Ferredoxin-dependent thioredoxin reductase was identified 35 y ago in the fermentative bacterium Clostridium pasteurianum [Hammel KE, Cornwell KL, Buchanan BB (1983) Proc Natl Acad Sci USA 80: 3681-3685]. The enzyme, a flavoprotein, was strictly dependent on ferredoxin as reductant and was inactive with either NADPH or NADH. This early work has not been further pursued. We have recently reinvestigated the problem and confirmed that the enzyme, here designated ferredoxin-dependent flavin thioredoxin reductase (FFTR), is a flavoprotein. The enzyme differs from ferredoxin-thioredoxin reductase (FTR), which has a signature [4Fe-4S] cluster, but shows structural similarities to NADP-dependent thioredoxin reductase (NTR). Comparative amino acid sequence analysis showed that FFTR is present in a number of clostridial species, some of which lack both FTR and an archetypal NTR. We have isolated, crystallized, and determined the structural properties of FFTR from a member of this group, Clostridium acetobutylicum, both alone and in complex with Trx. The structures showed an elongated FFTR homodimer, each monomer comprising two Rossmann domains and a non-covalently bound FAD cofactor that exposes the isoalloxazine ring to the solvent. The FFTR structures revealed an alternative domain organization compared with NTR that enables the enzyme to accommodate Fdx rather than NADPH. The results suggest that FFTR exists in a range of conformations with varying degrees of domain separation in solution and that the stacking between the two redox-active groups for the transfer of reducing equivalents results in a profound structural reorganization. A mechanism in accord with the findings is proposed.

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FADmolecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

Thioredoxins (Trxs) are key components of the redox system that regulates the activity of a spectrum of target proteins through dithiol-disulfide exchange reactions. Trxs are reduced by members of the Trx reductase (TR) family (Jacquot et al., 2009Jacquot J.-P. Eklund H. Rouhier N. Schürmann P. Structural and evolutionary aspects of thioredoxin reductases in photosynthetic organisms.Trends Plant Sci. 2009; 14: 336-343Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). NADP-dependent thioredoxin reductases (NTRs), the most common type, belong to the family of dimeric pyridine nucleotide disulfide oxidoreductase flavoproteins that use NADPH as the source of reducing equivalents. In oxyphotosynthetic organisms, in particular, NTRs coexist with the ferredoxin/thioredoxin system (FTS), composed of ferredoxin (Fdx), ferredoxin:thioredoxin reductase (FTR), and a Trx. FTRs convert the electron signal obtained from photoreduced Fdx to a thiol signal via a 4Fe-4S center and a redox-active disulfide catalytic center. FTR, in turn, reduces Trx. In cyanobacteria and chloroplasts, the FTS is closely associated with the regulation of enzymes of the Calvin-Benson cycle and associated processes; e.g., the oxidative pentose phosphate pathway (Balsera et al., 2014Balsera M. Uberegui E. Schürmann P. Buchanan B.B. Evolutionary development of redox regulation in chloroplasts.Antioxid. Redox Signal. 2014; 21: 1327-1355Crossref PubMed Scopus (70) Google Scholar). In contrast to other oxygenic photosynthetic organisms, the ancient cyanobacterium Gloeobacter and the ocean-dwelling green oxyphotobacteria Prochlorococcus lack an FTR gene. This observation raises the question of how these photosynthetic organisms link the Calvin-Benson cycle and related metabolic processes to light and other changing environmental conditions. To gain information on this point, we have conducted a comparative analysis of NTR-like TR protein sequences and identified an enzyme common to Gloeobacter and green oxyphotobacteria that possibly functions in this connection. The enzyme, provisionally named DTR for deeply rooted bacterial thioredoxin reductase, shows high similarity to NTR but exhibits unique structural features (Figure 1). The enzyme is present in other bacteria, including organisms within the deeply rooted bacterial lineages as well as marine algae (Supplemental Table 1 and Supplemental Figure 1). A multiple protein sequence alignment showed that Gloeobacter and Prochlorococcus DTRs harbor the conserved FAD-binding motif and the two active-site Cys residues typical of authentic NTRs (Figure 1A). Major differences were noted, however, in the amino acids responsible for pyridine nucleotide binding (GxGxxA/G and HRRxxR) (Figure 1A). These included substitutions of crucial residues for coordination to the pyrophosphate group and the absence of specific positively charged amino acids needed for electrostatic interactions with the 2′-phosphate group of the adenosine. Additional variations included a C-terminal extension with a conserved aromatic residue and a fused N-terminal redoxin domain that is restricted to a few bacteria (gray box; Supplemental Figure 1). The visible-UV absorption spectrum of purified Gloeobacter DTR (GvDTR) showed features typical of a flavoprotein, including absorption maxima at 391 and 459 nm (continuous black line, Figure 1B) (Prongay and Williams, 1992Prongay A.J. Williams C.H. Oxidation-reduction properties of Escherichia coli thioredoxin reductase altered at each active site cysteine residue.J. Biol. Chem. 1992; 267: 25181-25188Abstract Full Text PDF PubMed Google Scholar). In contrast to NTRs, the Gloeobacter flavoprotein failed to show activity in the assay of the enzyme in which the oxidation of NAD(P)H is coupled to the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Figure 1C) (Holmgren and Björnstedt, 1995Holmgren A. Björnstedt M. Thioredoxin and thioredoxin reductase.Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (816) Google Scholar). The apparent lack of activity could be due to either of two reasons. One, NAD(P)H reduces FAD, but electrons are not further shuttled to its homolog Trx and thus to DTNB; nonetheless, the activity of Gloeobacter Trx in the coupled assay was demonstrated with Escherichia coli NTR (Figure 1C). Alternatively, the flavoprotein does not function with NAD(P)H. To explore the second possibility, we applied isothermal titration calorimetry (ITC) as the most direct approach for assessing protein-ligand binding. For these experiments, we included 3-acetylpyridine adenine dinucleotide phosphate (AADP), a non-hydrolyzable analog of NADPH, that binds tightly to NAD(P)-dependent enzymes. In contrast to E. coli NTR (EcNTR), which binds NADP+ and AADP+ with a Kd in the micromolar range, the Gloeobacter protein failed to bind either ligand in vitro (Figure 1D and Supplemental Figure 3). The lack of affinity for pyridine nucleotides resembles TR from the archeon Thermoplasma acidophilum (TaTR; Hernandez et al., 2008Hernandez H. Jaquez O. Hamill M. Elliott S. Drennan C. Thioredoxin reductase from Thermoplasma acidophilum: a new twist on redox regulation.Biochemistry. 2008; 47: 9728-9737Crossref PubMed Scopus (21) Google Scholar), although structural differences were detected between the two proteins. GvDTR displayed a modified GxGxxA/G structural pattern for nucleotide binding (Supplemental Figure 4, red box), and contained a C-terminal extension missing in TaTR (Supplemental Figure 4, green box). To confirm that the Gloeobacter flavoprotein fulfills the function of a TR, we determined whether it could reduce Trx using dithionite as an artificial electron donor. Trx m (also known as TrxA) is the only canonical Trx form in Gloeobacter and Prochlorococcus (Florencio et al., 2006Florencio F. Pérez-Pérez M. López-Maury L. Mata-Cabana A. Lindahl M. The diversity and complexity of the cyanobacterial thioredoxin systems.Photosynth. Res. 2006; 89: 157-171Crossref PubMed Scopus (64) Google Scholar, Balsera et al., 2014Balsera M. Uberegui E. Schürmann P. Buchanan B.B. Evolutionary development of redox regulation in chloroplasts.Antioxid. Redox Signal. 2014; 21: 1327-1355Crossref PubMed Scopus (70) Google Scholar). We observed that dithionite was able to slowly reduce the flavoenzyme anaerobically as measured by a decrease in absorbance at 459 nm (Figure 1B). Partial reoxidation of the enzyme was observed immediately after adding Gloeobacter Trx as seen by recovery of the main features of the visible-UV spectrum. These results provide evidence that the Gloeobacter flavoprotein has TR activity and functions independently of pyridine nucleotides. To gain information on the molecular properties of DTR, we determined a high-resolution structure of the Gloeobacter protein by X-ray crystallography at 1.9 Å resolution (PDB: 5J60; Supplemental Table 2). The crystal structure revealed that GvDTR is a homodimer and that each monomer adopts a typical NTR fold (Figure 1E). Despite the high similarity between the FAD- and pseudo-NADPH-binding domains with those of authentic NTRs, the relative orientation of the domains in the crystal structure differed from the flavin-oxidizing (FO) and flavin-reducing (FR) conformations found in NTRs (Lennon and Williams, 1997Lennon B.W. Williams C.H. Reductive half-reaction of thioredoxin reductase from Escherichia coli.Biochemistry. 1997; 36: 9464-9477Crossref PubMed Scopus (53) Google Scholar) (Supplemental Figure 5). No direct interaction was observed between amino acids of the pseudo-NADPH-binding domains, as detected between the two corresponding domains in the NTR dimer both in FO and FR conformations, suggesting that the two functional domains in DTRs are flexible relative to each other. FAD in GvDTR was found to be oxidized based on the planar conformation and the yellow color of the crystal. The redox-active Cys residues were reduced and distant from the re face of the isoalloxazine ring instrumental for electron transfer (Figure 1E). Coordination to the FAD cofactor is well conserved compared with NTRs of known 3D structure (Supplemental Figure 6). However, analysis of the NADPH-binding pocket revealed significant structural deviations from NTRs (Supplemental Figure 7). One of the most striking variations is related to the substitution of the second strictly conserved Gly in the GxGxxA/G motif in EcNTR by an Asn in GvDTR (Figure 1A, red box). GvDTR structure showed that the substrate binding pocket is blocked by the side chain carboxamide of Asn (N154; Supplemental Figure 7E), supporting the finding that the enzyme does not bind pyridine nucleotides. Remarkably, the aromatic side chain of a tryptophan (W315) in one monomer stacks over the central portion of the FAD isoalloxazine ring of the second monomer on its re face (Figure 1E). This position is generally occupied by either the pyridine nucleotide or the disulfide bridge in NTRs. The tryptophan position is highly conserved in the protein sequences of the DTR family, including early branching bacteria, cyanobacteria, and marine algae, replaced by a tyrosine residue only in firmicutes and chlorobi (Supplemental Figure 1), and suggests a potential physiological role for this residue. As displayed here, the C-terminal tail has not previously been reported and is a unique feature of DTR. Relative to WT, deletion of the C-terminal motif in Gloeobacter DTR (GvDTR_Δt) was accompanied by ca. 7–9 nm shift in the flavin absorption maxima in the blue region of the spectrum (Figure 1B, lower panel). More interestingly, removal of the C-terminal tail increased the rate of flavin reduction by dithionite (∼10-fold) to levels comparable with those with the E. coli enzyme (Figure 1B). Collectively, these results suggest that the unique C-terminal module on DTR has a direct influence on enzyme activity and might constitute a mechanism of regulation not previously reported. The C-terminal extension of DTR resembles the C-terminal subdomain of certain ferredoxin-NADP reductase enzymes (FNRs) in which an aromatic residue protrudes over the re side of FAD, thereby requiring its displacement for a productive interaction between the pyridine nucleotide and FAD (Ceccarelli et al., 2004Ceccarelli E.A. Arakaki A.K. Cortez N. Carrillo N. Functional plasticity and catalytic efficiency in plant and bacterial ferredoxin-NADP(H) reductases.Biochim. Biophys. Acta. 2004; 1698: 155-165Crossref PubMed Scopus (127) Google Scholar). Among the different types of FNRs, structural similarities were detected between DTR and TR-type FNRs. These FNRs harbor conserved FAD and NADPH-binding motifs but lack the redox-active Cys. An aromatic residue in a short α helix at the C terminus protrudes over the re side of FAD (Supplemental Figure 8). Mutational studies in TR-type FNR enzymes have shown that replacement of the aromatic residue on the re face of the flavin perturbs binding with its redox partner (Seo et al., 2014Seo D. Asano T. Komori H. Sakurai T. Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP+ oxidoreductase from Bacillus subtilis.Plant Physiol. Biochem. 2014; 81: 143-148Crossref PubMed Scopus (11) Google Scholar). Following a molecular mechanism similar to these FNRs, displacement of the C-terminal extension of DTR would allow access of the redox disulfide to the re face of FAD likely in a regulatory manner. As for NTRs, conformational flexibility is expected to play an important role in the mechanism of enzyme action. Thus, DTR in solution may exist in equilibrium between the structure observed in the crystal and other configurations in which the donor molecule (and/or the disulfide) is in close contact with the flavin group. In light of the high structural similarity with NTRs and the finding that DTR lacks the archetypal NADPH-binding site, the physiological donor is thought to be a cofactor acting similar to pyridine nucleotides. Other types of molecules such as small redox carrier proteins can, however, not be excluded. To this point, a ferredoxin-linked flavoprotein enzyme that reduces Trx has been isolated from clostridia (Hammel et al., 1983Hammel K.E. Cornwell K.L. Buchanan B.B. Ferredoxin/flavoprotein-linked pathway for the reduction of thioredoxin.Proc. Natl. Acad. Sci. USA. 1983; 80: 3681-3685Crossref PubMed Scopus (29) Google Scholar). However, only identification of the physiological electron donor will clarify the catalytic mechanism of enzyme reduction and its physiological importance in the Trx system. In the present study, we have provided evidence for the diversity of Trx systems in bacteria and for variation in the ability of the reductases to deliver electrons to Trx. Our results support the view that the redox systems consist of structural modules and regulatory elements that different organisms have conveniently combined during evolution to create proteins capable of adapting to specific metabolic and environmental situations. They also provide a structural foundation for future exploration of the mechanism of action and open the door to understanding the evolutionary development of different flavin reductases in relation to particular metabolic demands. B.B.B. acknowledges support from an Alexander von Humboldt Research Award that catalyzed the launching of this project at the Ludwig-Maximilians-Universität in Munich. R.M.B. is supported by a Ramón y Cajal contract from the Spanish Ministerio de Economía y Competitividad. L.L.-M. is supported by a postdoctoral contract from Universidad de Sevilla.

The reversible oxidation of cysteine (Cys) residues is commonly used to regulate a range of cellular processes throughout biology. Modification of regulatory Cys alters protein solubility and activity and, under control conditions, ultimately results in the fine-tuning of processes in response to metabolic demands. In oxyphotosynthetic organisms, the reversible modification of protein thiols is a major mechanism for direct regulation of chloroplast enzymes in a light-dependent manner and in response to different types of stress. The main redox regulatory system in chloroplasts is the ferredoxin-dependent thioredoxin system (FTS) which coordinates metabolic pathways of oxygenic photosynthesis via thioredoxin-mediated dithiol/ disulfide (SH/S-S) transitions. NADP-linked thioredoxin reductase C and the glutathione/ glutaredoxin systems complement the FTS in maintaining redox balance under certain environmental conditions.

Rubén M. Buey, Sergio Galindo-Trigo, Luis López-Maury, Adrián VelázquezCampoy, José Luis Revuelta, F. Javier Florencio, José M. de Pereda, Peter Schürmann, Bob B. Buchanan, Monica Balsera Dept. Microbiología y Genética. Universidad de Salamanca, Salamanca, Spain; Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Salamanca, Spain; Instituto de Bioquímica Vegetal y Fotosíntesis (CSIC-Universidad de Sevilla), Sevilla, Spain; Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain; Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain; Laboratoire de Biologie Moléculaire et Cellulaire, Université de Neuchâtel, Neuchâtel, Switzerland; Department of Plant & Microbial Biology, University of California, Berkeley, USA; Corresponding author: monica.balsera@csic.es

SIGNIFICANCE The post-translational modification of thiol groups stands out as a key strategy that cells employ for metabolic regulation and adaptation to changing environmental conditions. Nowhere is this more evident than in chloroplasts-the O2-evolving photosynthetic organelles of plant cells that are fitted with multiple redox systems, including the thioredoxin (Trx) family of oxidoreductases functional in the reversible modification of regulatory thiols of proteins in all types of cells. The best understood member of this family in chloroplasts is the ferredoxin-linked thioredoxin system (FTS) by which proteins are modified via light-dependent disulfide/dithiol (S-S/2SH) transitions. RECENT ADVANCES Discovered in the reductive activation of enzymes of the Calvin-Benson cycle in illuminated chloroplast preparations, recent studies have extended the role of the FTS far beyond its original boundaries to include a spectrum of cellular processes. Together with the NADP-linked thioredoxin reductase C-type (NTRC) and glutathione/glutaredoxin systems, the FTS also plays a central role in the response of chloroplasts to different types of stress. CRITICAL ISSUES The comparisons of redox regulatory networks functional in chloroplasts of land plants with those of cyanobacteria-prokaryotes considered to be the ancestors of chloroplasts-and different types of algae summarized in this review have provided new insight into the evolutionary development of redox regulation, starting with the simplest O2-evolving organisms. FUTURE DIRECTIONS The evolutionary appearance, mode of action, and specificity of the redox regulatory systems functional in chloroplasts, as well as the types of redox modification operating under diverse environmental conditions stand out as areas for future study.

Uncovered in studies on photosynthesis 35 years ago, redox regulation has been extended to all types of living cells. We understand a great deal about the occurrence, function, and mechanism of action of this mode of regulation, but we know little about its origin and its evolution. To help fill this gap, we have taken advantage of available genome sequences that make it possible to trace the phylogenetic roots of members of the system that was originally described for chloroplasts—ferredoxin, ferredoxin:thioredoxin reductase (FTR), and thioredoxin as well as target enzymes. The results suggest that: (1) the catalytic subunit, FTRc, originated in deeply rooted microaerophilic, chemoautotrophic bacteria where it appears to function in regulating CO2 fixation by the reverse citric acid cycle; (2) FTRc was incorporated into oxygenic photosynthetic organisms without significant structural change except for addition of a variable subunit (FTRv) seemingly to protect the Fe–S cluster against oxygen; (3) new Trxs and target enzymes were systematically added as evolution proceeded from bacteria through the different types of oxygenic photosynthetic organisms; (4) an oxygenic type of regulation preceded classical light–dark regulation in the regulation of enzymes of CO2 fixation by the Calvin–Benson cycle; (5) FTR is not universally present in oxygenic photosynthetic organisms, and in certain early representatives is seemingly functionally replaced by NADP-thioredoxin reductase; and (6) FTRc underwent structural diversification to meet the ecological needs of a variety of bacteria and archaea.

Trx-z is a chloroplastic thioredoxin, exhibiting a usual WCGPC active site, but whose biochemical properties are unknown. We demonstrate here that Trx-z supports the activity of several plastidial antioxidant enzymes, such as thiol-peroxidases and methionine sulfoxide reductases, using electrons provided by ferredoxin-thioredoxin reductase. Its disulfide reductase activity requires the presence of both active site cysteines forming a catalytic disulfide bridge with a midpoint redox potential of -251 mV at pH7. These in vitro biochemical data suggest that, besides its decisive role in the regulation of plastidial transcription, Trx-z might also be involved in stress response.

Thioredoxins (Trxs) are small oxidoreductases that are involved in redox homeostasis and are found in large numbers in the subcellular compartments of eukaryotic plant cells, including the chloroplasts. Also present in chloroplasts are two forms of thioredoxin reductase (TR), which use either NADPH or ferredoxin as an electron donor. In other compartments, two additional TR forms also use NADPH: one is distributed in all photosynthetic organisms and is similar to prokaryotic enzymes, whereas the other is restricted to algae and is similar to mammalian selenoproteins. Here, we review current knowledge of the different forms of TRs across organisms and discuss the possible evolutionary fate of this class of enzymes, which provide an example of convergent functional evolution.

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca2+ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.

Ferredoxin:thioredoxin reductase catalyzes the reduction of thioredoxins in plant chloroplasts using the [Fe2S2] ferredoxin as a one-electron donor and as such plays a central role in light regulation of oxygenic photosynthesis. The active-site comprises a [Fe4S4] cluster next to a redox-active disulfide that is cleaved in sequential one-electron steps and the combination of spectroscopic and crystallographic studies have revealed a catalytic mechanism involving novel site specific cluster chemistry in the oxidized, one-electron- and two-electron-reduced redox states. Histidine-86 has emerged as a potential proton donor/acceptor in the catalytic mechanism based on redox-related changes in the positioning of the imidazole ring during redox cycling and greatly decreased activity for the H86Y variant. Here we report on spectroscopic and redox characterization of the [Fe4S4] center in Synechocystis sp. PCC 6803 H86Y ferredoxin:thoredoxin reductase in the accessible redox states of both the as purified and N-ethylmaleimide-modified forms, using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism, EPR, resonance Raman and Mössbauer spectroscopies. The results demonstrate that His86 is required for formation of the partially valence-localized [Fe4S4]2+ cluster that is the hallmark of two-electron-reduced intermediate. Taken together with the available structural data, the spectroscopic results indicate a functional role for His86 in protonation/deprotonation of the cluster-interacting thiol and anchoring the cluster interacting thiol in close proximity to the cluster in the two-electron-reduced intermediate.

Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determi- nations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a sig- nal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machin- ery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca 2 has a dramatic effect on

In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.

Forty years ago, ferredoxin (Fdx) was shown to activate fructose 1,6-bisphosphatase in illuminated chloroplast preparations, thereby laying the foundation for the field now known as "redox biology." Enzyme activation was later shown to require the ubiquitous protein thioredoxin (Trx), reduced photosynthetically by Fdx via an enzyme then unknown-ferredoxin:thioredoxin reductase (FTR). These proteins, Fdx, FTR, and Trx, constitute a regulatory ensemble, the "Fdx/Trx system." The redox biology field has since grown beyond all expectations and now embraces a spectrum of processes throughout biology. Progress has been notable with plants that possess not only the plastid Fdx/Trx system, but also the earlier known NADP/Trx system in the cytosol, endoplasmic reticulum, and mitochondria. Plants contain at least 19 types of Trx (nine in chloroplasts). In this review, we focus on the structure and mechanism of action of members of the photosynthetic Fdx/Trx system and on biochemical processes linked to Trx. We also summarize recent evidence that extends the Fdx/Trx system to amyloplasts-heterotrophic plastids functional in the biosynthesis of starch and other cell components. The review highlights the plant as a model system to uncover principles of redox biology that apply to other organisms.

Ferredoxin:thioredoxin reductase (FTR), catalyzes the two-electron reduction of thioredoxins in chloroplasts and cyanobacteria, using reduced ferredoxin as the electron donor. Reduced thioredoxins then play important roles in redox regulation. FTR, a heterodimer with a unique [4Fe-4S] cluster as its sole prosthetic group, has a single binding site for ferredoxin and a separate single binding site for thioredoxin. NMR spectroscopy was used to map the binding site on ferredoxin for FTR in a 1:1 complex of the two proteins. A mono-gallium analog of this [2Fe- 2S] ferredoxin was obtained by reconstituting apo-ferredoxin in a gallium-containing refolding buffer. The use of this diamagnetic Ga structural analog eliminates the paramagnetic broadening of NMR resonances of amino acids in the vicinity of the [2Fe-2S] cluster in native ferredoxin. This has allowed the first complete mapping of the interaction interface of a [2Fe-2S] ferredoxin for a target enzyme. NMR spectroscopy was also used to map the interaction domain for FTR on thioredoxin m in a 1:1 complex of the two proteins. Both similarities and differences are seen in the thioredoxin m interaction domain for FTR in the non-covalent complex examined by NMR and in a disulfide-linked covalent complex of FTR and thioredoxin m for which an X-ray crystal structure has been obtained. NMR has also been used to characterize a ternary complex between ferredoxin, FTR and thioredoxin m in solution, confirming the presence of separate binding sites on FTR for its two substrates.

Light not only provides the energy for carbon assimilation in the chloroplast, it is also an important regulatory factor of carbon metabolism. The activities of several of its key enzymes are linked to light. This enables the chloroplasts to switch between biosynthetic pathways in the light and catabolic processes in the dark.

The X-ray structures of ferredoxin–thioredoxin reductase (FDR) in its one- and two-electron-reduced intermediate states and four complexes in the pathway are solved, including the ternary ferredoxin–FTR–thioredoxin complex. These results provide a structural framework for understanding the mechanism of disulphide reduction by an iron-sulphur enzyme.