Proteomics were used to identify the proteins from the eukaryotic unicellular green alga Chlamydomonas reinhardtii that can be reduced by thioredoxin. These proteins were retained specifically on a thioredoxin affinity column made of a monocysteinic thioredoxin mutant able to form mixed disulfides with its targets. Of a total of 55 identified targets, 29 had been found previously in higher plants or Synechocystis, but 26 were new targets. Biochemical tests were performed on three of them, showing a thioredoxindependent activation of isocitrate lyase and isopropylmalate dehydrogenase and a thioredoxin-dependent deactivation of catalase that is redox insensitive in Arabidopsis. In addition, we identified a Ran protein, a previously uncharacterized nuclear target in a photosynthetic organism. The metabolic and evolutionary implications of these findings are discussed.O ur knowledge on redox regulation of various physiological processes through thiol-disulfide interchange with proteins of the thioredoxin (TRX) superfamily is rapidly progressing as genome-wide approaches and functional genomics studies are expanding. In the plant biology domain, thioredoxin-dependent regulation has been first discovered for chloroplastic enzymes involved in carbon assimilation (1). The completion of the sequencing of the Arabidopsis genome revealed that TRXs constituted a multigene family and led to the assumption that numerous TRX targets were still to be discovered (2-4). Thus, proteomic approaches aimed at identifying the largest possible number of TRX targets have been developed. The first attempts took advantage of the reaction mechanism of TRXs with their targets, in which a transient heterodisulfide forms between the reduced TRX and the oxidized protein, followed by the release of the reduced target due to the attack of the mixed disulfide by the second cysteine of TRX. Model studies showed that when this second cysteine was mutated, the heterodisulfide was stabilized (5, 6). This property was applied in vivo by transforming a TRX-null mutant yeast with a monocysteinic TRX and allowed isolating a peroxiredoxin (PRX) (7). Subsequently, monocysteinic TRX mutants were used in affinity chromatography to trap interacting proteins (8-11). Native TRX affinity columns also allowed retaining some targets by electrostatic interaction (12). Another technique was also developed that consists of a reduction of a crude soluble protein extract with reduced TRX followed by a separation of the proteins on a 2D gel after derivatization of the newly appeared thiols either with a fluorescent (13) or with a radioactive (14) reagent. In one case, both techniques have been applied in parallel (15), yielding similar results.Most of the disulfide proteomics have been led with higher plants: spinach (8, 10, 12), Arabidopsis (11, 14), or cereal grains (12). A single study was led with cyanobacteria and revealed targets mostly different from those of higher plants (16). The unicellular eukaryotic green alga Chlamydomonas reinhardtii possesses a TRX syste...

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...