s Abstract Thioredoxins, the ubiquitous small proteins with a redox active disulfide bridge, are important regulatory elements in plant metabolism. Initially recognized as regulatory proteins in the reversible light activation of key photosynthetic enzymes, they have subsequently been found in the cytoplasm and in mitochondria. The various plant thioredoxins are different in structure and function. Depending on their intracellular location they are reduced enzymatically by an NADP-dependent or by a ferredoxin (light)-dependent reductase and transmit the regulatory signal to selected target enzymes through disulfide/dithiol interchange reactions. In this review we summarize recent developments that have provided new insights into the structures of several components and into the mechanism of action of the thioredoxin systems in plants.

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (E m ) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values g 7.0, but with two components being present at pH values <7.0. The slopes of plots of E m versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i.e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the E m versus pH profile for PRK shows three regions, consistent with the presence of pK a values for the two regulatory cysteines in the region between pH 7.5 and 9.0.The ferredoxin/thioredoxin system of oxygenic photosynthetic organisms plays an important role in the regulation of the carbon metabolism of these organisms (1-3). The initial step in the thioredoxin regulatory pathway, which has been extensively characterized in spinach and pea chloroplasts, involves the reduction of ferredoxin:thioredoxin reductase (hereafter abbreviated FTR 1 ) by the reduced ferredoxin generated during light-driven noncyclic electron flow (1-3). Spinach leaf FTR, the best characterized of these enzymes, is a 25.6 kDa heterodimeric protein located in the chloroplast stroma (1-3). FTR contains a unique cluster that serves to stabilize the one-electron-reduced intermediate formed after the first electron donation by ferredoxin, during the two-electron reduction of the activesite disulfide of the oxidized enzyme to the two cysteine thiols present in reduced FTR (4, 5). FTR reduces thioredoxin in a reaction in which the two cysteines at the active site of the reduced enzyme become oxidized to a cystine disulfide, while the active-site disulfide of the oxidized thioredoxin becomes reduced to two cysteine thiols (1-5). FTR reduces both of the thioredoxins found in chloroplasts, thioredoxin f and thioredoxin m (monomeric proteins with molecular masses of ∼12 kDa that contain a conserved -WCGPCactive site), with equal efficiency. However, the two chloroplast thioredoxins display differential but overlapping reactivities among the array of identified target proteins (1-3). Although regulatory reduction by thioredoxin m appears to be restricted to glucose-6-phosph...

No abstract

NMR solution structures of a cytosolic plant thioredoxin h (112 amino acids, 11.7 kDa) from the green alga Chlamydomonas reinhardtii have been calculated on the basis of 1904 NMR distance restraints, which include 90 distances used to restrain 45 hydrogen bonds, and 44 4 dihedral restraints. The structure of C. reinhardtii thioredoxin h was solved in its oxidised form, and the ensemble of 23 converged structures superpose to the geometric average structure with an atomic rmsd of 0.080 nm ? 0.016 for the (N, Ca, C) backbone atoms of residues 4 -110. Comparisons with other thioredoxins, such as thioredoxin from the bacterium Escherichia coli, thioredoxin 2 from a cyanobacterium of the Anahaena genus, and human thioredoxin, showed that thioredoxin h models share more structural features with human thioredoxin than with other bacterial thioredoxins. Examination of the accessible surface around the redoxactive peptide sequence indicates that a potent thioredoxin-h -substrate interaction could be similar to the vertebrate thioredoxin-substrate interactions.

After the discovery of the light-activation properties of the chloroplastic NADP-dependent malate dehydrogenase in 1970, the elements of the activation pathway were identified and shown to consist of the stromal proteins of the ferredoxin/thioredoxin system. It was further demonstrated that the activation was a reductive process during which disulfides were reduced into dithiols by reduced thioredoxin. Sequence alignments with the permanently active NAD-malate dehydrogenases revealed N- and C-terminal extensions specific for the light-regulated form. A regulatory disulfide was identified in the amino-terminal extension by chemical derivatisation: its reduction was correlated to the activation of the enzyme. The use of site-directed mutagenesis techniques revealed the complexity of the intramolecular activation mechanism, showing that two different disulfides were reduced per subunit of this homodimeric enzyme: one located in the N-terminal extension, the other in the C-terminal extension. A model was proposed where the C-terminal extension locks the access to the active site, whereas the N-terminal extension governs the conformation of the active site. The identification of the catalytic histidine allowed us to test the accessibility of the active site and to demonstrate the validity of the proposed model.

The cytosolic isocitrate dehydrogenases (NADP-IDH) were purified to homogeneity from pea roots and green leaves with a high yield by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephacryl S-200 gel filtration, Matrex red-A affinity chromatography and phenyl-Superose HR 5/5 HPLC.Both isoenzymes were dimeric proteins, consisting of two apparently identical 41 -kDa subunits, having similar secondary structures with an a-helical content of 20% and a P-pleated sheet content of 43%. Similarly immunoassays suggested that the two isoenzymes were closely related in terms of antigenic determinants. However, the two proteins were distinguishable by their electrophoretic mobilities and amino acid compositions.The profiles of the two isoenzymes as a function of pH were similar and exhibited a broad pH optimum from 8.5 to 9.0 with Mg2+ as cofactor and 8.0 to 8.5 when Mn2+ was used. Compared to the root isoenzyme, the leaf NADP-IDH appeared to be more heat-labile. However, these isoenzymes exhibited similar behavior for thermal denaturation in the presence of bovine serum albumin and were stabilized upon addition of substrate, metal and coenzyme. Their values of activation energy were estimated as 47 kJ/mol. When using Mn2+ as cofactor, the two isoenzymes displayed identical @", eLL-isocitrate and CAD' values, which were calculated to be 2.1 pM, 5.7 pM and 2.7 pM respectively. With Mg2+ as cofactor, their egzt eL-isocitrate and CAD' values were also not statistically different, being 34.0 pM, 15.2 pM and 2.6 pM for the root NADP-IDH, and 29.0 pM, 20.3 pM and 3.1 pM for the leaf isoenzyme.From the above data it can be concluded that although the cytosolic NADP-IDH in pea roots and leaves are organ-specific isozymes, their similar physicochemical and kinetic properties suggest that the two isozyines might be involved in identical metabolic functions.

No abstract

Cells have developed defense mechanisms against heavy metals, which are highly toxic compounds. In animals and fungi small Cys-rich proteins called metallothioneins are induced by heavy metals (Thiele, 1992). These proteins can coordinate heavy metals by thiol bonds and confer heavymetal tolerance on these organisms. Similar proteins and genes have been identified in plants but their function remains unclear (Zenk, 1996; Foley et al., 1997). Moreover, plants contain another class of metal-binding ligands called phytochelatins. Phytochelatins are poly(␥-glutamylcysteinyl)glycines synthesized from GSH and are clearly involved in detoxification processes (for review, see Zenk, 1996). A recent study has shown that TRX-like genes can confer heavy-metal tolerance on Escherichia coli-sensitive