Protein S-nitrosylation in photosynthetic organisms: A comprehensive overview with future perspectives.

BACKGROUND: The free radical nitric oxide (NO) and derivative reactive nitrogen species (RNS) play essential roles in cellular redox regulation mainly through protein S-nitrosylation, a redox post-translational modification in which specific cysteines are converted to nitrosothiols. SCOPE OF VIEW: This review aims to discuss the current state of knowledge, as well as future perspectives, regarding protein S-nitrosylation in photosynthetic organisms. MAJOR CONCLUSIONS: NO, synthesized by plants from different sources (nitrite, arginine), provides directly or indirectly the nitroso moiety of nitrosothiols. Biosynthesis, reactivity and scavenging systems of NO/RNS, determine the NO-based signaling including the rate of protein nitrosylation. Denitrosylation reactions compete with nitrosylation in setting the levels of nitrosylated proteins in vivo. GENERAL SIGNIFICANCE: Based on a combination of proteomic, biochemical and genetic approaches, protein nitrosylation is emerging as a pervasive player in cell signaling networks. Specificity of protein nitrosylation and integration among different post-translational modifications are among the major challenges for future experimental studies in the redox biology field. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Biochemical basis of sulphenomics: how protein sulphenic acids may be stabilized by the protein microenvironment.

Among protein residues, cysteines are one of the prominent candidates to ROS-mediated and RNS-mediated post-translational modifications, and hydrogen peroxide (H2 O2 ) is the main ROS candidate for inducing cysteine oxidation. The reaction with H2 O2 is not common to all cysteine residues, being their reactivity an utmost prerequisite for the sensitivity towards H2 O2 . Indeed, only deprotonated Cys (i.e. thiolate form, S- ) can react with H2 O2 leading to sulphenic acid formation (SOH), which is considered as a major/central player of ROS sensing pathways. However, cysteine sulphenic acids are generally unstable because they can be further oxidized to irreversible forms (sulphinic and sulphonic acids, SO2 H and SO3 H, respectively), or alternatively, they can proceed towards further modifications including disulphide bond formation (SS), S-glutathionylation (SSG) and sulphenamide formation (SN). To understand why and how cysteine residues undergo primary oxidation to sulphenic acid, and to explore the stability of cysteine sulphenic acids, a combination of biochemical, structural and computational studies are required. Here, we will discuss the current knowledge of the structural determinants for cysteine reactivity and sulphenic acid stability within protein microenvironments.

A case of autochthonous human Dirofilaria infection, Germany, March 2014.

In March 2014, an infection with the nematode Dirofilaria repens was diagnosed in a German citizen in the federal state of Saxony-Anhalt. The patient had developed an itching subcutaneous nodule containing a female worm, which was identified as D. repens by 12S ribosomal ribonucleic acid (rRNA) gene sequencing. Autochthonous human D. repens infections have not been described in Germany so far, but this finding is consistent with the recent detection of D. repens in mosquitoes from east Germany.

[Bilateral acetabulum fracture after suffering sport trauma]. / Beidseitige Acetabulumfraktur nach erlittenem Sporttrauma.

This case study describes a 37-year-old male who suffered a bilateral transverse acetabulum fracture with a fracture of the posterior wall and a double-sided dorsal hip dislocation in combination with a left-sided femoral head fracture (Pipkin IV) while skiing in a "fun park". The accurate diagnosis and presurgical planning was made by means of a computed tomography (CT) scan and a subsequent 3D reconstruction. After a primarily executed shielded repositioning of the bilateral hip dislocationearly secondary and anatomical reconstruction of the double-sided acetabulum fracture was possible using the Kocher-Langenbeck approach. A consistent physiotherapy as well as rehabilitation finally led to a positive clinical result for the patient.

Molecular mechanism of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-regulated, NAD(P)H-dependent enzyme involved in plant photosynthetic carbon reduction. Unlike lower photosynthetic organisms, which only contain A(4)-GAPDH, the major GAPDH isoform of land plants is made up of A and B subunits, the latter containing a C-terminal extension (CTE) with fundamental regulatory functions. Light-activation of AB-GAPDH depends on the redox state of a pair of cysteines of the CTE, which can form a disulfide bond under control of thioredoxin f, leading to specific inhibition of the NADPH-dependent activity. The tridimensional structure of A(2)B(2)-GAPDH from spinach chloroplasts, crystallized in the oxidized state, shows that each disulfide-containing CTE is docked into a deep cleft between a pair of A and B subunits. The structure of the CTE was derived from crystallographic data and computational modeling and confirmed by site-specific mutagenesis. Structural analysis of oxidized A(2)B(2)-GAPDH and chimeric mutant [A+CTE](4)-GAPDH revealed that Arg-77, which is essential for coenzyme specificity and high NADPH-dependent activity, fails to interact with NADP in these kinetically inhibited GAPDH tetramers and is attracted instead by negative residues of oxidized CTE. Other subtle changes in catalytic domains and overall conformation of the tetramers were noticed in oxidized A(2)B(2)-GAPDH and [A+CTE](4)-GAPDH, compared with fully active A(4)-GAPDH. The CTE is envisioned as a redox-sensitive regulatory domain that can force AB-GAPDH into a kinetically inhibited conformation under oxidizing conditions, which also occur during dark inactivation of the enzyme in vivo.

Purification of cytochrome b-561 from bean hypocotyls plasma membrane. Evidence for the presence of two heme centers.

The high potential, ascorbate-reducible b-type cytochrome of plant plasma membranes, named cytochrome b-561, has been purified to homogeneity from etiolated bean hypocotyls. The pure protein migrated in denaturing electrophoresis as a broad band of approximately 55 kDa, and was found to be glycosylated. Optical redox titrations of partially purified cytochrome b-561 indicated that it contains two hemes with similar spectral features, but distinct midpoint redox potentials (E(m7)+135 mV and +206 mV, respectively). The presence of two heme centers in cytochrome b-561 is consistent with its role in electron transfer across plant plasma membranes.

Crystal structure of the non-regulatory A(4 )isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP.

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 A resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis. The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A(4)-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2'-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes. Although the overall structure of A(4)-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.

NAD(P)H:(Quinone-Acceptor) Oxidoreductase of Tobacco Leaves Is a Flavin Mononucleotide-Containing Flavoenzyme.

The soluble NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR, EC 1.6.99.2] of Nicotiana tabacum L. leaves and roots has been purified. NAD(P)H-QR contains noncovalently bound flavin mononucleotide. Pairs of subunits of 21.4 kD are linked together by disulfide bridges, but the active enzyme is a homotetramer of 94 to 100 kD showing an isoelectric point of 5.1. NAD(P)H-QR is a B-stereospecific dehydrogenase. NADH and NADPH are electron donors of similar efficiency with Kcat:Km ratios (with duroquinone) of 6.2 x 107 and 8.0 x 107 m-1 s-1, respectively. Hydrophilic quinones are good electron acceptors, although ferricyanide and dichlorophenolindophenol are also reduced. The quinones are converted to hydroquinones by an obligatory two-electron transfer. No spectral evidence for a flavin semiquinone was detected following anaerobic photoreduction. Cibacron blue and 7-iodo-acridone-4-carboxylic acid are inhibitory. Tobacco NAD(P)H-QR resembles animal DT-diaphorase in some respects (identical reaction mechanism with a two-electron transfer to quinones, unusually high catalytic capability, and donor and acceptor substrate specificity), but it differs from DT-diaphorase in molecular structure, flavin cofactor, stereospecificity, and sensitivity to inhibitors. As in the case with DT-diaphorase in animals, the main NAD(P)H-QR function in plant cells may be the reduction of quinones to quinols, which prevents the production of semiquinones and oxygen radicals. The enzyme appears to belong to a widespread group of plant and fungal flavoproteins found in different cell compartments that are able to reduce quinones.

Dissecting the Diphenylene Iodonium-Sensitive NAD(P)H:Quinone Oxidoreductase of Zucchini Plasma Membrane.

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) in zucchini (Cucurbita pepo L.) hypocotyls. In the presence of NADPH, lipophilic ubiquinone homologs with up to three isoprenoid units were reduced by intact PM vesicles with a Km of 2 to 7 [mu]M. Affinities for both NADPH and NADH were similar (Km of 62 and 51 [mu]M, respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I in gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers NADH over NADPH, it is markedly sensitive to the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound to the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present in the same tissue. Following purification of peak I, however, the latter gave rise to a quinone oxidoreductase of the soluble type (peak II), based on substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, G. Tedeschi, and P. Trost [1996] Plant Physiol. 112:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones.

NADH:Fe(III)-chelate reductase of maize roots is an active cytochrome b5 reductase.

Microsomal NADH:Fe(III)-chelate reductase (NFR) of maize roots has been purified as a monomeric flavoprotein of 32 kDa with non-covalently bound FAD. In the presence of NADH, NFR efficiently reduced the physiological iron-chelate Fe(III)-citrate (K[cat]/K[m](Fe(III)-citrate) = 6.0 X 10[6] M[-1] S[-1]) with a sequential reaction mechanism. Purified NFR was totally inhibited by the sulfhydryl reagent PHMB at 10(-9) M, and it could use cyt b5 as alternative electron acceptor with a maximal reduction rate as high as with Fe(III)-citrate. We conclude that in maize roots the reduction of Fe(III)-citrate is chiefly performed by a cytochrome b5 reductase, mostly associated with intracellular membranes and in part with the plasma membrane.

Cloning and heterologous expression of NAD(P)H:quinone reductase of Arabidopsis thaliana, a functional homologue of animal DT-diaphorase.

In higher plants, NAD(P)H:quinone reductase (NQR) is the only flavoreductase known to reduce quinone substrates directly to hydroquinones by a two-electron reaction mechanism. This enzymatic activity is believed to protect aerobic organisms from the oxidative action of semiquinones. For this reason plant NQR has recently been suggested to be related to animal DT-diaphorase. A cDNA clone for NQR of Arabidopsis thaliana was identified, expressed in Escherichia coli, purified and characterized. Its amino acid sequence was found related to a number of putative proteins, mostly from prokaryotes, with still undetermined function. Conversely, in spite of the functional homology, sequence similarity between plant NQR and animal DT-diaphorase was limited and essentially confined to the flavin binding site.

Arguments against a close relationship between non-phosphorylating and phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

Non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.9) from spinach leaves was purified to homogeneity using an improved purification procedure. Thus, a major contaminant with molecular mass and ion-exchange properties similar to non-phosphorylating GAPDH was eliminated. Using this pure non-phosphorylating GAPDH, cofactor stereospecificity was determined by 1H NMR. Analysis of the NADPH formed from the hydride transfer from glyceraldehyde-3-phosphate to [4-2H]NADP showed that the enzyme belongs to the A-stereospecific dehydrogenase family. This stereospecificity is the same as that described for the aldehyde dehydrogenase (ALDH) superfamily and opposite to that of the phosphorylating GAPDH. Moreover, results from peptide sequencing analysis suggest a similarity in sequence between the non-phosphorylating GAPDH and ALDHs. Thus, the results taken all together strongly suggest that non-phosphorylating GAPDH belongs to the ALDH family and has no close relationship to the phosphorylating GAPDH class.

Comparative sensitivity of 13 species of pathogenic bacteria to seven chemical germicides.

BACKGROUND: The relative resistance of diverse human bacterial pathogens to commonly used germicidal agents has not been established. METHODS: We measured by titration the survival of thirteen different bacteria after exposure to glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate, sodium hypochlorite, or phenol. RESULTS: Our comparative experiments allowed classification of the organisms' survival into four groups: (a) Pseudomonas aeruginosa and Staphylococcus aureus showed the most resistance, (b) Clostridium perfringens, Salmonella typhimurium, Staphylococcus epidermidis, and Escherichia coli O157:H7 showed intermediate resistance, (c) Listeria monocytogenes, Shigella sonnei, and Vibrio parahaemolyticus survived some treatments with chemical agents only in the presence of protecting protein (serum albumin), and (d) Vibrio cholerae, Vibrio vulnificus, Bacillus cereus, and Yersinia enterocolitica did not survive any of the treatments applied. CONCLUSION: We found species that more frequently survived exposure to germicidal agents were also those most commonly reported in association with hospital infections. Our findings suggest that resistance to disinfectants may be more important than pathogenicity in determining the relative prominence of an organism as an agent responsible for nosocomial infections.

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms.

Regulation of the Calvin-Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin-Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A(2)B(2)-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A(4)-GAPDH. GapB subunits of A(2)B(2)-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A(2)B(2)-GAPDH into strongly inactivated A(8)B(8)-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A(4)-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A(2)B(2)-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

Inhibition of spinach D-glyceraldehyde 3-phosphate: NADP+ oxidoreductase (nonphosphorylating) by adenylate compounds: the effect of dead-end inhibitors on a steady state random reaction mechanism.

D-Glyceraldehyde 3-phosphate: NADP+ oxidoreductase, nonphosphorylating (GNR; EC 1.2.1.9) purified from spinach leaves was investigated by initial velocity analysis. The hyperbolic saturation curves became nonhyperbolic when NADP+ was varied at elevated D-glyceraldehyde 3-phosphate (G3P) concentrations (sigmoidicity) or when G3P was varied at low NADP+ concentrations (pseudo-substrate inhibition), suggesting a random bi bi mechanism (Scagliarini et al. Plant Physiol. 94, 1337-1344, 1990). Free ATP was a linear competitive inhibitor of both NADP+ with KI 0.5 +/- 0.2 mM (SD) and G3P with KI 3.2 +/- 0.2 mM as determined by data in the hyperbolic range of responses when the nonvaried substrate was saturating. Similarly ADP inhibited competitively with KI 1.9 +/- 0.4 mM (NADP+) and 3.5 +/- 0.5 mM (G3P). Inhibition was mixed-type when the nonvaried substrate was below saturation. ATP, but not ADP, tended to enhance the nonhyperbolic behavior of GNR, resulting in potentiated inhibition at high [G3P]/[NADP+] ratios. The Mg-chelated form of ATP was less effective. The rate equation of a steady state random bi bi reaction mechanism in the presence of a dead-end inhibitor was derived. Suitable values of the rate constants were chosen to fit the kinetic data for the uninhibited enzyme. These values and the measured inhibition constants inserted in the rate equation can satisfactorily account for the nonhyperbolic inhibition patterns of ATP and ADP. The generalized model represents a possible alternative to allosteric models in interpreting nonlinear kinetics and dead-end inhibition of two-substrate enzymes.

Fiber bundle based scanning detection system for automated DNA sequencing.

High-throughput DNA sequencing techniques are under rapid development currently, mainly triggered by the Human Genome Project. At the present time, slab gel based automated DNA sequencing is the standard procedure, utilizing fluorophore labeling and laser-induced fluorescence detection with scanning technology. In this paper, a novel, fiber-optic bundle based detection system is introduced, where a central illuminating fiber is used for the excitation of the electrophoretically separated fluorophore-labeled DNA sequencing fragments, along with several collecting fibers disposed around the illuminating fiber to collect the emitted fluorescent signal. As a model system, Cy5-labeled DNA sequencing fragments were separated on an ultrathin polyacrylamide slab gel and detected by the fiber bundle based laser-induced fluorescence detection system. A 640-nm diode laser was used to generate the illumination beam, and the emitted light collected by the fiber bundle was detected by a solid-state avalanche photodiode.

[Vitamin E deficiency and anaemia in premature infants (author's transl)]. / Zur Bedeutung des Vitamin E-Mangels bei der Frühgeburtenanämie.

The effect of a prophylactic treatment with tocopherol, early iron substitution and administration of polyunsaturated fatty acids in different quantities on the plasma concentration of vitamin E, hemolysis in the peroxide test and hematological parameters during the first two months of life was determined in 23 premature infants with birth weights of less than 1800 gms. In no patient in any of the different treatment groups a tocopherol deficiency (plasma concentration less than 0.50 mg/100ml) was observed and accordingly no hemolytic anemia attributable to E hypovitaminosis was found. The mean concentration of tocopherol at birth (0.51 mg/100 ml) was already above the critical limit, and no single value was found below the latter after the 10th day of life. For that reason, the amount of vitamin E included in formulas generally used in this country and its intestinal absorption have to be considered as adequate even for small premature children. The different treatments of the test groups had no significant influence on the clinical state or the hematological findings. The infants with vitamin E substitution and those without had similar hemoglobin levels. Almost all children had a distinct reticulocytosis and thrombocytosis during the second month of their life. This seems to occur naturally. The peroxide test proved to be no reliable indicator of a tocopherol deficiency in the individual case. It is not yet clear which additional factors cause vitamin E deficiency, infrequently seen in small premature infants.

Glyceraldehyde 3-Phosphate:NADP Reductase of Spinach Leaves : Steady State Kinetics and Effect of Inhibitors.

The steady state kinetics of glyceraldehyde 3-phosphate:NADP(+) oxidoreductase (GNR) (EC 1.2.1.9) have been investigated. The enzyme exhibits hyperbolic behavior over a wide range of substrate concentrations. Double-reciprocal plots are nearly parallel or distantly convergent with limiting K(m) values of 2 to 5 micromolar for NADP(+) and 20 to 40 micromolar for D-glyceraldehyde 3-phosphate (G3P). The velocity response to NADP(+) as the varied substrate is however sigmoidal if G3P concentration exceeds 10 micromolar, whereas the response to G3P may show inhibition above this concentration. This ;G3P-inhibited state' is alleviated by saturating amounts of NADP(+) or NADPH. Product inhibition patterns indicate NADPH as a potent competitive inhibitor to NADP(+) (K(i) 30 micromolar) and mixed inhibitor towards G3P, and 3-phosphoglycerate (3PGA) as mixed inhibitor to both NADP(+) and G3P (K(i) 10 millimolar). The data, and those obtained with dead-end inhibitors, are consistent with a nonrapid equilibrium random mechanism with two alternative kinetic pathways. Of these, a rapid kinetic sequence (probably ordered with NADP(+) binding first and G3P binding as second substrate) is dominant in the range of hyperbolic responses. A reverse reaction with 3PGA and NADPH as substrates is unlikely, and was not detected. Of a number of compounds tested, erythrose 4-phosphate (K(i) 7 micromolar) and Pi (K(i) 2.4 millimolar) act as competitive inhibitors to G3P (uncompetitive towards NADP(+)) and are likely to affect the in vivo activity. Ribose 5-phosphate, phosphoenolpyruvate, ATP, and ADP are also somewhat inhibitory. Full GNR activity in the leaf seems to be allowed only under high photosynthesis conditions, when levels of several inhibitors are low and substrate is high. We suggest that a main function of leaf GNR is to supply NADPH required for photorespiration, the reaction product 3PGA being cycled back to chloroplasts.

Ascorbate-independent electron transfer between cytochrome b561 and a 27 kDa ascorbate peroxidase of bean hypocotyls.

Cytochrome b561 (cyt b561) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)H:quinone reductase activity. In bean hypocotyl PM, juglonol-reduced cyt b561 was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cyt b561 was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cyt b561 used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cyt b561, the peroxidase interacting with cyt b561, and H2O2, in this order, constitute an artificial electron transfer chain in which cyt b561 is indirectly reduced by NADPH and indirectly oxidized by H2O2.

Purification and properties of NAD(P)H: (quinone-acceptor) oxidoreductase of sugarbeet cells.

NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR], a plant cytosolic protein, was purified from cultured sugarbeet cells by a combination of ammonium sulfate fractionation, FPLC Superdex 200 gel filtration, Q-Sepharose anion-exchange chromatography, and a final Blue Sepharose CL-6B affinity chromatography with an NADPH gradient. The subunit molecular mass is 24 kDa and the active protein (94 kDa) is a tetramer. The isoelectric point is 4.9. The enzyme was characterized by ping-pong kinetics and extremely elevated catalytic capacity. It prefers NADPH over NADH as electron donor (kcat/Km ratios of 1.7 x 10(8) M-1 S-1 and 8.3 x 10(7) M-1 S-1 for NADPH and NADH, respectively, with benzoquinone as electron acceptor). The acridone derivative 7-iodo-acridone-4-carboxylic acid is an efficient inhibitor (I0.5 = 5 x 10(-5) M), dicumarol is weakly inhibitory. The best acceptor substances are hydrophilic, short-chain quinones such as ubiquinone-0 (Q-0), benzoquinone and menadione, followed by duroquinone and ferricyanide, whereas hydrophobic quinones, cytochrome c and oxygen are reduced at negligible rates at best. Quinone acceptors are reduced by a two-electron reaction with no apparent release of free semiquinonic intermediates. This and the above properties suggest some relationship of NAD(P)H-QR to DT-diaphorase, an animal flavoprotein which, however, has distinct structural properties and is strongly inhibited by dicumarol. It is proposed that NAD(P)H-QR by scavenging unreduced quinones and making them prone to conjugation may act in plant tissues as a functional equivalent of DT-diaphorase.