Biomedical subjects

F Blasco

Publications and source records attributed to F Blasco.

At least 37 records · Page 2Linked to original sources

PubMed1998

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli.

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.

Cell Fractionation↗

PubMed1997

Heme axial ligation by the highly conserved His residues in helix II of cytochrome b (NarI) of Escherichia coli nitrate reductase A.

Optical spectroscopy and EPR studies confirm the existence of two b-type hemes in the NarI subunit (cytochrome bnr) of the membrane-bound nitrate reductase (NarGHI) of Escherichia coli. Replacement of His-56 by Arg and His-66 by Tyr results in the loss of the high-potential heme and of the low-potential heme, respectively. These data support the assignment of the axial ligands to the low-potential heme (His-66 and His-187) and to the high-potential heme (His-56 and His-205). This pairing is consistent with the model proposed for NarI of the nitrate reductase of Thiosphaera pantotropha (Berks, B. C., Page, M. D., Richardson, D. J. , Reilly, A., Cavill, A., Outen, F., and Ferguson, S. J. (1995) Mol. Microbiol. 15, 319-331) in which the two bis-histidine ligated hemes are coordinated by conserved His residues of helix II and V. EPR and optical studies suggest that the low-potential heme (Em,7 = +17 mV) and the high-potential heme (Em,7 = +122 mV) are located near the periplasmic side and the cytoplasmic side of the membrane, respectively. Moreover, correct insertion of both hemes into NarI requires anchoring to NarGH.

Cold Temperature↗

PubMed1997

Organization of the nar genes at the chlZ locus.

The two membrane-bound respiratory nitrate reductases of Escherichia coli are encoded by distinct operons at two different loci, chlC and chlZ, on the chromosome. The chlZ locus includes a narK homologue, narU, encoding a nitrite extrusion protein, and narZYWV encoding nitrate reductase Z. No apparent homologue to the narXL operon has been found. Homology between narU and narK on the one hand and narZYWV and narGHJI on the other hand is limited to the coding regions.

Chromosomes, Bacterial↗

PubMed1997

Simultaneous spectrophotometric determination of calcium and magnesium in mineral waters by means of multivariate partial least-squares regression.

A method for simultaneous spectrophotometric determination of calcium and magnesium in mineral waters using multivariate calibration methods is proposed. The method is based on the development of the reaction between the analytes and Methylthymol Blue at pH 11. Two operational modes were used: static (spectral information) and flow injection (FI) (spectral and kinetic information). The selection of variables was studied. A series of synthetic solutions containing different concentrations of calcium and magnesium were used to check the prediction ability of the partial least-squares models. The method was applied to the analysis of mineral waters and the results were compared with those obtained by complexometry. No significant differences at the 95% confidence level were found. The proposed method is simple, accurate and reproducible, and it could be easily adapted as a portable (static mode) or automatic (FI) method.

Calcium↗

PubMed1997

Characterization by electron paramagnetic resonance of the role of the Escherichia coli nitrate reductase (NarGHI) iron-sulfur clusters in electron transfer to nitrate and identification of a semiquinone radical intermediate.

We have used Escherichia coli cytoplasmic membrane preparations enriched in wild-type and mutant (NarH-C16A and NarH-C263A) nitrate reductase (NarGHI) to study the role of the [Fe-S] clusters of this enzyme in electron transfer from quinol to nitrate. The spectrum of dithionite-reduced membrane bound NarGHI has major features comprising peaks at g = 2.04 and g = 1.98, a peak-trough at g = 1.95, and a trough at g = 1.87. The oxidized spectrum of NarGHI in membranes comprises an axial [3Fe-4S] cluster spectrum with a peak at g = 2.02 (g(z)) and a peak-trough at g = 1.99 (g(xy)). We have shown that in two site-directed mutants of NarGHI which lack the highest potential [4Fe-4S] cluster (B. Guigliarelli, A. Magalon, P. Asso, P. Bertrand, C. Frixon, G. Giordano, and F. Blasco, Biochemistry 35:4828-4836, 1996), NarH-C16A and NarH-C263A, oxidation of the NarH [Fe-S] clusters is inhibited compared to the wild type. During enzyme turnover in the mutant enzymes, a distinct 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive semiquinone radical species which may be located between the hemes of NarI and the [Fe-S] clusters of NarH is observed. Overall, these studies indicate (i) the importance of the highest-potential [4Fe-4S] cluster in electron transfer from NarH to the molybdenum cofactor of NarG and (ii) that a semiquinone radical species is an important intermediate in electron transfer from quinol to nitrate.

Benzoquinones↗

PubMed1996

Complete coordination of the four Fe-S centers of the beta subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters.

The beta subunit of the nitrate reductase A from Escherichia coli contains four groups of cysteine residues (I-IV) which are thought to bind the four iron-sulfur centers (1-4) of the enzyme. The fourth Cys residue of each group was replaced by Ala by site-directed mutagenesis, which led to the C26A, C196A, C227A, and C263A mutants. Physiological and biochemical effects of the mutations were investigated on both the membrane-bound and the soluble forms of the enzyme. In addition, detailed redox titrations of the mutants were monitored by EPR spectroscopy. The C196A and C227A mutations resulted in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzymes. In contrast, the C26A and C263A mutants retained significant nitrate reductase activities. The EPR analysis showed that the highest redox potential [4Fe-4S] cluster (center 1) was selectively removed by the C263A mutation and that the C26A replacement likely eliminated the lowest potential [4Fe-4S] cluster (center 4). In both mutants, the three remaining Fe-S clusters kept the same spectral and redox properties as in the wild type enzyme. These results enabled the determination of the Cys ligands of center 1 to be completed and led to a proposed model for the coordination of the four Fe-S centers by the four Cys groups of the beta subunit. In this model, the four clusters are organized in two pairs, (center 1, center 4) and (center 2, center 3), which is in good agreement with the magnitude of intercenter magnetic interactions observed by EPR and with the stability of the different mutants. The possible implications on the intramolecular electron transfer pathway are discussed.

Cysteine↗

PubMed1996

Cobalt(II), nickel(II), and copper(II) complexes of sulfanilamide derivatives: synthesis, spectroscopic studies, and antibacterial activity. Crystal structure of [Co(sulfacetamide)2(NCS)2].

The synthesis and characterization of new coordination compounds of Co(II), Ni(II), and Cu(II) with sulfacetamide (N-[4-(amino-fenil)sulfonil]acetamide) is reported. The complex [Co(sulfacetamide)2(NCS)2] crystallizes in the triclinic space group P-1. The cell dimensions are a = 7.80(2) A, b = 8.327(9) A, c = 9.568(3) A, alpha = 90.5(1) degrees, beta = 90.5(1) degrees, gamma = 97.8(2) degrees, V = 616(1) A3, Z = 2, and Dx = 1.689 g/cm3. The final conventional R-factor = 0.039 (Rw = 0.039) for 3535 "observed" reflections and 173 variables. The Co(II) is surrounded in a regular octahedral arrangement by two Nthyocianato from the NCS, two Namino and two Oacetamido atoms from the sulfacetamide. Each sulfacetamide, acting as a bidentate ligand, chelates two Co(II) ions as a bridge through the Namino and the Oacetamido atoms. IR, Reflectance Diffuse, EPR, and magnetic properties of the obtained complexes are discussed. The complexes were screened for their activity against E. Coli and S. aureus, showing an appreciable antimicrobial activity compared with the ligands.

Anti-Bacterial Agents↗

PubMed1996

Specific serological response by active immunization with GD3-bearing liposomes.

GD3 is the most prominent ganglioside on the surface of human melanoma cells, and therefore it has been considered by several investigators as a potential tool for active immunotherapy of melanoma. The main obstacle to this goal is that GD3 is poorly immunogenic in mice and in humans. Several approaches have been described for increasing the GD3 immunogenicity. Here, the immunogenicity of GD3 ganglioside was investigated by vaccination of Balb/c x C57B1/6 F1 mice with several types of GD3-bearing liposomes. The humoral immune response was analyzed by ELISA and tumor cell recognition. Several liposome formulations were assayed in order to increase the immunogenicity of GD3. We also tested vaccinations with GD3-Salmonella minnesota, Freund's complete adjuvant and Bordetella pertussis antigen. Immunization of mice with sphingomyelin:cholesterol:dicetyl-phosphate:GD3, molar ratio 40:40:10:10, liposomes resulted in good IgM and IgG3 anti-GD3 response, with a maximum titer of 1:1200 and absence of significant cross-reactivity with other gangliosides. In immunofluorescence assays, the antisera induced showed high capacity of recognition of melanoma cells with no reactivity against other tumor cells. The results clearly showed a high positive correlation between liposomes with sphingomyelin and GD3 immunoreactivity. The incorporation of muramyl-dipeptide or Lipid A in the liposome formulation increased the secretion of IgM and IgG as well as the nonspecificity of the response.

Adjuvants, Immunologic↗

PubMed1995

Kinetic studies of a soluble alpha beta complex of nitrate reductase A from Escherichia coli. Use of various alpha beta mutants with altered beta subunits.

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.

Benzyl Viologen↗

PubMed1994

Synthesis and spectroscopy studies of copper(II) nitrate of sulfacetamide drug. Crystal structure of [Cu(sulfacetamide)2(NO3)2]. Antibacterial studies.

The structural spectroscopic, and thermal properties of a complex of sulfacetamide (Hsacm) with Cu(II) have been investigated. The complex [Cu(Hsacm)2(NO3)2] crystallizes in the monoclinic system, space group P2(1)/n. The cell dimensions are a = 7.696(7) A, b = 8.017(7) A, c = 19.230(10), beta = 110.80(1) degree, V = 1109(1) A3, Z = 2, and Dx = 1.84 g/cm3. The structure was refined to R = 0.0776. Cu(Hsacm)2(NO3)2 molecules form a long polymeric chain extended along the b-axis. The copper(II) coordinated geometry is tetragonally distorted octahedral with two amino nitrogens from Hsacm and two oxygens from nitrato anions in the basal plane and two acetamido oxygens from neighbor Hsacm molecules in the apical position. Each sulfacetamide, acting as a bidentate ligand, links two Cu(II) ions as a bridge through the Namino and the Oacetamido atoms. The complex proved to possess higher bacteriostatic activity than the corresponding ligand.

Chemical Phenomena↗

PubMed1993

Removal of the high-potential [4Fe-4S] center of the beta-subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutated enzymes.

The beta-subunit of the nitrate reductase of Escherichia coli contains four groups of Cys residues (I-IV) which are thought to bind the single [3Fe-4S] center and the three [4Fe-4S] centers. The first or second Cys residue of group I was substituted by site-directed mutagenesis with Ala or Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. With small variations, the properties of these mutant enzymes do not differ from one another. They were found to be as abundant and as stably bound to the membrane as the native enzyme, provided the gamma-subunit was present. Although physiological activity was reduced, it was sufficient to allow growth on nitrate. The study of variations in EPR intensity as a function of the redox potential indicated that these enzymes only contained three iron-sulfur centers instead of the usual four in the native enzyme. Spectral EPR analysis showed that the [4Fe-4S] center of high redox potential (center 1, +80 mV) was missing. The loss of this center did not affect the stable integration of the other three centers. The data presented here are in total contrast to those we have reported for each of the other three centers (centers 2-4), the loss of which was detrimental to the integration of all centers and to the integration of the molybdenum cofactor (Augier et al., in press). Taken together, our results demonstrated that the first and second Cys residues of group I are the ligands of the [4Fe-4S] center (center 1, +80 mV) and that this center participates in electron transfer, but is dispensable. On the basis of these results, it is proposed that the [3Fe-4S] center (center 2, +60 mV) also plays a biological role and that in the native enzyme both high-potential centers, centers 1 and 2, contribute independently and in parallel to the electron transfer to the molybdenum cofactor.

Cell Membrane↗

PubMed1993

Site-directed mutagenesis of conserved cysteine residues within the beta subunit of Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of the mutated enzymes.

We have used site-directed mutagenesis to alter the ligands to the iron-sulfur centers of Escherichia coli nitrate reductase A. The beta subunit of this enzyme contains four Cys groups which are thought to accommodate the single [3Fe-4S] center and the three [4Fe-4S] centers involved in the electron-transfer process from quinol to nitrate. The third Cys group (group III) contains a Trp at a site occupied by a Cys residue in typical ferredoxin arrangements or in the DmsB subunit of dimethyl sulfoxide (DMSO) reductase. In an attempt to determine the coordination site of the different iron-sulfur centers in the amino acid sequence, we have changed the Trp of group III to Cys, Ala, Phe, and Tyr and the first Cys residue of groups II-IV to Ala and Ser. Physiological, biochemical, and EPR studies were performed on the mutated enzymes. Substitution of Ala for either Cys184, Cys217, or Cys244 results in the full loss of all four iron-sulfur centers present in the wild-type enzyme. These inactive enzymes still possess the alpha,beta, and gamma polypeptides associated in a membrane-bound complex. These Cys have important structural roles and are very likely involved in the coordination of the iron-sulfur centers. Substitution of Cys184 with a Ser residue produces an enzyme containing the four iron-sulfur centers, but displaying reduced activity. EPR studies suggest that Cys184 is a ligand of the [4Fe-4S] center whose midpoint potential is -200 mV in the native enzyme. All substitutions performed in this study on Trp220 lead to mutant enzymes harboring the four iron-sulfur centers and a nitrate reductase activity close to that of the wild-type. In spite of the high similarity between the NarH and DmsB subunits, the Trp220-->Cys substitution does not allow the conversion of the [3Fe-4S] center of the nitrate reductase into a [4Fe-4S] center. Therefore, Trp220 does not seem to play any major role in the beta subunit.

Amino Acid Sequence↗

PubMed1993

Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis.

A 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced. Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon. The encoded proteins (MoaA-MoaE) have predicted molecular weights of 37,346, 18,665, 17,234, 8843 and 16,981 respectively. Examination of subclones of the whole locus in an expression system demonstrated the predicted products. N-terminal amino acid sequences for the moaA, B, C and E products confirmed the translational starts. Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E. Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified. Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively. The moa locus is orientated clockwise at 17.7 minutes in the chromosome.

Amino Acid Sequence↗

PubMed1992

EPR and redox characterization of iron-sulfur centers in nitrate reductases A and Z from Escherichia coli. Evidence for a high-potential and a low-potential class and their relevance in the electron-transfer mechanism.

The redox properties of the iron-sulfur centers of the two nitrate reductases from Escherichia coli have been investigated by EPR spectroscopy. A detailed study of nitrate reductase A performed in the range +200 mV to -500 mV shows that the four iron-sulfur centers of the enzyme belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled, leading to a broad featureless spectrum. The redox behaviour of the high-pH EPR signal given by the molybdenum cofactor was also studied. The iron-sulfur centers of the second nitrate reductase of E. coli, nitrate reductase Z, exhibit essentially the same characteristics than those of nitrate reductase A, except that the midpoint potentials of the high-potential centers appear negatively shifted by about 100 mV. From the comparison between the redox centers of nitrate reductase and of dimethylsulfoxide reductase, a correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed.

Cell Membrane↗

PubMed1992

Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli.

Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same alpha beta gamma, subunits composition and have similar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcloning the different genes and expressing them from separate transcriptional units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantly but independently can meet efficiently and associate to form active enzymes, and (ii) that the alpha subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yield an active membrane-bound heterologous enzyme. One such heterologous enzyme, alpha A beta Z gamma Z, has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heterologous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.

Blotting, Western↗

PubMed1992

Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli.

Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli. Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase. When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble. When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble. When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane. It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity. The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.

Blotting, Western↗

PubMed1991

Nucleotide sequence and molecular characterization of pnlA, the structural gene for damage-inducible pectin lyase of Erwinia carotovora subsp. carotovora 71.

In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.

Amino Acid Sequence↗

PubMed1990

Purification and further characterization of the second nitrate reductase of Escherichia coli K12.

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.

Coenzymes↗