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In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP + reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.0), NaCl concentrations (0-200 mM), and temperatures (19-28 °C) for mimicking physiological conditions in chloroplasts. Energetically unfavorable endothermic enthalpy changes were accompanied by Fd:FNR complexation at all conditions. This energetic cost was compensated by favorable entropy changes, balanced by conformational and hydrational entropy. Increases in the NaCl concentration and pH weakened interprotein affinity due to the less contribution of favorable entropy change regardless of energetic gains from enthalpy changes, suggesting that entropy drove complexation and modulated affinity. Effects of temperature on binding thermodynamics were much smaller than those of pH and NaCl. NaCl concentration and pH-dependent enthalpy and heat capacity changes provided clues for distinct binding modes. Moreover, decreases in the enthalpy level in the Hammond's postulate-based energy landscape implicated kinetic advantages for FNR activity. All these energetic interplays were comprehensively demonstrated by the driving force plot with the enthalpy-entropy compensation which may serve as an energetic buffer against outer stresses. We propose that high affinity at pH 6.0 may be beneficial for protection from proteolysis of Fd and FNR in rest states, and moderate affinity at pH 8.0 and proper NaCl concentrations with smaller endothermic enthalpy changes may contribute to increase FNR activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å. Mutational analysis of a total of seven basic residues of SiR distributed widely at the interface of the complex showed their importance for supporting an efficient Fd-dependent activity and a strong physical binding to Fd. These combined results suggest that the productive electron transfer complex of SiR and Fd could be formed through multiple processes of the electrostatic intermolecular interaction and this implication is discussed in terms of the multi-functionality of Fd in various redox metabolisms., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

It is known that plants contain ferredoxin (Fd)-dependent nitrite reductase (NiR) and glutamate synthase (GOGAT). The Fd-NiR reaction produces ammonia from nitrite, and the activity is usually measured by nitrite disappearance. The Fd-GOGAT reaction forms two glutamates of different origin, from glutamine and 2-oxoglutarate, and the activity is measured by the oxidation of reductant (NADPH) or by formation of total glutamate. Here, a quantitative probe of the products and efficiency of the process was conducted using (15)N tracing techniques on these reactions in vitro. We quantified the reduction of (15)N-labeled [Formula: see text] to [Formula: see text] and the formation of [(15)N]glutamate and [(14)N]glutamate from [5-(15)N-amide]glutamine plus 2-oxoglutarate by NiR and GOGAT, respectively, with the reductant-Fd-NADP(+) oxidoreductase (FNR)-Fd system as the sequential electron donors. The supply of dithionite or NADPH to recombinant cyanobacterial NiR led to electron donation system-dependent formation of [(15)N]ammonium from [(15)N]nitrite. Addition of 20 mM NaCl and 20 mM Na-ascorbate accelerated nitrite reduction under high concentrations of NADPH. A sufficient supply of NADPH to recombinant Zea mays Fd-GOGAT generated complete GOGAT activity (transferring the [5-(15)N]amide of glutamine to 2-oxoglutarate to form [(15)N]glutamate), whereas a shortage of NADPH resulted in glutaminase activity only, which removed the amide from glutamine and released ammonia and [(14)N]glutamate. We conclude that although the recombinant Fd-GOGAT enzyme has two forms of glutamate synthesis, the first by glutaminase (ammonia release by glutamine amidotransferase) and the second by glutamate synthase (coupling of the ammonia and exogenously applied 2-oxoglutarate), the first works without NADPH, while the second is strictly dependent on NADPH availability., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys(83) on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg(72) and Tyr(71). Removal of a positive charge at Arg(72) did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr(71) with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr(71) and Arg(72) with ascorbate. However, their mechanistic roles were distinctly different from that of Lys(83), as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

Ferredoxin (Fd), which plays a pivotal role in photosynthesis as an electron carrier, forms a transient complex with various Fd-dependent enzymes, such as nitrite reductase (NiR), to achieve efficient intermolecular electron transfer. We studied the protein-protein interaction of Fd and NiR by NMR spectroscopy and determined three acidic regions of Fd to be major sites for the interaction with NiR, indicating that the complex is stabilized through electrostatic interaction. During this study, we found Fds from higher plant and cyanobacterium, in spite of their high structural similarities including the above acidic regions, differ remarkably in the interaction with cyanobacterial NiR. In activity assay of NiR, K(m) value for maize Fd (74.6 µM) was 9.6 times larger than that for Leptolyngbya boryana Fd (7.8 µM). The two Fds also showed a similar difference in binding assay to NiR-immobilized resin. Comparative site-specific mutagenesis of two Fds revealed that their discriminative ability for the interaction with NiR is attributed mainly to non-charged residues in the peripheral region of [2Fe-2S] cluster. These non-charged residues are conserved separately between Fds of plant and cyanobacterial origins. Our data highlight that intermolecular force(s) other than electrostatic attraction is(are) also crucial for the molecular interaction between Fd and partner enzyme.

The aim of the present study was to investigate the effects of hydroxypropyl-distarch phosphate (HDP) supplementation on postprandial energy metabolism and glucose-dependent insulinotropic polypeptide (GIP) in human subjects. A total of ten healthy male subjects, with a mean BMI of 23·6 (SEM 1·3) kg/m(2), age 35·2 (SEM 1·9) years and body weight 71·1 (SEM 4·0) kg, participated in a randomised, cross-over, intervention study with two different test meals (1673·6 kJ) containing either waxy maize starch or HDP from waxy maize starch (degree of substitution 0·154, P content 0·004 %). Resting energy expenditure (REE) and blood concentrations of various biomarkers were measured at fasting and up to 180 min postprandially. Indirect calorimetry showed that the HDP meal caused higher REE (P < 0·05) and fat utilisation (P < 0·001) than the waxy maize starch meal. The HDP meal led to significantly lower postprandial glucose (P < 0·05), insulin (P < 0·05) and GIP (P < 0·05) responses than the waxy maize starch meal. Both postprandial REE (R - 0·576, P < 0·01) and fat utilisation (R - 0·514, P < 0·05) were negatively correlated with the postprandial GIP response, but not with the glucose and insulin responses. In conclusion, dietary supplementation with HDP lowers postprandial GIP and increases postprandial REE and fat utilisation in healthy humans. An HDP-rich diet may therefore have beneficial implications in weight management. Further studies are required to confirm the efficacy in overweight or obese subjects, and to determine the precise mechanisms.

Cytochromes b(561), a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic transmembrane alpha-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b(561) obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z. mays cytochrome b(561) as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys(83) residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys(83) might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser(118) and Trp(122) do not have major roles for the redox events on the intravesicular side.

We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADH-GOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L. The plants responded to a light/dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ. The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase. We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP(+) oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor. The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf. The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates. Taken together, we conclude that the GS1/ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization.

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (Em) values of the siroheme and [4Fe-4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.

In higher plants, ferredoxin (Fd):NADPH oxidoreductase (FNR) catalyzes reduction of NADP+ in the final step of linear photosynthetic electron transport and is also implicated in cyclic electron flow. We have identified three leaf FNR isoenzymes (LFNR1, LFNR2, and LFNR3) in maize (Zea mays) chloroplasts at approximately equivalent concentrations. Fractionation of chloroplasts showed that, while LFNR3 is an exclusively soluble enzyme, LFNR1 is only found at the thylakoid membrane and LFNR2 has a dual location. LFNR1 and LFNR2 were found to associate with the cytochrome b6f complex following its partial purification. We cloned LFNR3 and produced all three isoenzymes as stable, soluble proteins. Measurement of Fd reduction ability showed no significant differences between these recombinant enzymes. Column chromatography revealed variation between the interaction mechanisms of LFNR1 and LFNR2 with Fd, as detected by differential dependence on specific intermolecular salt bridges and variable sensitivity of interactions to changes in pH. A comparison of LFNR transcripts in leaves of plants grown on variable nitrogen regimes revealed that LFNR1 and LFNR2 transcripts are relatively more abundant under conditions of high demand for NADPH. These results are discussed in terms of the functional differentiation of maize LFNR isoenzymes.

Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E(m) values have been determined for the siroheme and [4Fe-4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe-4S] cluster significantly perturbs the spectrum of the reduced siroheme group of the enzyme. The effects of siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined.

Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.

All oxygenic photosynthetically derived reducing equivalents are utilized by combinations of a single multifuctional electron carrier protein, ferredoxin (Fd), and several Fd-dependent oxidoreductases. We report the first crystal structure of the complex between maize leaf Fd and Fd-NADP(+) oxidoreductase (FNR). The redox centers in the complex--the 2Fe-2S cluster of Fd and flavin adenine dinucleotide (FAD) of FNR--are in close proximity; the shortest distance is 6.0 A. The intermolecular interactions in the complex are mainly electrostatic, occurring through salt bridges, and the interface near the prosthetic groups is hydrophobic. NMR experiments on the complex in solution confirmed the FNR recognition sites on Fd that are identified in the crystal structure. Interestingly, the structures of Fd and FNR in the complex and in the free state differ in several ways. For example, in the active site of FNR, Fd binding induces the formation of a new hydrogen bond between side chains of Glu 312 and Ser 96 of FNR. We propose that this type of molecular communication not only determines the optimal orientation of the two proteins for electron transfer, but also contributes to the modulation of the enzymatic properties of FNR.

In the C(4) plant maize (Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle-sheath cells, respectively. cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2-9 strains expressing Fd I and Fd II. The growth of TM202 was retarded under high light (130 micromol/m(2)/s), whereas KM2-9 grew at a normal rate but exhibited a nitrogen-deficient phenotype. Measurement of photosynthetic O(2) evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2-9. After starvation of the cells in darkness, the P700 oxidation level under far-red illumination increased significantly in TM202. However, it remained low in KM2-9, indicating an active cyclic electron flow. In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP(+) decreased in KM2-9 as compared with TM202. These results demonstrated that the two cell type-specific ferredoxins differentially modulate electron flow around photosystem I.

In higher plants ferredoxin (Fd):NADP(+) oxidoreductase (FNR) and Fd are each distributed in photosynthetic and non-photosynthetic organs as distinct isoproteins. We have cloned cDNAs for leaf FNR (L-FNR I and L-FNR II) and root FNR (R-FNR) from maize (Zea mays L.), and produced recombinant L-FNR I and R-FNR to study their enzymatic functions through kinetic and Fd-binding analyses. The K(m) value obtained by assay for a diaphorase activity indicated that R-FNR had a 10-fold higher affinity for NADPH than L-FNR I. When we assayed for NADPH-cytochrome c reductase activity using maize photosynthetic Fd (Fd I) and non-photosynthetic Fd (Fd III), the R-FNR showed a marked difference in affinity between these two Fd isoproteins; the K(m) for Fd III was 3.0 microM and that for Fd I was 29 microM. Consistent with this, the dissociation constant for the R-FNR:Fd III complex was 10-fold smaller than that of the R-FNR:Fd I complex. This differential binding capacity was confirmed by an affinity chromatography of R-FNR on Fd-sepharose with stronger binding to Fd III. L-FNR I showed no such differential interaction with Fd I and Fd III. These data demonstrated that R-FNR has the ability to discriminate between these two types of Fds. We propose that the stronger interaction of R-FNR with Fd III is crucial for an efficient electron flux of NADPH-FNR-Fd cascade, thus supporting Fd-dependent metabolism in non-photosynthetic organs.

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor. Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems. Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots. The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively. The molar ratio of Fd III to SiR in root plastids was approximately 3:1. Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR. In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I. Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested. NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner. These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction. In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of -423 and -406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

A genomic clone encoding the precursor of a non-photosynthetic ferredoxin (Fd III) from maize has been isolated and characterized. In comparison with the corresponding cDNA, the gene (fedIII) was found to be interrupted in the 5'-untranslated region by an intron consisting of 3,037 bp. About 80% of the total region of this intron is organized into four classes of tandemly repeated sequences with monomeric lengths of about 200 bp, 320 bp, 130 bp, and 90 bp. This unique intron organization is present in the fedIII gene of three related maize cultivars examined.

We have identified and characterized a nitrate-inducible ferredoxin (Fd) in maize (Zea mays L.) roots by structural analysis of the purified protein and by cloning of its cDNA and gene. In maize Fd isoproteins are encoded by a small multigene family, and the nitrate-inducible Fd was identified as a novel isoprotein, designated Fd VI, which differed from an Fd I to Fd V identified to date. In the roots of seedlings cultured without nitrate, Fd VI was undetectable. However, during the induction of the capacity for nitrate assimilation, the amount of Fd VI increased markedly within 24 h. Concurrently, the level of transcript for Fd VI increased, but more quickly, reaching a maximal level within 2 h with kinetics similar to those of nitrite reductase and Fd-NADP+ reductase. Fd III was constitutively expressed in roots, and no such changes at the protein and mRNA levels were observed during the nitrate induction. In the 5' flanking region of the gene for Fd VI only, we identified NIT-2 motifs, which are widely found in genes for enzymes related to nitrogen metabolism. These data indicate that Fd VI is co-induced with the previously characterized enzymes involved in nitrate assimilation, and they suggest that the novel Fd isoprotein, distinct from the constitutively expressed Fd, might play an important role as an electron carrier from NADPH to nitrite reductase and other Fd-dependent enzymes in root plastids.

In maize, a small multigene family encodes the cytosolic isoforms of glutamine synthetase (GS), and five cDNAs, designated pGS1a, pGS1b, pGS1c, pGS1d, and pGS1e, have been cloned (Sakakibara, H., Kawabata, S., Takahashi, H., Hase, T., and Sugiyama, T. (1992) Plant Cell Physiol. 33, 49-58; Li, M., Villemur, R., Hussey, P. J., Silflow, C. D., Gantt, J. S., and Snustad, D. P. (1993) Plant Mol. Biol. 23, 401-407). This report describes the identification and enzymatic characterization of the cytosolic isoforms of GS in maize roots, namely GS1 and GSr. The purified isoforms, as well as recombinant enzymes that had been overexpressed in Escherichia coli, were analyzed by capillary liquid chromatography/electrospray ionization-mass spectrometry, and GS1 and GSr were identified as the products of the GS1a/GS1b and GS1c/GS1d genes, respectively. Upon the addition of ammonia to the culture medium, significant amounts of GSr accumulated and a preferential increase in GS synthetase activity, as compared to GS transferase activity, was found in the root extract. Assays with the purified recombinant enzymes confirmed that the specific biosynthetic and synthetase activities of GSr were 1.6-fold higher than those of GS1. Marked differences in stability were also found between the two isoforms: GSr was more sensitive to heat than GS1 and octameric aggregates of the subunits of GSr were easily dissociated to monomers than those of GS1 at low concentrations of Mn2+ and Mg2+ ions. These characteristics of the ammonia-induced isoform of GS seem to be physiologically important for the primary assimilation of external ammonia by roots.

The sequence of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mRNA from maize has been determined. Complementary DNAs were isolated from a cDNA library of light-induced leaf poly(A)+ RNA constructed in an expression vector. An open reading frame beginning at an ATG codon at nucleotide 328 of the longest cDNA (5617-bases long) encoded 1616 amino acid residues. The amino terminus of the purified mature enzyme coincided with the cysteine residue at position 98 of the predicted sequence. This enzyme is homologous with the large subunit of Escherichia coli NADPH-dependent glutamate synthase having about 42% identical residues between the two proteins. The enzyme also contains a short region similar to a potential FMN-binding region of yeast flavocytochrome b2. The cDNA hybridizes to an RNA band about 5.5 kilobases whose steady-state level is markedly increased upon illumination of etiolated maize seedlings. Analysis of genomic DNA indicates the presence of a single-copy gene for ferredoxin glutamate synthase in maize.