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Query: EC:1.7.1.2 (nitrate reductase)
 3,861 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.
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PMID:Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter. 373 17

Nitrobacter agilis, which contains a very active nitrite dehydrogenase, was studied in vivo under anaerobic conditions by the 15N NMR technique. When incubated with equimolar 15NO3- and unlabeled nitrite (or 15NO2- and unlabeled nitrate) the bacterium catalyzed an isotope exchange reaction at rates about 10% those observed in the nitrite oxidase assay. When incubated with 18O-labeled 15NO2- and 18O-labeled 15NO3-, the 18O was observed to exchange at similar rates from both species into water. Finally, when incubated with equimolar [18O]nitrate and 15NO2-, intermolecular 18O transfer was observed to result in formation of double labeled nitrate and nitrite at similar rates. 18O was transferred from nitrate to a 15N species or to water at approximately equal rates under the conditions of the experiments. It is argued that the enzyme responsible for these exchange reactions is nitrite dehydrogenase and not nitrate reductase. This work and the related experiments of DiSpirito and Hooper (DiSpirito, A.A., and Hooper, A.B. (1986) J. Biol. Chem. 261, 10534-10537) represent the first demonstrations of intermolecular oxygen atom transfer among oxotransferases. Mechanistic implications are discussed.
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PMID:Catalysis of intermolecular oxygen atom transfer by nitrite dehydrogenase of Nitrobacter agilis. 373 18

During growth in high concentrations of iron nitrate, H. influenzae produces compounds reactive in biochemical assays for hydroxamates. Mixing experiments established that nitrate was responsible for inducing these compounds. Analysis by 1H and 13C NMR and high resolution mass spectrometry identified the active species as 2,2-bis(3'-indolyl)indoxyl. Bacterial production of the latter compound has been previously observed only in Pseudomonas aureofaciens. A mutant defective in the production of 2,2-bis(3'-indolyl)indoxyl was constructed by marker insertion. The formation of indole and 2,2-bis (3'-indolyl)indoxyl was quantitated by reverse-phase high pressure liquid chromatography during growth in high concentrations of nitrate. The mutant produced high concentrations of indole, but only minimal amounts of 2,2-bis(3'-indolyl)indoxyl, and also proved to be defective in nitrate reduction. These data suggest that indole may function as an electron donor for nitrate reductase in H. influenzae.
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PMID:Production and oxidation of indole by Haemophilus influenzae. 781 18

The family of b5-like cytochromes encompasses, besides cytochrome b5 itself, hemoprotein domains covalently associated with other redox proteins, in flavocytochrome b2 (L-lactate dehydrogenase), sulfite oxidase and assimilatory nitrate reductase. A comparison of about 40 amino acid sequences deposited in data banks shows that eight residues are invariant and about 15 positions carry strongly conservative substitutions. Examination of the location of these invariant and conserved positions in the light of the three-dimensional structures of beef cytochrome b5 and S cerevisiae flavocytochrome b2 suggests a strongly conserved protein structure for the b5-like heme-binding domain throughout evolution. Numerous NMR studies have demonstrated the existence of a positional isomerism for the heme, which involves both a 180 degree-rotation around the heme alpha,gamma-meso carbon atoms and a rotation through an axis normal to the heme plane at the iron. NMR studies did not detect significant differences in protein structure between reduced and oxidized states, or between species. The role of a number of side chains was probed by site-directed mutagenesis. Studies of complex formation and of electron transfer rates between cytochrome b5 and redox partners have led to the idea that complexation is driven by electrostatic forces, that it is generally the exposed heme edge which makes contact with electron donors and acceptors, but that there are multiple overlapping sites within this general area. For the bi- and trifunctional members of the family, extrapolation of available data would suggest a mobile heme-binding domain within a complex structure. In these cases the existence of a single interaction area for both electron donor and acceptor, or of two different ones, remains open to discussion.
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PMID:The cytochrome b5-fold: an adaptable module. 789 19

The sequential transphosphorylation from autophosphorylated nitrate-sensing protein (NarX) to the transcriptional regulator protein (NarL), both operating in signal transduction to control the expression of the respiratory nitrate reductase (nar) operon in E. coli, was demonstrated with an in vitro reconstructed system to function similarly to other bacterial two-component regulatory systems. Over-expression system established by means of the pT7 promoter/polymerase provided both NarX and NarL proteins to reconstruct the in vitro transphosphorylation system. The phosphorylated NarL was detected, and the unstable phosphorylated group was directly assigned to acyl phosphate in the in vitro system by 31P-NMR spectroscopy.
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PMID:Studies on phosphorylated transcriptional regulator (NarL) for E. coli nar operon by 31P-NMR spectroscopy. 826 Sep 40

Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.
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PMID:Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase. 869 50

Nitrogen metabolism is a highly regulated process in Neurospora crassa. The structural genes that encode nitrogen catabolic enzymes are subject to nitrogen metabolite repression, mediated by the positive-acting NIT2 protein and by the negative-acting NMR protein. NIT2, a globally acting factor, is a member of the GATA family of regulatory proteins and has a single Cys2/Cys2 zinc finger DNA-binding domain. The negative-acting NMR protein interacts via specific protein-protein binding with two distinct regions of the NIT2 protein, a short alpha-helical motif within the NIT2 DNA-binding domain and a second motif at its carboxy terminus. Deletions of segments of NIT2 throughout most of its length result in truncated proteins, which are still functional for activating gene expression; most of these mutant NIT2 proteins still allow proper nitrogen repression of nitrate reductase synthesis. In contrast, deletions or certain amino acid substitutions within the zinc finger and the carboxy-terminal tail result in a loss of nitrogen metabolite repression. Those mutated forms of NIT2 that are insensitive to nitrogen repression have also lost one of the NIT2-NMR protein-protein interactions. These results provide compelling evidence that the specific NIT2-NMR interactions have a regulatory function and play a central role in establishing nitrogen metabolite repression.
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PMID:Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa. 942 2

A water soluble truncated heme domain (a tetramer of MW = 45 kDa) of the tetrameric nitrate reductase complex from the green alga Chlorella vulgaris has been overexpressed and purified. This truncated heme domain with four identical subunits has a high redox potential (midpoint potential E1/2 = +16 mV) as compared with other heme-containing flavoproteins. We have undertaken a determination of the detailed configuration of the heme moiety in order to understand the unique electrochemical property of the heme moiety of this enzyme. We report here the study of the heme prosthetic group of the truncated heme domain by the use of 2D 1H and 13C NMR techniques. A complete signal assignment of the heme has been achieved. Our observations suggest that the heme configuration is similar to that of the crystal structure of the membrane-bound bovine liver cytochrome b5.
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PMID:1H and 13C NMR studies of a truncated heme domain from Chlorella vulgaris nitrate reductase: signal assignment of the heme moiety. 950 89

In Neurospora crassa, the expression of the nit-3 gene (nitrate reductase) is dependent upon nitrogen derepression and nitrate induction and is regulated by two positive-acting transcription factors, NIT2 and NIT4, and a negative regulator, NMR. The presence of a tightly linked cluster of NIT2 and NIT4 binding sites suggested that their close spacing might be required for a synergistic interaction of the NIT2 and NIT4 proteins. We show here that the NIT2 and NIT4 binding sites can be separated without affecting either the expression level or the precise regulation of the nit-3 gene. Studies conducted on the NIT2 site II, which contains only a single GATA element and yet plays a major role in nit-3 gene expression, showed that nucleotides both 5' and 3' of the GATA sequence were important for strong DNA binding in vitro and its activation function in vivo. The nit-3 promoter contains two long AT-rich sequences, one of which is located just upstream of the transcription start sites and is required for optimal promoter function. The nit-3 transcript contains eight TACC repeats in its 5' noncoding region which appear to be involved in mRNA instability. Deletion of these TACC repeats led to a significant increase in the stability of nit-3 mRNA.
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PMID:Analysis of a distal cluster of binding elements and other unusual features of the promoter of the highly regulated nit-3 gene of Neurospora crassa. 969 10

A strain of Acinetobacter with potential for bioremediation of heavy metal-contaminated waters was isolated from a wastewater-treatment plant operating an enhanced biological phosphate removal process. NMR and extractive methods showed that polyphosphate accumulated aerobically was degraded under anaerobic conditions both in the presence and absence of cadmium or uranium (0.2-0.5 mM). NMR showed that free phosphate was formed at the expense of polyphosphate, and an extractive technique indicated that this reaction could be stimulated by the presence of UO(2)2+ under these conditions. Energy-dispersive X-ray microanalysis demonstrated that only cadmium could enter the cells, and co-localized with intra-cellular granules containing phosphate and other divalent metals. The effects of other environmental parameters on the anaerobic phosphate metabolism were also investigated. Between pH 5.5 and 8.0, phosphate release increased with increasing pH. Between 4 degrees C and 37 degrees C, phosphate release increased with increasing temperature. The presence of nitrate at concentrations of 10 mM and above inhibited anoxic phosphate release, but supplying tungstate in the growth medium prior to anoxic incubation reduced the production of active nitrate reductase and alleviated this effect.
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PMID:The effect of heavy metals and other environmental conditions on the anaerobic phosphate metabolism of Acinetobacter johnsonii. 1043 10


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