On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions.

Nitrite reductase (cytochrome $\text{cd}$) from $\text{T. denitrificans}$ has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from $\text{P. aeruginosa}$. EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both $\text{c}$- and $\text{d}$-hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a ¹⁴N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant.To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.     

Nitrate reductase,nitrite reductase activities and leaf diffusive resistance in wheat under water deficit

The flag and second leaves of wheat showed physiological and biochemical differences in the normal and water stressed plants. Although the flag leaf had lower enzyme activities in the control plants, in stressed plants it could continue nitrate assimilation better than the second and other leaves. The flag leaf also exhibited a higher tendency to resume normal metabolic activity after the release of stress as is indicated by higher NRA, NiRa, lower r_ad and r_ab.     

Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR: ferredoxin NADP+ reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions

Among the 118 genes upregulated by Pseudomonas aeruginosa in response to iron starvation [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287], we focused on the products of the two genes encoding electron transfer proteins, as a means of identifying the redox partners of the heme oxygenase (pa-HO) expressed under low-iron stress conditions. Biochemical and spectroscopic investigations demonstrated that the bfd gene encodes a 73-amino acid protein (pa-Bfd) that incorporates a [2Fe-2S]2+/+ center, whereas the fpr gene encodes a 258-residue NADPH-dependent ferredoxin reductase (pa-FPR) that utilizes FAD as a cofactor. In vitro reconstitution of pa-HO catalytic activity with the newly characterized proteins led to the surprising observation that pa-FPR efficiently supports the catalytic cycle of pa-HO, without the need of a ferredoxin. In comparison, electron transfer from pa-Bfd to pa-HO is sluggish, which strongly argues against the possibility that the seven electrons needed by pa-HO to degrade biliverdin are transferred from NADPH to pa-HO in a ferredoxin (Bfd)-dependent manner. Given that pa-HO functions to release iron from exogenous heme acquired under iron-starvation conditions, the use of a flavoenzyme rather than an iron-sulfur center-containing protein to support heme degradation is an efficient use of resources in the cell. The crystal structure of pa-FPR (1.6 A resolution) showed that its fold is comparable that of the superfamily of ferredoxin reductases and most similar to the structure of Azotobacter vinelandii FPR and Escherichia coli flavodoxin reductase. The latter two enzymes interact with distinct redox partners, a ferredoxin and a flavodoxin, respectively. Hence, findings reported herein extend the range of redox partners recognized by the fold of pa-FPR to include a heme oxygenase (pa-HO).     

Kinetics of redox interaction between substituted quinones and ascorbate under aerobic conditions.

One-electron reduction of quinones (Q) by ascorbate (AscH ); (1) AscH + Q --> Q*- + Asc*- + H+, followed by the oxidation of semiquinone (Q*-) by molecular oxygen; (2) Q*- + O2 --> Q + O2*-, results in the catalytic oxidation of ascorbate (with Q as a catalyst) and formation of active forms of oxygen. Along with enzymatic redox cycling of Q. this process may be related to Q cytotoxicity and underlie an antitumor activity of some Qs. In this work, the kinetics of oxygen consumption accompanied the interaction of ascorbate with 55 Qs including substituted 1,4- and 1,2-benzoquinones, naphthoquinones and other quinoid compounds were studied in 50 mM sodium phosphate buffer, pH 7.40, at 37 degrees C by using the Clark electrode technique. The capability of Q to catalyze ascorbate oxidation was characterized by the effective value of kEFF calculated from the initial rate of oxygen consumption (R(OX)) by the equation R(OX) = kEFF[Q][AscH-] as well as by a temporary change in R(OX). The correlation of kEFF with one-electron reduction potential, E(Q/Q*-), showed a sigma-like plot, the same for different kinds of Qs. Only the Qs which reduction potential E(Q/Q*-) ranged from nearly -250 to + 50 mV displayed a pronounced catalytic activity, kEFF increased with shifting E(Q/Q*-) to positive values. The following linear correlation between kEFF (in M (-1) s(-1)) and E(Q/Q*-) (in mV) might be suggested for these Qs: lg(kEFF)= 3.91 + 0.0143E(Q/Q*-). In contrast, Qs with E(Q/Q*-) < - 250 mV and E(Q/Q*-) > + 50 mV showed no measurable catalytic activity. The Qs studied displayed a wide variety in the kinetic regularities of oxygen consumption. When E(Q/Q*-) was more negative than - 100 mV, Q displayed a simple ('standard') kinetic behavior--R(OX) was proportional to [AscH-][Q] independently of concentration of individual reagents, [AscH-] and [Q]; R(OX) did not decrease with time if [AscH-] was held constant: Q recycling was almost reversible. Meanwhile, Qs with E(Q/Q*-) > - 100 mV demonstrated a dramatic deviation from the 'standard' behavior that was manifested by the fast decrease in R(OX) with time and non-linear dependence of even starting values of R(OX) on [Q] and [AscH-]. These deviations were caused basically by the participation of Q*- in side reactions different from (2). The above findings were confirmed by kinetic computer simulations. Some biological implications of Q-AscH- interaction were discussed.     

Purification and general properties of spinach leaf nitrite reductase

Nitrite reductase was isolated from spinach leaves. The enzymewas purified 168-fold by a procedure involving extraction withphosphate buffer, gel filtration on Sephadex G-200, ion-exchangechromtography on DEAE-Sephadex A-50, and adsorption on hydroxyapatite.The preparation was homogeneous in the ultracentrifuge withsedimentation coefficient at infinite dilution (s?_20,w) of 4.57S. Disc electrophoresis revealed some small bands together witha major protein band. The molecular weight of the spinach nitritereductase was estimated to be 60,000 by gel filtration on SephadexG-100 while a molecular weight of 72,000 was obtained from thesedimentation-diffusion coefficients of the protein. Resultsof sodium dodecyl sulfate gel electrophoresis suggested thatthe enzyme molecule consists of two subunits of molecular sizeof 37,000. After close examination of assay systems based onsodium dithioniteviologen dye procedures, we developed a moreelaborate, improved chemical assay method. Some enzymatic propertiesof the purified nitrite reductase were examined. ¹This work was reported in part at the Annual Meeting of JapaneseSociety of Plant Physiologists, April 6–8, 1972. (Received November 16, 1972; )     

Methylation and demethylation of mercury under controlled redox, pH and salinity conditions.

In estuarine sediments, the microbially mediated processes of methylation, demethylation, and volatilization determine the state and overall toxicity of mercury pollutants. The effects of redox potential (Eh) and salinity on the above microbial processes were investigated in reactors constructed to allow for continuous monitoring and adjustment of the pH (6.8) and Eh of freshly collected estuarine sediments. For measurements of methylation and demethylation activity, sediment slurries adjusted to appropriate salinity were spiked with HgCl2 or CH3HgCl, respectively, and were incubated in the reactors. Methylmercury was measured by gas chromatography. Volatilized elemental mercury (Hg0) was trapped and determined by cold vapor atomic absorption spectrometry. Volatilization of Hg0 and CH3HgCH3 were found to be minimal. Methylation of Hg2+ was favored at Eh-220 mV as compared to +110 mV. At -220 mV, high salinity (2.5%) inhibited methylation, and low salinity (0.4%) favored it. At +110 mV, the salinity effect was less pronounced. Demethylation of CH3HgCl was favored at +110 mV regardless of the salinity level. Low redox potential under low salinity conditions inhibited demethylation, but high salinity reversed this inhibition. These findings are helpful for interpreting and predicting the behavior of mercury pollutants in estuarine sediments.     

Measurement of nitrite reductase in leaf tissue of Vigna mungo

The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH₃) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.     

Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light

Nitrite reductase has been purified almost 3000-fold, in 35% yield, to a specific activity of 77 units (mg protein)-1 from wheat leaves using a multi-step procedure with affinity chromatography on ferredoxin-Sepharose as the final step. The purified enzyme, although not homogeneous, exhibited absorption maxima at 278, 390, 568 and 687 nm. Minor contaminants were removed by gel filtration in the presence of sodium dodecyl sulphate to yield a single polypeptide of Mr 60 500 as judged by polyacrylamide gel electrophoresis. Antibodies raised against this polypeptide were shown to cross-react with native nitrite reductase and were used to study the synthesis of nitrite reductase in vivo and in vitro. The increase in nitrite reductase activity following exposure of dark-grown plants to nitrate and light was shown by immunodecoration of Western blots to be due to synthesis de novo. Poly(A)-rich RNA isolated from plants actively synthesising nitrite reductase was shown to direct the synthesis in a rabbit reticulocyte lysate of a polypeptide of Mr 64000 which was immunoprecipitated by antibodies to nitrite reductase.     

The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin

1. NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into two enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. 2. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithionite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. 3. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. 4. Nitrite reductase had K(m) 0.15mm for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. 5. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Optical biosensor based on nitrite reductase immobilised in controlled pore glass.

The increasing concentration of nitrite in groundwater, rivers and lakes brings serious risks to the public health and to the environment. The aim of this work was the development of an optical biosensor for quantifying nitrite based on the activity of cytochrome cd(1) nitrite reductase immobilised in controlled pore glass (CPG) beads. The developed biosensor operates by measuring the optical reflectance of nitrite reductase, which shows spectroscopic changes when nitrite reversibly binds to the reduced form and oxidizes the enzyme. The optimisation of the immobilisation procedure showed that the immobilisation efficiency is highly dependent on the pH, being very low at basic pH, and that the maximum capacity of the CPG for the immobilisation of cd(1) was estimated in 57+/-10 mg cd(1)/g CPG. The CPG/cd(1) specific activity remained stable at 4 degrees C, decreasing only 10% in 15 days. No observed effects of the immobilisation on the enzyme characteristics were detected, regarding both the red/ox absorbance spectra and the enzyme specific activity, since the red/ox spectra are in good agreement with similar ones obtained for cd(1) in solution, and the specific activity at time zero (0.6 micromoles of NO(2)(-) reduced min(-1) mg of protein(-1)) is similar to that found for the soluble enzyme. The biosensor shows a sensitive response to increasing concentrations of nitrite in solution, especially at 460 nm, at which it showed higher sensitivity. The corresponding detection limit of 0.93 microM is well below the maximum admissible concentration imposed by European Community norms, of 2.2 microM.     

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.     

Homology predicted structure and functional interaction of ferredoxin from the eukaryotic alga Chlamydomonas reinhardtii with nitrite reductase and glutamate synthase

Ferredoxin (Fd) from Chlamydomonas reinhardtii is composed of 94 amino-acid residues and a [2Fe-2S] cluster. The homology modelling technique has been used to predict the tertiary structure of C. reinhardtii Fd. The overall structure shows the typical fifth-stranded beta-grasp plus two additional beta-sheets and three alpha-helices. Site-directed mutagenesis of recombinant Fd has allowed us to obtain four point mutants and one double mutant--all mutations being located in the short alpha-helix at the carboxy-terminal segment as well as a triple mutant affected on helix alpha1. Crosslinking studies and measurement of enzymatic activities reveal that the residues changed are critical for the interaction of Fd with glutamate synthase (GOGAT) and nitrite reductase (NiR). Potentiometric analyses of the Fd mutants show that the replacement of glutamate in position 91 drastically changes the redox potential value (70 mV), thereby suggesting that such a glutamate can modulate the reactivity of Fd towards its reaction partners. According to results herein presented, the reported mutations modify the electrostatic interactions within the complex formed between Fd and GOGAT or NiR.     

Intracellular localization of nitrate reductase, nitrite reductase, and glutamic Acid dehydrogenase in green leaf tissue

Greenhouse grown seedlings of corn (Zea mays L.) and foxtail (Setaria faberii Herrm.) were used as source material in determining the intracellular localization of nitrate reductase, nitrite reductase, and glutamic acid dehydrogenase, Nonaqueous and aqueous isolation techniques were used to establish that nitrite reductase is localized within the chloroplasts, but that nitrate reductase and glutamic acid dehydrogenase are not. Nonaqueous isolation gives distribution patterns of nitrite reductase which are the same as those observed for NADP-dependent 3-phosphoglyceraldehyde dehydrogenase but which differ drastically from the patterns observed for pyruvic acid kinase. The distribution patterns for nitrate reductase are the same as those of pyruvic acid kinase. The techniques used do not eliminate the possibility that nitrate reductase and pyruvic acid kinase are localized on the external chloroplast membrane.

The data obtained establish that glutamic acid dehydrogenase of green leaves is localized within the mitochondria.

     

Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.