated that expression of the niiA and niaD genes is controlled at the level of mRNA accumulation and that mutations within the nirA and areA regulatory genes, as well as certain mutations within niaD itself or cnxE (for its molybdenum cofactor), markedly affect niiA and niaD transcript levels.

Aspergillus nidulans

Change in the ratio of cytochrome oxidase activity to nitrite reductase activity of Pseudomonas aeruginosa nitrite reductase with the kind of C-type cytochrome used as an electron donor.

The ratio between the nitrite reductase and cytochrome oxidase activities of Pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2.] varies with kind of C-type cytochrome used as the electron donor. Withe cytochrome c-548, 554 (Micrococcus sp.), the nitrite reductase activity is greater than the cytochrome oxidase activity, while the former is smaller than the latter with cytochrome c-554 (Navicula pelliculosa). The aerobic oxidation catalyzed by this enzyme of denitrifying bacterial ferrocytochrome c is greatly accelerated on addition of nitrite, while that of the algal ferrocytochrome c is not affected or is even depressed by the salt. An accelerative effect of nitrite is generally observed with many kinds of C-type cytochromes which react with the enzyme very or fairly rapidly. The difference in the ratio of the two activities of the enzyme seems to arise according to whether or not nitrite affects the interaction of C-type cytochrome with the enzyme.

Cytochrome c Group

The purification of a cd1-type nitrite reductase from, and the absence of a copper-type nitrite reductase from, the aerobic denitrifier Thiosphaera pantotropha; the role of pseudoazurin as an electron donor.

Thiosphaera pantotropha has been reported to contain a copper-type nitrite reductase on the basis that the copper chelator diethyldithiocarbamate inhibited the overall process of denitrification. It is now shown that nitrous oxide reduction is 100% inhibited by 10 mM diethyldithiocarbamate or 100 microM azide. We also found that both these inhibitors partially inhibited nitrite reduction in this organism. We purified the nitrite reductase of T. pantotropha and found that it was of the cytochrome cd1 type, contrary to the published report of it being a copper-type nitrite reductase. This is of importance since T. pantotropha is capable of aerobic nitrite reduction. The only detectable nitrite reductase in anaerobically or aerobically grown cells is the cd1 type. We also purified a small copper-containing protein, pseudoazurin. Pseudoazurin was found to be capable of donating electrons to the cd1-type nitrite reductase in vitro, and its copper centre was chelated by diethyldithiocarbamate. Since nitrite reduction is partially inhibited by diethyldithiocarbamate, it is thought that pseudoazurin is an electron donor to nitrite reductase in vivo.

Aerobiosis

Nitrite reductase gene from Synechococcus sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite reductases.

The gene encoding nitrite reductase (nir) from the cyanobacterium Synechococcus sp. PCC 7942 has been identified and sequenced. This gene comprises 1536 nucleotides and would encode a polypeptide of 56,506 Da that shows similarity to nitrite reductase from higher plants and to the sulfite reductase hemoprotein from enteric bacteria. Identities found at positions corresponding to those amino acids which in the above-mentioned proteins hold the Fe4S4-siroheme active center suggest that nitrite reductase from Synechococcus bears an active site much alike that present in those reductases. The fact that the Synechococcus and higher-plant nitrite reductases are homologous proteins gives support to the endosymbiont theory for the origin of chloroplasts.

Amino Acid Sequence