Ferredoxin-thioredoxin System Research Articles

Inter-Organelle NAD Metabolism Underpinning Light Responsive NADP Dynamics in Plants.

Upon illumination, photosystem I in chloroplasts catalyzes light-driven electron transport from plastocyanin to ferredoxin, followed by the reduction of NADP+ to NADPH by ferredoxin:NADP+ reductase for CO2 fixation. At the beginning of photosynthesis, NADP+ supply control is dominated by de novo NADP+ synthesis rather than being recycled from the Calvin cycle. Importantly, ferredoxin distributes electrons to NADP+ as well as to thioredoxins for light-dependent regulatory mechanisms, to cyclic electron flow for more adenosine triphosphate (ATP) production, and to several metabolites for reductive reactions. We previously demonstrated that the NADP+ synthesis activity and the amount of the NADP pool size, namely the sum of NADP+ and NADPH, varies depending on the light conditions and the ferredoxin-thioredoxin system. In addition, the regulatory mechanism of cytoplasmic NAD+ supply is also involved in the chloroplastic NADP+ supply control because NAD+ is an essential precursor for NADP+ synthesis. In this mini-review, we summarize the most recent advances on our understanding of the regulatory mechanisms of NADP+ production, focusing on the interactions, crosstalk, and co-regulation between chloroplasts and the cytoplasm at the level of NAD+ metabolism and molecular transport.

Overexpression of thioredoxin m in tobacco chloroplasts inhibits the protein kinase STN7 and alters photosynthetic performance.

The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.

Malate valves: old shuttles with new perspectives.

Malate valves act as powerful systems for balancing the ATP/NAD(P)H ratio required in various subcellular compartments in plant cells. As components of malate valves, isoforms of malate dehydrogenases (MDHs) and dicarboxylate translocators catalyse the reversible interconversion of malate and oxaloacetate and their transport. Depending on the co‐enzyme specificity of the MDH isoforms, either NADH or NADPH can be transported indirectly. Arabidopsis thaliana possesses nine genes encoding MDH isoenzymes. Activities of NAD‐dependent MDHs have been detected in mitochondria, peroxisomes, cytosol and plastids. In addition, chloroplasts possess a NADP‐dependent MDH isoform. The NADP‐MDH as part of the ‘light malate valve’ plays an important role as a poising mechanism to adjust the ATP/NADPH ratio in the stroma. Its activity is strictly regulated by post‐translational redox‐modification mediated via the ferredoxin‐thioredoxin system and fine control via the NADP +/NADP(H) ratio, thereby maintaining redox homeostasis under changing conditions. In contrast, the plastid NAD‐MDH (‘dark malate valve’) is constitutively active and its lack leads to failure in early embryo development. While redox regulation of the main cytosolic MDH isoform has been shown, knowledge about regulation of the other two cytosolic MDHs as well as NAD‐MDH isoforms from peroxisomes and mitochondria is still lacking. Knockout mutants lacking the isoforms from chloroplasts, mitochondria and peroxisomes have been characterised, but not much is known about cytosolic NAD‐MDH isoforms and their role in planta. This review updates the current knowledge on MDH isoforms and the shuttle systems for intercompartmental dicarboxylate exchange, focusing on the various metabolic functions of these valves.

Multi-level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance.

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ-subunit through the ferredoxin-thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild-type levels as irradiance was increased. This effect was caused by an altered redox state of the γ-subunit under low, but not high, light. The low light-specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non-photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin-thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.

Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.

Two different thiol redox systems exist in plant chloroplasts, the ferredoxin-thioredoxin (Trx) system, which depends on ferredoxin reduced by the photosynthetic electron transport chain and, thus, on light, and the NADPH-dependent Trx reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested, therefore, that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of Trx f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis (Arabidopsis thaliana) mutants, combined, but not single, deficiencies of Trx f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson cycle activity and starch accumulation. Light activation of key enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH-NADP(+) and ATP-ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation, and light vulnerability of photosystem I core proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both colocalized in the same chloroplast substructure. Results provide genetic evidence that light- and NADPH-dependent thiol redox systems interact at the level of Trx f1 and NTRC to coordinately participate in the regulation of the Calvin-Benson cycle, starch metabolism, and growth in response to varying light conditions.

Inhibition of CO2 fixation by iodoacetamide stimulates cyclic electron flow and non-photochemical quenching upon far-red illumination

The Benson-Calvin cycle enzymes are activated in vivo when disulfide bonds are opened by reduction via the ferredoxin-thioredoxin system in chloroplasts. Iodoacetamide reacts irreversibly with free -SH groups of cysteine residues and inhibits the enzymes responsible for CO2 fixation. Here, we investigate the effect of iodoacetamide on electron transport, when infiltrated into spinach leaves. Using fluorescence and absorption spectroscopy, we show that (i) iodoacetamide very efficiently blocks linear electron flow upon illumination of both photosystems (decrease in the photochemical yield of photosystem II) and (ii) iodoacetamide favors cyclic electron flow upon light excitation specific to PSI. These effects account for an NPQ formation even faster in iodoacetamide under far-red illumination than in the control under saturating light. Such an increase in NPQ is dependent upon the proton gradient across the thylakoid membrane (uncoupled by nigericin addition) and PGR5 (absent in Arabidopsis pgr5 mutant). Iodoacetamide very tightly insulates the electron current at the level of the thylakoid membrane from any electron leaks toward carbon metabolism, therefore, providing choice conditions for the study of cyclic electron flow around PSI.

Analysis of the Chloroplast Protein Kinase Stt7 during State Transitions

State transitions allow for the balancing of the light excitation energy between photosystem I and photosystem II and for optimal photosynthetic activity when photosynthetic organisms are subjected to changing light conditions. This process is regulated by the redox state of the plastoquinone pool through the Stt7/STN7 protein kinase required for phosphorylation of the light-harvesting complex LHCII and for the reversible displacement of the mobile LHCII between the photosystems. We show that Stt7 is associated with photosynthetic complexes including LHCII, photosystem I, and the cytochrome b 6 f complex. Our data reveal that Stt7 acts in catalytic amounts. We also provide evidence that Stt7 contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved Cys that are critical for its activity and state transitions. On the basis of these data, we propose that the activity of Stt7 is regulated through its transmembrane domain and that a disulfide bond between the two lumen Cys is essential for its activity. The high-light–induced reduction of this bond may occur through a transthylakoid thiol–reducing pathway driven by the ferredoxin-thioredoxin system which is also required for cytochrome b 6 f assembly and heme biogenesis.

Photosynthetic Regulation of the Cyanobacterium Synechocystis sp. PCC 6803 Thioredoxin System and Functional Analysis of TrxB (Trx x) and TrxQ (Trx y) Thioredoxins

The expression of the genes encoding the ferredoxin-thioredoxin system including the ferredoxin-thioredoxin reductase (FTR) genes ftrC and ftrV and the four different thioredoxin genes trxA (m-type; slr0623), trxB (x-type; slr1139), trxC (sll1057) and trxQ (y-type; slr0233) of the cyanobacterium Synechocystis sp. PCC 6803 has been studied according to changes in the photosynthetic conditions. Experiments of light-dark transition indicate that the expression of all these genes except trxQ decreases in the dark in the absence of glucose in the growth medium. The use of two electron transport inhibitors, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), reveals a differential effect on thioredoxin genes expression being trxC and trxQ almost unaffected, whereas trxA, trxB, and the ftr genes are down-regulated. In the presence of glucose, DCMU does not affect gene expression but DBMIB still does. Analysis of the single TrxB or TrxQ and the double TrxB TrxQ Synechocystis mutant strains reveal different functions for each of these thioredoxins under different growth conditions. Finally, a Synechocystis strain was generated containing a mutated version of TrxB (TrxBC34S), which was used to identify the potential in-vivo targets of this thioredoxin by a proteomic analysis.

C3 photosynthesis in silico

A computer model comprising light reactions, electron-proton transport, enzymatic reactions, and regulatory functions of C3 photosynthesis has been developed as a system of differential budget equations for intermediate compounds. The emphasis is on electron transport through PSII and PSI and on the modeling of Chl fluorescence and 810 nm absorptance signals. Non-photochemical quenching of PSII excitation is controlled by lumenal pH. Alternative electron transport is modeled as the Mehler type O2 reduction plus the malate-oxaloacetate shuttle based on the chloroplast malate dehydrogenase. Carbon reduction enzymes are redox-controlled by the ferredoxin-thioredoxin system, sucrose synthesis is controlled by the fructose 2,6-bisphosphate inhibition of cytosolic FBPase, and starch synthesis is controlled by ADP-glucose pyrophosphorylase. Photorespiratory glycolate pathway is included in an integrated way, sufficient to reproduce steady-state rates of photorespiration. Rate-equations are designed on principles of multisubstrate-multiproduct enzyme kinetics. The parameters of the model were adopted from literature or were estimated from fitting the photosynthetic rate and pool sizes to experimental data. The model provided good simulations for steady-state photosynthesis, Chl fluorescence, and 810 nm transmittance signals under varying light, CO2 and O2 concentrations, as well as for the transients of post-illumination CO2 uptake, Chl fluorescence induction and the 810 nm signal. The modeling shows that the present understanding of photosynthesis incorporated in the model is basically correct, but still insufficient to reproduce the dark-light induction of photosynthesis, the time kinetics of non-photochemical quenching, 'photosynthetic control' of plastoquinone oxidation, cyclic electron flow around PSI, oscillations in photosynthesis. The model may find application for predicting the results of gene transformations, the analysis of kinetic experimental data, the training of students.

Ferredoxin-Thioredoxin System Plays a Key Role in Plant Response to Oxidative Stress

Ferredoxin (Fd) plays a central role in the physiology of the plant cell, distributing the reducing equivalents generated during photosynthetic electron transport to the electron-consuming reactions of the chloroplast. Fd can also function to eliminate excess reducing power and prevent uncontrolled

Redox regulation of carbon storage and partitioning in response to light and sugars

Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.

Molecular characterization and redox regulation of phosphoribulokinase from the cyanobacterium Synechococcus sp. PCC 7942.

We isolated and characterized a gene encoding phosphoribulokinase (PRK) from Synechococcus sp. PCC 7942. The isolated sequence consisted of a 999 bp open reading frame encoding 333 amino acid residues of PRK. The PRK contained a pair of cysteinyl residues corresponding to Cys16 and Cys55 of spinach PRK regulated by a ferredoxin-thioredoxin system. However, there were seventeen amino acid residues lacking between the two cysteinyl residues compared with those of the chloroplastic enzyme in higher plants. The recombinant PRK of Synechococcus sp. PCC 7942 accounted for about 6-13% of the total soluble protein in the Escherichia coli. The specific activity of the enzyme was 230 micro mol min(-1) (mg protein)(-1). The enzyme activity was completely inactivated by treatment with 5,5'-dithiobis (2-nitrobenzoic acid) (cysteinyl residue-specific oxidant) or was decreased by treatment with H(2)O(2), but was more tolerant to oxidation than that of chloroplast. The oxidized PRK was fully activated by treatment with excessive dithiothreitol. Furthermore, incubation with 3 mM ATP protected the oxidation of the enzyme by either 5,5'-dithiobis (2-nitrobenzoic acid) or H(2)O(2). These results suggest Synechococcus sp. PCC 7942 PRK can be regulated by reversible oxidation/reduction in vitro, but might be resistant to oxidative inactivation in vivo.

Translation of chloroplast psbA mRNA is regulated by signals initiated by both photosystems II and I.

Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intrachloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic psbA mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast psbA mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin-thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.

Complex regulation of Photosystem II protein phosphorylation via redox state of chloroplast

Phosphorylation of photosystem II (PSII) core and light harvesting II complex (LHCII) proteins is regulated in vivo by complex redox systems involving reduction of plastoquinone and cytochrome b6f complex as well as thiol redox state in chloroplasts. This redox regulation is universal in all tested plant species including a model plant, Arabidopsis thaliana. CP43 protein was highly phosphorylated in darkness in several plant species, but some phosphorylated D1 and D2 proteins may also be detected in dark-adapted plants. The shift of leaves from darkness to very dim light best induced dephosphorylation of PSII core phosphoproteins, while further increase in irradiance up to the growth level significantly increased the number of PSII centres with phosphorylated core proteins. Both in vitro and in vivo experiments indicated that the kinase(s) for PSII core phosphoproteins is regulated via reduction of plastoquinone pool that strongly depends on metabolic state of plant leaf. Redox regulation of LHCII protein phosphorylation differs from that of PSII core proteins. The LHCII kinase is activated via reduction of plastoquinone and the cytochrome b6f complex. High redox state of chloroplast, on the other hand, inactivates the phosphorylation of LHCII, disclosing a second regulatory mechanism for LHCII phosphorylation. This novel regulation of LHCII phosphorylation is likely to be mediated by the ferredoxin-thioredoxin system. A cooperative model for the function of these two regulation mechanisms that determine the phosphorylation level of the LHCII proteins in vivo will be presented.

Cooperative regulation of light-harvesting complex II phosphorylation via the plastoquinol and ferredoxin-thioredoxin system in chloroplasts.

Light induces phosphorylation of photosystem II (PSII) proteins in chloroplasts by activating the protein kinase(s) via reduction of plastoquinone and the cytochrome b(6)f complex. The recent finding of high-light-induced inactivation of the phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the PSII antenna in floated leaf discs, but not in vitro, disclosed a second regulatory mechanism for LHCII phosphorylation. Here we show that this regulation of LHCII phosphorylation is likely to be mediated by the chloroplast ferredoxin-thioredoxin system. We present a cooperative model for the function of the two regulation mechanisms that determine the phosphorylation level of the LHCII proteins in vivo, based on the following results: (i) Chloroplast thioredoxins f and m efficiently inhibit LHCII phosphorylation. (ii) A disulfide bond in the LHCII kinase, rather than in its substrate, may be a target component regulated by thioredoxin. (iii) The target disulfide bond in inactive LHCII kinase from dark-adapted leaves is exposed and easily reduced by external thiol mediators, whereas in the activated LHCII kinase the regulatory disulfide bond is hidden. This finding suggests that the activation of the kinase induces a conformational change in the enzyme. The active state of LHCII kinase prevails in chloroplasts under low-light conditions, inducing maximal phosphorylation of LHCII proteins in vivo. (iv) Upon high-light illumination of leaves, the target disulfide bond becomes exposed and thus is made available for reduction by thioredoxin, resulting in a stable inactivation of LHCII kinase.

Translation of chloroplast psbA mRNA is modulated in the light by counteracting oxidizing and reducing activities.

Light has been proposed to stimulate the translation of Chlamydomonas reinhardtii chloroplast psbA mRNA by activating a protein complex associated with the 5' untranslated region of this mRNA. The protein complex contains a redox-active regulatory site responsive to thioredoxin. We identified RB60, a protein disulfide isomerase-like member of the protein complex, as carrying the redox-active regulatory site composed of vicinal dithiol. We assayed in parallel the redox state of RB60 and translation of psbA mRNA in intact chloroplasts. Light activated the specific oxidation of RB60, on the one hand, and reduced RB60, probably via the ferredoxin-thioredoxin system, on the other. Higher light intensities increased the pool of reduced RB60 and the rate of psbA mRNA translation, suggesting that a counterbalanced action of reducing and oxidizing activities modulates the translation of psbA mRNA in parallel with fluctuating light intensities. In the dark, chemical reduction of the vicinal dithiol site did not activate translation. These results suggest a mechanism by which light primes redox-regulated translation by an unknown mechanism and then the rate of translation is determined by the reduction-oxidation of a sensor protein located in a complex bound to the 5' untranslated region of the chloroplast mRNA.

Activation of the Potato Tuber ADP-glucose Pyrophosphorylase by Thioredoxin

The potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, P(i). It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045-25052) that the enzyme was activated by reduction of the Cys(12) disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 microM) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 microM for reduced thioredoxin f and m, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys(12) is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch.

Thylakoid protein phosphorylation and the thiol redox state.

Illumination of thylakoid membranes leads to the phosphorylation of a number of photosystem II-related proteins, including the reaction center proteins D1 and D2 as well as the light-harvesting complex (LHCII). Regulation of light-activated thylakoid protein phosphorylation has mainly been ascribed to the redox state of the electron carrier plastoquinone. In this work, we show that this phosphorylation in vitro is also strongly influenced by the thiol disulfide redox state. Phosphorylation of the light-harvesting complex of photosystem II was found to be favored by thiol-oxidizing conditions and strongly downregulated at moderately thiol-reducing conditions. In contrast, phosphorylation of the photosystem II reaction center proteins D1 and D2 as well as that of other photosystem II subunits was found to be stimulated up to 2-fold by moderately thiol-reducing conditions and kept at a high level also at highly reducing conditions. These responses of the level of thylakoid protein phosphorylation to changes in the thiol disulfide redox state are reminiscent of those observed in vivo in response to changes in the light intensity and point to the possibility of a second loop of redox regulation of thylakoid protein phosphorylation via the ferredoxin-thioredoxin system.

Roles and Microenvironments of Tryptophanyl Residues of Spinach Phosphoribulokinase

Phosphoribulokinase is one of several Calvin cycle enzymes that are light-regulated via the ferredoxin–thioredoxin system (R. A. Wolosiuk and B. B. Buchanan, 1978,Arch. Biochem. Biophys.189, 97–101). Substitution of the only two Trp residues of the enzyme was prompted by the following goals: to identify each tryptophanyl residue with respect to prior classifications as exposed and buried (C. A. Ghironet al.,1988,Arch. Biochem. Biophys.260, 267–272); to explore the possible active-site location and function of conserved Trp155, as suggested by sequence proximity to catalytic Asp160 (H. A. Charlieret al.,1994,Biochemistry33, 9343–9350); and to determine if fluorescence of a Trp residue can serve as a gauge of conformational differences between the reduced (active) and the oxidized (inactive) forms of the enzyme. Emission spectra and acrylamide quenching data demonstrate that Trp155 is solvent exposed, while Trp241 is buried. Kinetic parameters of the W241F mutant are not significantly altered relative to those of wild-type enzyme, thereby discounting any requirement for Trp at position 241. While substitution of Trp155 with Phe or Ala has little impact onVmax, theKmfor Ru5P and ATP are increased substantially; the diminished affinity for ATP is particularly pronounced in the case of the Ala substitution. In further support of an active-site location of Trp155, its fluorescence emission is subject to quenching by nucleotides. Fluorescence quenching of reduced W241F by ATP gives a dissociation constant (Kd) of 37 μM, virtually identical with itsKmof 46 μM, and provides for the first time a direct measurement of the interaction of the kinase with product ADP (Kdof 1.3 mM). Fluorescence quenching of oxidized W241F by ATP reveals aKdof 28 mM; however, this weakened binding does not reflect an altered microenvironment of Trp155, as its steady-state emission and fluorescence lifetimes are unaffected by the oxidation state.

Cysteine-153 is required for redox regulation of pea chloroplast fructose-1,6-bisphosphatase

Chloroplastic fructose-1,6-bisphosphatases are redox regulatory enzymes which are activated by the ferredoxin thioredoxin system via the reduction/isomerization of a critical disulfide bridge. All chloroplastic sequences contain seven cysteine residues, four of which are located in, or close to, an amino acid insertion region of approximately 17 amino acids. In order to gain more information on the nature of the regulatory site, five cysteine residues (Cys 49, Cys 153, Cys 173, Cys 178 and Cys 190) have been modified individually into serine residues by site-directed mutagenesis. While mutations C173S and C178S strongly affected the redox regulatory properties of the enzyme, the most striking effect was observed with the C153S mutant which became permanently active and redox independent. On the other hand, the C190S mutant retained most of the properties of the wild-type enzyme (except that it could now also be partially activated by the NADPH/NTR/thioredoxin h system). Finally, the C49S mutant is essentially identical to the wild-type enzyme. These results are discussed in the light of recent crystallographic data obtained on spinach FBPase [Villeret et al. (1995) Biochemistry 34, 4299–4306].