From the photosynthetic green sulfur bacterium Chlorobium tepidum (pro synon. Chlorobaculum tepidum), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome c 554 . These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome c 551 , composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in Escherichia coli cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome c 554 reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose sox gene cluster sequences have been deposited so far and ranging over the Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae, etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.

Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18 -27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys 330 residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography. Malate dehydrogenase (MDH)3 catalyzes the reversible reaction of oxaloacetate to malate utilizing the NAD ϩ /NADH or NADP ϩ /NADPH cofactor system and is known to occur ubiquitously in mammals, plants, and most prokaryotes. NAD-dependent MDHs (EC 1.1.1.37) generally exist as a homodimer with a subunit of 32-37 kDa (1, 2), whereas NADP-dependent MDH (EC 1.1.1.82) possesses a larger subunit of ϳ42 kDa (3, 4). In plants, MDH isoforms have been reported in the cytosol and in organelles and have been found to carry out important roles in a number of metabolic pathways (5). In general, MDH proteins are grouped into five classes according to their location within the cell; (i) cytosolic NAD-dependent MDH (cytMDH); (ii) mitochondrial NAD-dependent MDH, which is part of the tricarboxylic acid cycle; (iii) glyoxisomal and peroxisomal NAD-dependent MDHs, which are involved in photorespiration; (iv) chloroplast NADPdependent MDH, which is required for the transfer of reducing equivalents from chloroplast stroma to cytosol; and (v) chloroplast NAD-dependent MDH (6).Among the classes listed, chloroplast NADP-dependent MDH is a thiol enzyme that has been subject of intense study (4,(7)(8)(9)(10)(11)(12) and that uses NADP ϩ for catalysis. Chloroplast NADPdependent MDH possesses both N-and C-terminal extensions, both of which harbor a redox-sensitive cysteine pair (4,(12)(13)(14). Central to the regulation of chloroplast MDH, ferredoxin-thioredoxin reductase sources electrons from photosynthesis to reduce chloroplast thioredoxins (Trx) (8). Trxs are an important class of regulatory proteins that modulate the activity of a wide variety of thiol-containing chloroplast enzymes by the reduction of specific disulfide bonds (15). In the light, excess NADPH (produced by photosynthesis but surplus to CO 2 fixation requirements) induces the conversion of oxaloacetate to malate, r...

Thioredoxin affinity chromatography can be used to recognize the target proteins of thioredoxin or thioredoxin-related proteins in whole cells or certain cellular compartments. In the last couple of years, many potential target proteins have been identified from various organelles and organisms by this method. Based on the information on the target proteins provided by these studies, the complete thioredoxin-related redox networks can now be efficiently described.

Thioredoxins are a ubiquitous family of redox equivalent mediators, long considered to possess a limited number of target enzymes. Recent progress in proteomic research has allowed the identification of a wide variety of candidate proteins with which this small protein may interact in vivo. Moreover, the activity of thioredoxin itself has been recently found to be subject to regulation by posttranslational modifications, adding an additional level of complexity to the function of this intriguing enzyme family. The current review charts the technical progress made in the continuing discovery of the numerous and diverse roles played by these proteins in the regulation of redox networks in plant cells.

Thioredoxin (Trx) is a small ubiquitous protein involved in the disulfide-dithiol exchange reaction occurring in cells and organelles. In vivo, Trx is reduced by Trx reductase using NADPH or photosynthetically produced reducing equivalents, and the reduced form Trx takes on the physiological functions. In the cyanobacterium Synechocystis sp. PCC6803, two Trx reductases, ferredoxin-Trx reductase (FTR) and NADPH-Trx reductase (NTR), and four typical Trx isoforms have been identified by genomic analysis. Based on analysis of the physiological features of the Trx reductase disruptants, we found that the NTR-Trx pathway is important for the antioxidant system, whereas the FTR-Trx pathway may play a more important role in the control of cell growth rate. In addition, by quantification of Trx abundance in the wild-type and the disruptant Synechocystis cells, we found that the gene product of slr0623, the homolog of m-type Trx in higher plants, is the most abundant Trx, and that accumulation of Trx isoforms occurs dependent on the expression of the other redox-related proteins. A study of the binary reducing equivalent pathways in cyanobacterial cells is reported here.