Fluorescence spectroscopy has contributed to advances in biochemistry that we now take for granted. Many biological and chemical processes of great importance in both nature and technology were uncovered using this versatile technique. The finding of the jellyfish Aequorea victoria green fluorescent protein (GFP) has revolutionized cell labeling and molecular tagging (1). In the few years since its discovery, GFP has become a reporter for gene expression, protein localization, and protein dynamics in living cells. Given that we have learned much about a plethora of biological events using this green glowing marker, the Nobel Prize in Chemistry given in 2008 to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien rewarded their seminal research. Further developments in molecular biology led to proteins that glow cyan, blue, and yellow. Remarkably, many events in living cells are followed in real time because the chemical environment modulates the fluorescence of genetically encoded GFPs. On this basis, Sugiura et al. (2) now estimate intracellular redox changes via a variant of GFP prepared by the rational design of the GFP mechanism, an important aspect of cell metabolism that pervades a wide range of biological events, such as photosynthesis in plants and cancer in humans. In the present study, Sugiura et al. (2) describe a GFP-based redox sensor, FROG/B, with an absorption peak around 400 nm, but more importantly, an emission spectrum sensitive to the redox state of the molecule. The relevant feature of FROG/B is that the maturation level of the chromophore does not alter the signal of the sensor because one chromophore perceives redox changes. Although currently many other genetically encoded GFP-based indicators report on different cellular events, even the redox status (e.g., rxYFP and roGFPs) (3), FROG/B can be used to monitor the redox status in cell populations containing … [↵][1]1Email: rwolosiuk{at}leloir.org.ar. [1]: #xref-corresp-1-1

AbstractThe sections in this article areIntroductionThe Reactivity of the Sulfhydryl GroupProtein‐Disulfide Oxido‐ReductasesThioredoxinsGlutaredoxinsProtein‐Disulfide IsomerasesConcluding RemarksAcknowledgements

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous enzymes that have been implicated in peroxide-mediated signaling of markedly different processes, such as cancer and photosynthesis. A highly conserved C-terminal extension of eukaryotic homologues modulates both the overoxidation of cysteines and the formation of oligomers. Here, we reveal that the plant counterpart regulates the self-polymerization of 2-Cys Prx triggered by ATP and Mg(2+). This feature is of particular importance under oxidative stress because the interaction of ATP with 2-Cys Prx rapidly integrates nonredox chemistry of signaling pathways into a network hub governed by multiple redox transformations at cysteine residues.

2-Cys peroxiredoxins (2-Cys Prxs) are ubiquitous peroxidases with important roles in cellular antioxidant defense and hydrogen peroxide-mediated signaling. Post-translational modifications of conserved cysteines cause the transition from low to high molecular weight oligomers, triggering the functional change from peroxidase to molecular chaperone. However, it remains unclear how non-covalent interactions of 2-Cys Prx with metabolites modulate the quaternary structure. Here, we disclose that ATP and Mg2+ (ATP/Mg) promote the self-polymerization of chloroplast 2-Cys Prx (polypeptide 23.5 kDa) into soluble higher order assemblies (> 2 MDa) that proceed to insoluble aggregates beyond 5mM ATP. Remarkably, the withdrawal of ATP or Mg2+ brings soluble oligomers and insoluble aggregates back to the native conformation without compromising the associated functions. As confirmed by transmission electron microscopy, ATP/Mg drive the toroid-like decamers (diameter 13 nm) to the formation of large sphere-like particles (diameter similar to 30 nm). Circular dichroism studies on ATP-labeled 2-Cys Prx reveal that ATP/Mg enhance the proportion of beta-sheets with the concurrent decrease in the content of alpha-helices. In line with this observation, the formation of insoluble aggregates is strongly prevented by 2,2,2-trifluoroethanol, a cosolvent employed to induce alpha-helical conformations. We further find that the response of self-polymerization to ATP/Mg departs abruptly from that of the associated peroxidase and chaperone activities when two highly conserved residues, Arg(129) and Arg(152), are mutated. Collectively, our data uncover that non-covalent interactions of ATP/Mg with 2-Cys Prx modulate dynamically the quaternary structure, thereby coupling the non-redox chemistry of cell energy with redox transformations at cysteine residues.

Resumen es: La formacion, escision e isomerizacion de los puentes disulfuro juega un rol importante en la estructura y la funcion de las proteinas. Sin embargo, las ...

2‐Cys peroxiredoxins are peroxidases devoid of prosthetic groups that mediate in the defence against oxidative stress and the peroxide activation of signaling pathways. This dual capacity relies on the high reactivity of the conserved peroxidatic and resolving cysteines, whose modification embraces not only the usual thiol–disulfide exchange but also higher oxidation states of the sulfur atom. These changes are part of a complex system wherein the cooperation with other post‐translational modifications – phosphorylation, acetylation – may function as major regulatory mechanisms of the quaternary structure. More importantly, modern proteomic approaches have identified the oxyacids at cysteine residues as novel protein targets for unsuspected post‐translational modifications, such as phosphorylation that yields the unusual sulfi(o)nic–phosphoryl anhydride. In this article, we review the biochemical attributes of 2‐Cys peroxiredoxins that, in combination with complementary studies of forward and reverse genetics, have generated stimulating molecular models to explain how this enzyme integrates into cell signaling in vivo.

2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg2+ on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO2PO32-] and the sulfonic-phosphoryl [Prx-(Cys175)-SO3PO32-] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.

2-Cys peroxiredoxin (2-Cys Prx) is a large group of proteins that participate in cell proliferation, differentiation, apoptosis, and photosynthesis. In the prevailing view, this ubiquitous peroxidase poises the concentration of H2O2 and, in so doing, regulates signal transduction pathways or protects macromolecules against oxidative damage. Here, we describe the first purification of 2-Cys Prx from higher plants and subsequently we show that the native and the recombinant forms of rapeseed leaves stimulate the activity of chloroplast fructose-1,6-bisphosphatase (CFBPase), a key enzyme of the photosynthetic CO2 assimilation. The absence of reductants, the strict requirement of both fructose 1,6-bisphosphate and Ca2+, and the response of single mutants C174S and C179S CFBPase bring forward clear differences with the well-known stimulation mediated by reduced thioredoxin via the regulatory 170's loop of CFBPase. Taken together, these findings provide an unprecedented insight into chloroplast enzyme regulation wherein both 2-Cys Prx and the 170's loop of CFBPase exhibit novel functions.

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

Experiments initiated in the early 1960s on fermentative bacteria led to the discovery of ferredoxin-dependent alpha-ketocarboxylation reactions that were later found to be key to a new cycle for the assimilation of carbon dioxide in photosynthetic bacteria (the reductive carboxylic acid or reverse citric cycle). The latter finding set the stage for the discovery of a regulatory system, the ferredoxin/thioredoxin system, functional in photosynthesis in chloroplasts and oxygen-evolving photosynthetic prokaryotes. The chloroplast research led to a description of the extraplastidic NADP/thioredoxin system that is now known to function in heterotrophic plant processes such as seed germination and self-incompatibility. Extensions of the fundamental research have begun to open doors to the broad application of thioredoxin in technology and medicine.

Chloroplast Fru-1,6-bisphosphatase is a key enzyme of the Benson-Calvin cycle for photosynthetic COZ assimilation in higher plants. Although the capacity to hydrolyze Fru-1,6bisP is low in dark-adapted chloroplasts, it increases upon illumination. Numerous authors have identified the Fd-thioredoxin system (Fd, Fd-thioredoxin reductase, thioredoxin) as the mediator in enzyme activation (Wolosiuk et al., 1993). On the other hand, the concerted action of a reductant, a sugar bisphosphate, a bivalent cation, and either co-solvents, chaotropic anions, or high hydrostatic pressure stimulate in vitro the enzyme activity. Thus, the replacement of reduced thioredoxin with nonphysiological modulators disclosed the importance of appropriate conformations not only in the activation process but also for the assembly of the nucleaiencoded Fru-1,6-bisphosphatase in the chloroplast stroma (Lallicora and Wolosiuk, 1990). To analyze these processes, we isolated and sequenced a cDNA coding for the chloroplast Fru-1,6-bisphosphatase from rapeseed (Brassica napus) (Table I). Polyadenylated mRNA, isolated from 30-d-old green leaves, was used for the synthesis of cDNA, which was subsequently cloned between NotIlSalI sites of pSPORTl (BRL). The cDNA library was screened for Fru-1,6-bisphosphatase expression with a polyclonal antibody raised in rabbits against the spinach chloroplast enzyme. In positive dones, the presence of cDNA inserts coding for the enzyme was confirmed in a Southem blot assay using a DNA probe coding for the wheat counterpart (kindly provided by Dr. T. Dyer). The largest cDNA insert obtained was used for sequencing.

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.

In chloroplasts, the light-modulated fructose-1,6-bisphosphatase catalyzes the formation of fructose 6-bisphosphate for the photosynthetic assimilation of CO2 and the biosynthesis of starch. We report here the construction of a plasmid for the production of chloroplast fructose-1,6-bisphosphatase in a bacterial system and the subsequent purification to homogeneity of the genetically engineered enzyme. To this end, a DNA sequence that coded for chloroplast fructose-1,6-bisphosphatase of rapeseed (Brassica napus) leaves was successively amplified by PCR, ligated into the Ndel/EcoRI restriction site of the expression vector pET22b, and introduced into Escherichia coli cells. When gene expression was induced by isopropyl-β-D-thiogalactopyranoside, supernatants of cell lysates were extremely active in the hydrolysis of fructose 1,6-bisphosphate. Partitioning bacterial soluble proteins by ammonium sulfate followed by anion exchange chromatography yielded 10 mg of homogeneous enzyme per 1 of culture. Congruent with a preparation devoid of contaminating proteins, the Edman degradation evinced an unique N-terminal amino acid sequence [A-V-A-A-D-A-T-A-E-T-K-P-]. Gel filtration experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the (recombinant) rapeseed chloroplast fructose-1,6-bisphosphatases was a tetramer [160 kDa] comprised of four identical subunits. Like other chloroplast fructose-1,6-bisphosphatases, the recombinant enzyme was inactive at 1 mM fructose 1,6-bisphosphate and 1 mM Mg(2+) but became fully active after an incubation in the presence of either 10 mM dithiothreitol or 1 mM dithiothreitol and chloroplast thioredoxin. However, at variance with counterparts isolated from higher plant leaves, the low activity observed in absence of reductants was not greatly enhanced by high concentrations of fructose 1,6-bisphosphate (3 mM) and Mg(2+) (10 mM). In the catalytic process, all chloroplast fructose-1,6-bisphosphatases had identical features; viz., the requirement of Mg(2+) as cofactor and the inhibition by Ca(2+). Thus, the procedure described here should prove useful for the structural and kinetic analysis of rapeseed chloroplast fructose-1,6-bisphosphatase in view that this enzyme was not isolated from leaves.

The cDNA fragment coding for mature chloroplast pea fructose-1,6-bisphosphatase [Fru(1,6)P(2)ase] was introduced by PCR into the expression vector pET-3d resulting in the construction pET-FBP. After transformation of BL21(DE3) Escherichia coli cells by the pET-FBP plasmid and induction with isopropyl thio-beta-D-galactoside, high-level expression of the recombinant enzyme was achieved. The protein could be purified in three days by a simple procedure which includes heat treatment, ammonium sulfate fractionation, DEAE Sephacel and ACA 44 chromatographies with a yield of 20 mg/l culture. In every respect, the recombinant enzyme was similar to plant chloroplast Fru(1,6)P(2)ase and, in particular, its reactivity with Mg2+ and redox regulatory properties were conserved. In a second series of experiments based on three-dimensional modeling of the chloroplast protein and sequence alignments, two cysteine residues of the recombinant enzyme (Cys173 and Cys178) were mutated into serine residues. An active enzyme, which did not respond to thiol reagents and to light activation, was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplast enzyme.

Neutral salts enhanced the specific activity of chloroplast NADP-glyceraldehyde-3-phosphate dehydrogenase (d-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) from spinach leaves. The ordering of the respective anions, according to the concentration for maximal stimulation, yielded the lyotropic (Hofmeister) series [SCN− (0.05 m), ClO4− (0.08 m), Cl3CCO2− (0.24 m), I− (0.35 m), Br− (0.6 m), Cl− (1.0 m)]; the more chaotropic the anion the less its concentration for maximal activation. Neither the NAD-linked activity of the chloroplast enzyme nor glyceraldehyde-3-phosphate dehydrogenases originating from cyanobacteria and rabbit muscle were stimulated by neutral salts. Chaotropic anions also enhanced the catalytic capacity of the chloroplast enzyme at concentrations lower than those required for the activation process. In the presence of 0.12 m NaBr the rate of catalysis was maximum whereas the highest conversion from the inactive to an active form was observed at 0.6 m NaBr. On the other hand, nonstimulatory concentrations of chaotropic anions lowered the concentration of ATP, Pi, and NADPH required for maximum stimulation of the specific activity (concerted hysteresis). On the basis that the enhancement of NADP-glyceraldehyde-3-phosphate dehydrogenase (and other chloroplast enzymes) by chaotropic anions paralleled the effect of organic solvents and reduced thioredoxin, it appeared that the modification of hydrophobic (intramolecular) interactions participates in the mechanism of light-mediated regulation.

Studies in vitro have shown that the NADP-glyceraldehyde-3-P dehydrogenase activity is enhanced by components indigenous to chloroplasts: low molecular weight effectors and reduced thioredoxin (1–4). We recently showed that, as had been found for chloroplast fructose-l,6-bisphosphatase, the modification of the milieu constitutes another way for changing the kinetic properties of the enzyme (5–7); organic solvents miscible in water (cosolvents) are functional in increasing the NADP-linked activity. Moreover, the increase of enzyme specific activity correlates with the hydrophobic character of the respective organic solvent; high concentrations of cosolvent are required for maximal stimulation when low is the octanol/water partition coefficient. These results suggest that slight modifications of the enzyme hydrophobic interactions have substantial effects in its activity. To explore this possibility the enzyme was (i) subjected to treatments aimed at modifying its intramolecular interactions, and (ii) analyzed for kinetic changes. In this context we studied on chloroplast glyceraldehyde-3-P dehydrogenase the influence of salts and detergents that are often used for perturbing the balance of interactions in proteins (8,9).

1. IN'IRODUCTION Studies in vitro have shown that the NADP-glyceraldehyde-3-P dehydrogenase activity is enhanced by components indigenous to chloroplasts: low molecular weight effectors and reduced thioredoxin (1-4). We recently showed that, as had been found for chloroplast fructose-1,6-bisphosphatase, the modification cf the milieu constitu­ tes another way for changing the kinetic properties cf the enzyme (5-7); organic solvents miscible in water (cosolvents) are functional in increasing the NADP-linked activity. Moreover, the increase cf enzyme specific activity correlates with the hydrophobic character cf the respective organic solvent; high concentrations cf cosolvent are required for !lE.Ximal stirrulation when low is the octanollwater rar­ tition coefficient. These results suggest that slight modifications cf the enzyme hydrophobic interactions have substantial effects in its activity. Tb explore this possibility the enzyme was (i) subjected to treatments aimed at modifying its intramolecular int eract ions , ani (11) analyzed for kinetic changes. In this context we studied on chloroplast glyceraldehyde-3-P dehydrogenase the influence cf salts and detergents that are cften used for perturbing the mlance cf interactions in proteins (8,9). 2. EXPERIMENTAL PROCEDURES