Prosthetic group content and ligand-binding properties of spinach nitrite reductase

Chemical analysis of the ferredoxin-dependent native form (Mr = 85,000) of spinach nitrite reductase has demonstrated a siroheme content that approaches 2 mol of siroheme/mol of enzyme. A widely studied modified (Mr = 61,000) form of nitrite reductase, that has lost much of the native enzyme's ability to use ferredoxin as an electron donor, contains approximately 1 mol of siroheme/mol of enzyme. Quantitation of the high spin ferri-siroheme EPR signals and of nitrite-binding sites of the two preparations confirmed that the native enzyme's siroheme content is approximately twice that of the modified enzyme. Plots of nitrite and cyanide binding to the native enzyme versus ligand concentration are sigmoidal, with Hill coefficients of 1.6-1.8 and 2.3-2.8, respectively. Plots of enzyme activity versus nitrite concentration for the native enzyme are sigmoidal with a Hill coefficient of 2.4. Cyanide inhibition of enzymatic activity was shown to be not competitive. Addition of cyanide to the native enzyme resulted in a diminution of the high spin ferri-siroheme EPR signal and produced EPR signals with g values of 2.71, 2.33, and 1.49 due to low spin ferri-siroheme.     

Characterization of the siroheme active site in spinach nitrite reductase by resonance Raman spectroscopy

The resonance Raman spectra of various species of spinach nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) have been obtained with Soret excitation. These spectra allow for the vibrational properties of the unique siroheme chromophore at the enzyme's active site. The wholesale reordering of siroheme vibrational properties relative to those of protoporphyrins can be rationalized as resulting from a combination of symmetry lowering and bond order reductions within the siroheme macrocyle.     

The role of an iron-sulfur center and siroheme in spinach nitrite reductase.

Purified spinach nitrite reductase, a protein that contains siroheme, is characterized by absorption maxima in the visible region at 385 and 573 nm. On addition of the substrate nitrite, the bands shift to 360 and 570 nm. Dithionite also causes shifts in the maxima of the visible absorption region. Electron paramagnetic resonance studies show that the untreated enzyme contains a high-spin Fe³⁺ heme and that the addition of cyanide, an inhibitor that is competitive with nitrite, results in a spin-state change of the heme. Electron paramagnetic resonance analysis of the enzyme in the presence of dithionite or dithionite plus cyanide indicates the presence of a reduced iron-sulfur center with rhombic symmetry (g-values of 2.03, 1.94, and 1.91). In contrast, when the enzyme is treated with dithionite plus nitrite, the EPR spectrum of an NO-heme complex (g-values of 2.07 and 2.00) is observed. The presence of an iron-sulfur center has also been confirmed by chemical analyses of the nonheme iron and acid-labile sulfide in nitrite reductase. These results are discussed in terms of a mechanism for nitrite reduction that involves electron transfer between the iron-sulfur center and siroheme.     

Structure of spinach nitrite reductase: implications for multi-electron reactions by the iron-sulfur:siroheme cofactor

The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 alpha/beta domains with the siroheme-iron sulfur cofactor at the interface of the three domains. The Fe(4)S(4) cluster is coordinated by cysteines 441, 447, 482, and 486. The siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that could reflect sequence differences or could arise from alterations of the sulfite reductase structure that arise from the isolation of this subunit from the native complex. The implications of the nitrite reductase structure for understanding multi-electron processes are discussed in terms of differences in the protein environments of the cofactors.     

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; )     

Intracellular location of nitrate reductase and nitrite reductase in spinach and sunflower leaves

Summary Chloroplasts have been isolated from spinach and from sunflower which retain their outer membrane and their stroma protein as determined both by ability to fix CO₂ and evolve O₂ at high rates, and by appearance under the phase contrast microscope. Such chloroplasts contain both nitrate and nitrite reductase activity. However, calculations on the distribution of these enzymes, when compared with the distribution of pyruvate kinase and cytochrome c oxidase activity, demonstrate that the larger part of both nitrate and nitrite reductase is located outside of the chloroplast.Supported in part by the National Research Council of Canada.     

The role of tryptophan in the ferredoxin-dependent nitrite reductase of spinach

A system has been developed for expressing a His-tagged form of the ferredoxin-dependent nitrite reductase of spinach in Escherichia coli. The catalytic and spectral properties of the His-tagged, recombinant enzyme are similar, but not identical, to those previously observed for nitrite reductase isolated directly from spinach leaf. A detailed comparison of the spectral, catalytic and fluorescence properties of nitrite reductase variants, in which each of the enzyme’s eight tryptophan residues has been replaced using site-directed mutagenesis by either aromatic or non-aromatic amino acids, has been used to examine possible roles for tryptophan residues in the reduction of nitrite to ammonia catalyzed by the enzyme.     

Mechanism of spinach chloroplast ferredoxin-dependent nitrite reductase: spectroscopic evidence for intermediate states

Nitrite reductases found in plants, algae, and cyanobacteria catalyze the six-electron reduction of nitrite to ammonia with reduced ferredoxin serving as the electron donor. They contain one siroheme and one [4Fe-4S] cluster, acting as separate one-electron carriers. Nitrite is thought to bind to the siroheme and to remain bound until its complete reduction to ammonia. In the present work the enzyme catalytic cycle, with ferredoxin reduced by photosystem 1 as an electron donor, has been studied by EPR and laser flash absorption spectroscopy. Substrate depletion during enzyme turnover, driven by a series of laser flashes, has been demonstrated. A complex of ferrous siroheme with NO, formed by two-electron reduction of the enzyme complex with nitrite, has been shown to be an intermediate in the enzyme catalytic cycle. The same complex can be formed by incubation of free oxidized nitrite reductase with an excess of nitrite and ascorbate. Hydroxylamine, another putative intermediate in the reduction of nitrite catalyzed by nitrite reductase, was found to react with oxidized nitrite reductase to produce the same ferrous siroheme-NO complex, with a characteristic formation time of about 13 min. The rate-limiting step for this reaction is probably hydroxylamine binding to the enzyme, with the conversion of hydroxylamine to NO at the enzyme active site likely being much faster.     

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

Summary The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.     

Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO(2)(-)) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S](2+)/Fe(3+)), resonance Raman spectroscopy indicated that the siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO(2)(-) results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 +/- 0.01 s(-)(1). This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO(2)(-) to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusually heterogeneous, and (iii) the ligand is not nitric oxide, most likely NO(2)(-). The reaction of oxidized NiR with hydroxylamine (NH(2)OH), a putative intermediate, results in a ferrous siroheme-NO complex that is spectroscopically identical to the one observed during NiR turnover. Resonance Raman and absorption spectroscopy data show that the reaction of oxidized NiR ([4Fe-4S](2+)/Fe(3+)) with hydroxylamine is binding-limited, while the NH(2)OH conversion to nitric oxide is much faster.     

The subunit structure of nitrite reductase purified from the denitrifier Achromobacter cycloclastes

The copper-containing nitrite reductase of Achromobacter cycloclastes has been considered to be a homotrimer with three identical subunits both in the crystal and in solution. In this study, however, the enzyme was found to be a heterotrimer consisting of two subunits with molecular masses of 37 kDa and 36.2 kDa, and the 37 kDa subunit was 6 amino acid residues longer than the smaller subunit. Signal-peptide cleavage sites in its N-terminal region are discussed.     

Yeast PAPS reductase: properties and requirements of the purified enzyme

The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (M_R 80–85 k) is represented by a dimer which reduces 3-phosphoadenylyl sulphate to adenosine-3,5-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for bound-sulphite(s) as intermediate. V _max of the enriched enzyme was 4–7 nmol sulphite · min^-1 · mg^-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite · min^-1 · mg^-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The K _m of homologous thioredoxin was 0.6 · 10^-6 M, for the heterologous cosubstrate it increased to 1.4 · 10^-6 M. The affinity for PAPS remained practically unaffected (K _{m PAPS}: 19 · 10^-6 M in the homologous, and 21 · 10^-6 M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.Abbreviations APS adenylyl sulphate - DTE dithioerythritol - DTT dithiothreitol - HPLC high performance liquid chromatography - IEF isoelectric focusing - LSC liquid scintillation counting - 3,5-PAP adenosine-3,5-bisphosphate - PAPS 3-phosphoadenylyl sulphate - PEP phospho-(enol)pyruvate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - Tris 2-amino-2-hydroxymethyl-1,3-propanediol     

Analysis of cis-acting DNA elements mediating induction and repression of the spinach nitrite reductase gene

The expression of nitrite reductase (NiR; EC 1.7.7.1), the second enzyme in the nitrate assimilatory pathway, is regulated by nitrate as well as by end-products of nitrate assimilation, namely, glutamine (Gln) and asparagine (Asn). Nitrate induces expression of the NiR gene. Previously, using deletion analysis of the spinach (Spinacia oleracea L.) NiR gene promoter in transgenic tobacco (Nicotiana tabacum L.) and in-vivo dimethyl sulfate footprinting, we had identified the region between −230 bp and −180 bp as being critical for nitrate inducibility of this gene. In the present study, we show that the region from +1 to +67, which forms part of its untranslated leader, is important for minimal induction in the presence of nitrate. Electrophoretic mobility shift assays reveal concentration-dependent and competitive binding of a factor in tobacco nuclear extracts to this region. In the presence of Gln or Asn, the expression of spinach NiR is repressed. This repression is observed with the full-length NiR promoter (−3100 bp) as well as with the shortest promoter (−230 bp) that gives nitrate induction, which includes the +67 bp leader sequence. The repressed expression of the gene is not the result of reduced nitrate accumulation in the presence of the nitrogen metabolites. Received: 2 December 1997 / Accepted: 20 January 1998     

Characterization of partially purified glutathione reductase from cold-hardened and nonhardened spinach leaf tissue

Glutathione reductase (GR) (EC 1.6.4.2) was studied in crude and partially purified extracts from nonhardened (25/20 °C D/N) and hardened (5/5 °C D/N) spinach-leaf tissue. Crude extracts of hardened tissue showed a 66% increase in glutathione reductase activity over that of nonhardened tissue. The enzyme was purified by ammonium sulfate precipitation, Sephadex G-150 chromatography, 2′, 5′ ADP-Sepharose affinity chromatography, and DEAE-Sephadex A-50 ion-exchange chromatography. The partially purified enzyme from the two sources showed different kinetic characteristics, heat inactivation, freezing inactivation, and electrophoretic mobilities. Hardened leaves contain different forms of glutathione reductase than do nonhardened leaves. GR from hardened spinach has greater stability against freezing and a higher affinity for substrates at low temperature than does GR from nonhardened spinach.     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.