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.

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.

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.

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.

Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria.

NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica oleracea L., var. botrytis). The NAD-linked activity is accompanied by a minor NADP-linked activity. Some contaminant NADP-malic enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations. NAD-dependent malic enzyme has been purified 38-fold by ammonium sulfate fractionation and gel permeation chromatography, to a specific activity up to 2 micromoles per minute per milligram.The nature of the activating effect of coenzyme A and dithiothreitol has been investigated. Both compounds act by decreasing the apparent Michaelis constants for l-malate and NAD(+) (and NADP(+)), V(max) remaining approximately constant. However, enzyme fully activated by dithiothreitol can still be stimulated up to 2.4-fold by coenzyme A treatment.Velocity versus substrate responses show hyperbolic kinetics under present assay conditions (pH 7.5, 2 millimolar Mn(2+)), but biphasic kinetics have been observed with enzyme purified in the presence of 10 millimolar dithiothreitol, suggesting enzyme heterogeneity with respect to an activated state. This condition is reverted to linearity by treatment with coenzyme A. K(m) values do not vary with changing concentrations of the second substrate. Enzyme molecular weight is 400,000 in the completely activated state and 200,000 in the ;inactivated' state; intermediate forms are also found. All coenzyme A derivatives tested are effective activators, showing activation constants lower than for coenzyme A itself. The concentration dependence of the activation is sigmoidal.

Two forms of NADP-dependent malic enzyme in expanding maize leaves.

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a Km (L-malate) 0.3-0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a Km (L-malate) 0.10-0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5-1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar Km (NADP(+)), and the typical increase of Km for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min(-1) mg protein(-1) by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.

Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes.

An NAD(P)H dehydrogenase stimulated by quinone (P Pupillo, V Valenti, L de Luca, R Hertel 1986 Plant Physiol 80: 384-389) was solubilized from washed microsomes of zucchini squash hypocotyls (Cucurbita pepo L.) by use of 1% Triton X-100. The solubilized enzyme remained in solution in aqueous buffer and could be purified by a combination of Sepharose 6B chromatography and Blue Ultrogel chromatography. Of the three peaks of activity eluted from the latter column with a salt gradient, peak 3 had 50% or more of the activity and was almost pure enzyme. The preparation examined in SDS-gel electrophoresis consisted of two types of subunits, a (molecular weight 39,500) and b (37,000) in equal amounts. Peak 2 was less pure but had a similar polypeptide pattern. The active protein is proposed to be a heterotetramer (a(2)b(2)) having a molecular weight of about 150,000, as found by gel exclusion chromatography. The purified enzyme can reduce several quinones, DCPIP, cytochrome c, and with best efficiency ferricyanide, and is therefore a diaphorase. The kinetics for the substrates are negatively cooperative with Hill coefficients n(H) = 0.55 +/- 0.05 for NADPH and 0.22 +/- 0.04 for duroquinone. A weak inhibition by p-hydroxymercuric benzoate and mersalyl (stronger with microsomal preparations) suggests the presence of essential sulfhydryl group(s). The possibility is discussed that the dehydrogenase is an NAD(P)H-P450 reductase or similar flavoprotein, and that it is responsible for the NADPH-cytochrome c reductase activity of plant microsomes.

The NADH-dependent Fe(3+)-chelate reductases of tomato roots.

The NADH-dependent Fe(3+)-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe(3+)-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe(3+)-EDTA reductase assay, which was catalyzed by at least another reductase of 46 kDa. After high-speed centrifugation of tomato root membranes, high FeCitR activities were detected in pellets and lower activities in supernatants. After two-phase partitioning of microsomes, FeCitR activity (91 nmol.min-1.mg-1) was less active in the upper phase (plasma membrane) than in the lower phase (277 nmol.min-1.mg-1). However, only the activity of the plasma-membrane-associated NFCHR (FeCitR) was significantly enhanced (2.6-fold) in iron-deficient tomato plants, whereas that of NFCHR in non-plasma-membrane rich fractions was unaffected by this treatment. The NFCHR obtained from lysophosphatidylcholine-solubilized plasma membrane was present as a 200-kDa protein complex following fast protein liquid chromatography on Superdex 200, or as a 28-kDa form following Blue Sepharose CL-6B chromatography. Both preparations were more active following iron starvation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 28-kDa protein purified from solubilized tomato microsomes or supernatant fractions by a final Mono Q step consisted of a single band of 32 kDa. Tomato root NFCHR resembled the NFCHR of maize (a strategy II plant, P Bagnaresi and P Pupillo, 1995, J Exp Bot 46: 1497-1503) in several properties: relative molecular mass, hydrophilicity, chromatographic behaviour, sensitivity to mercurials, specificity for electron donors and acceptors (e.g. cytochrome c), and a ferricyanide reductase-to-FeCitR ratio of 2.5. Preincubation with NADH partially protected NFCHR from PHMB-induced inactivation. Our data show that strategy I and II plants seem to share similar NFCHR proteins, which appear to belong to the cytochrome b5 reductase flavoprotein group.

The reversible depolymerization of spinach chloroplast glyceraldehyde-phosphate dehydrogenase. Interaction with nucleotides and dithiothreitol.

The ligand-dependent dissociation of spinach chloroplast glyceraldehyde-phosphate dehydrogenase (Mr 600000) to protomers of Mr about 145000, previously shown by us in 1973, has been further characterized by the technique of velocity sedimentation in sucrose gradients. The process exhibits cooperativity and is accompanied by an increase of the apparent NADP+-dependent activity (reactivation) from a ratio of 0.1-0.2 to a ratio of 1 to 2 with respect to the NAD+-dependent activity. In addition to NADP+ and NADPH, most nucleotide triphosphates and, to some extent, Pi, act as dissociating agents. The enzyme is depolymerized and progressively inactivated in the presence of 2'-AMP. 2. Incubation with 20 mM dithiothreitol or 8-10 mM GTP increases the apparent NADP/H)-dependent activity, although addition of a small amount of a dissociating compound, such as 0.06 mM NADP+, is required for depolymerization. 3. NAD+, NADH and, to a lesser extent, glyceraldehyde 3-phosphate, NMN and cyclic AMP act as inhibitors of the dissociation and reactivation, however induced. They also favour the reassociation of protomers to tetramers. 4. The NADP(H)-linked activity is probably a property of the protomers only. The system described here resembles in many respects the light-dependent regulation of the NADP(H)-linked activity in vivo.

A possible plasma membrane particle containing malic enzyme activity.

A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This "malic enzyme particle" is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucrose gradients, in comparison with enzyme markers, and sluggish penetration by the sugar Metrizamide. Enzyme binding to the plasma membrane is stable and scarcely sensitive to salts and EDTA, although all activity is released to the supernatant in the presence of Triton-X-100 or under hypotonic conditions. The properties of bound enzyme are similar to those of free enzyme in cell extracts. It is proposed that osmotically sensitive plasma membrane vesicles, containing cytoplasm fragments, are formed during homogenization. Low malic enzyme activities are also associated with Cucurbita proplastids.

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.

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes.

Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K(m) of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting K(m) of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S(0.5) of about 30 micromolar and production of H(2)O and H(2)O(2), without superoxide involvement. The ratio NAD(P)H:O(2) averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to O(2) without release of intermediates.

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties.

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP(+) to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP(+). The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K(m) of NADP(+) being 2.5-fold higher for pI 4.3 form. Free ATP (K(m) = 0.64 millimolar) and ADP (K(m) = 1.13 millimolar) act as competitive inhibitors with respect to NADP(+) in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

Characterization of a novel NADH-specific, FAD-containing, soluble reductase with ferric citrate reductase activity from maize seedlings.

A novel NADH-dependent, soluble flavoreductase of 60 kDa, active toward ferric chelates and quinones, has been purified from maize seedlings. Two closely related isoforms were separated. The two isoforms are similar in several biochemical features, with the exception of the apparent molecular mass of their subunits (29 and 31 kDa, respectively). They are homodimers in the native state, they bind FAD as the prosthetic group and show strong preference for NADH over NADPH as the electron donor. Ferric chelates (chiefly ferric citrate, Km 3-5 x 10(-5) M; kcat/Km 3.4-3.7 x 10(5) M-1 s-1), and some quinones (benzoquinone, coenzyme Q-0, and juglone) are used as electron acceptors. Enzymatic reduction of benzoquinone occurs with formation of radical semiquinones. Both soluble ferric chelate reductase isoforms are strongly inhibited by p-hydroxymercuribenzoic acid (I50 5 nM) and by cibachron blue, the latter giving nonlinear inhibition. It is suggested that soluble ferric chelate reductase might be involved in the symplastic reduction of iron chelates which is required for the assembly of iron-containing macromolecules such as cytochromes and ferritin.

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.