Ferredoxin-thioredoxin Reductase Research Articles

Redox regulation in the chloroplast thylakoid lumen: a new frontier in photosynthesis research

Initially linked to photosynthesis, regulation by change in the redox state of thiol groups (S-S<-- -->2SH) is now known to occur throughout biology. Thus, in addition to serving important structural and catalytic functions, it is recognized that, in many cases, disulphide bonds can be broken and reformed for regulation. Several systems, each linking a hydrogen donor to an intermediary disulphide protein, act to effect changes that alter the activity of target proteins by change in the thiol redox state. Pertinent to the present discussion is the chloroplast ferredoxin/thioredoxin system, comprised of photoreduced ferredoxin, a thioredoxin, and the enzyme ferredoxin-thioredoxin reductase, that occur in the stroma. In this system, thioredoxin links the activity of enzymes to light: those enzymes functional in biosynthesis are reductively activated by light via thioredoxin (S-S-->2SH), whereas counterparts acting in degradation are deactivated under illumination conditions and are oxidatively activated in the dark (2SH-->S-S). Recent research has uncovered a new paradigm in which an immunophilin, FKBP13, and potentially other enzymes of the chloroplast thylakoid lumen are oxidatively activated in the light (2SH-->S-S). The present review provides a perspective on this recent work.

The plant thioredoxin system

Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cell. In plants, the thioredoxin system is particularly complex since at least 20 thioredoxin isoforms are found in the plant model Arabidopsis thaliana. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. Different pathways allowing thioredoxin reduction also coexist in the plant involving ferredoxin-thioredoxin reductase, thioredoxin reductases and the glutathione/glutaredoxin system. This review discusses the literature of plant thioredoxins with emphasis on recent findings in the field.

A Novel NADPH Thioredoxin Reductase, Localized in the Chloroplast, Which Deficiency Causes Hypersensitivity to Abiotic Stress in Arabidopsis thaliana

Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress.

Characterization of Ferredoxin:Thioredoxin Reductase Modified by Site-directed Mutagenesis

Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.

Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe–4S] Cluster Chemistry

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe-2S] ferredoxin as the electron donor and an active site comprising a [4Fe-4S] cluster and a redox-active disulfide. This review summarizes structural and spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) and a chemically modified form, termed NEM-FTR, which provides a stable analog of the one-electron reduced catalytic intermediate. Detailed spectroscopic characterization of FTR and NEM-FTR using absorption, EPR, electron-nuclear double resonance, variable-temperature magnetic circular dichroism, resonance Raman and Mössbauer spectroscopies indicate that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation of a [4Fe-4S](2+) cluster to yield a unique type of S= 1/2 [4Fe-4S](3+) cluster with two cysteine residues ligated at a specific Fe site. The results provide the basis for a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific [4Fe-4S] cluster chemistry. A similar mechanism is proposed for direct [4Fe-4S]-mediated cleavage of the CoM-S-S-CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.

Characterization of Arabidopsis Mutants for the Variable Subunit of Ferredoxin:thioredoxin Reductase

The ferredoxin/thioredoxin reductase (FTR) is the key enzyme of a light dependent redox regulatory system controlling enzyme activities in oxygenic photosynthetic cells. It is composed of two dissimilar subunits. The catalytic subunit contains a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site. The function of the second subunit, named the variable subunit because it has less conserved primary sequence and length, is not yet known. In order to get insights into the physiological role and importance of FTR, we studied two Arabidopsis mutant lines in which one of two genes encoding FTRA subunit was disrupted by T-DNA insertion. In FTRA1 mutants, the absence of the corresponding transcript was not compensated by the increase in the level of FTRA2 mRNA. Mutant plants exhibited phenotypic perturbations when compared with wild-type plants. Disruptants were found significantly more sensitive to oxidative stress as imposed under high light or in the presence of paraquat. Mutants were further characterized at the biochemical level. Despite the fact that no difference was found by immunodetection of FTR polypeptides, evidence for an impaired FTR system occurring in the mutants was obtained by measuring the endogenous activation rate of one of its targets. In the leaves of mutants placed under normal culture conditions, NADP-dependent malate dehydrogenase (NADP-MDH) activation rate was abnormally low. A partially compensating increase of the enzyme activity was found as well as a higher amount of 2-cys-peroxiredoxin. Our results provide in planta confirmation of the antioxidant role previously proposed for some of the plastidial thioredoxins from Arabidopsis thaliana. The variable subunit of the FTR proved to be important, but its precise role remains to be established.

The universally conserved HCF101 protein is involved in assembly of [4Fe-4S]-cluster-containing complexes in Arabidopsis thaliana chloroplasts.

The seedling-lethal nuclear Arabidopsis hcf101 (high chlorophyll fluorescence) mutant is impaired in photosynthesis and complemented by the wild-type HCF101 cDNA. Photosystem I (PSI) activity is abolished, and PSI core complexes fail to accumulate in hcf101, whereas levels of other thylakoid membrane proteins are unaffected. Northern and in vivo labelling analyses as well as studies on polysome loading show that PSI transcript levels and translation rates of proteins, which belong to PSI, are normal in hcf101. PSI-specific fluorescence at 77 K is shifted from 735 to 728 nm in hcf101, indicating that exitons cannot efficiently be transferred to the PSI reaction centre, whereby the PSI antenna is almost unaffected. Mutant plants not only fail to accumulate mature PSI, which contains three [4Fe-4S]clusters (FSCs), but also are characterized by reduced levels of the soluble FSC-containing complex ferredoxin-thioredoxin reductase (FTR) in the stroma. Inhibited FTR maturation is not a secondary effect stemming from lack of PSI because the mutant hcf145, which also lacks PSI, accumulates FTR at normal levels. Levels of the [2Fe-2S] cluster-containing soluble and membrane proteins, ferredoxin and PetC, respectively, were unchanged in hcf101 plants. These data suggest a specific role of HCF101 in FSC biogenesis. HCF101 is plastid localized and belongs to an ancient and universally conserved family of P-loop ATPases previously designated as the 'MRP' (metGrelated protein) family. The function identified for HCF101 suggests a new designation, FSC, for this family.

N-terminal truncation of the variable subunit stabilizes spinach ferredoxin:thioredoxin reductase

The variable subunit of spinach ferredoxin:thioredoxin reductase (FTR) has an extended N-terminus compared to FTRs from other sources and this was proposed to contribute to the instability of the protein. We constructed two N-terminal truncation mutants of recombinant FTR by removing 16 or 24 residues from the variable subunit. The mutant proteins are readily expressed and show half-saturation values ( S 0.5) for ferredoxin and thioredoxin f comparable to WT. However, truncation increases significantly their stability. Using the stabilized FTR an exposed Cys on its thioredoxin contact surface could be substituted without altering its properties, whereas the replacement of an active site Cys by Ser completely destabilized the protein.

Spectroscopic evidence for site specific chemistry at a unique iron site of the [4Fe-4S] cluster in ferredoxin:thioredoxin reductase.

Ferredoxin:thioredoxin reductase (FTR) catalyzes the reduction of the disulfide in thioredoxin in two one-electron steps using an active site comprising a [4Fe-4S] in close proximity to a redox active disulfide. Mössbauer spectroscopy has been used to investigate the ligation and electronic properties of the [4Fe-4S] cluster in as-prepared FTR which has the active-site disulfide intact and in the N-ethylmaleimide (NEM)-modified form which provides a stable analogue of the one-electron-reduced heterodisulfide intermediate and has one of the cysteines of the active-site disulfide alkylated with NEM. The results reveal novel site-specific cluster chemistry involving weak interaction of the active-site disulfide with a unique Fe site of the [4Fe-4S]2+ cluster in the resting enzyme and cleavage of the active-site disulfide with concomitant coordination of one of the cysteines to yield a [4Fe-4S]3+ cluster with a five-coordinate Fe site ligated by two cysteine residues in the NEM-modified enzyme. The results provide molecular-level insight into the catalytic mechanism of FTR and other Fe-S-cluster-containing disulfide reductases, and suggest a possible mechanism for the reductive cleavage of S-adenosylmethionine by the radical SAM family of Fe-S enzymes.

Proteomics gives insight into the regulatory function of chloroplast thioredoxins.

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (f and m) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins f and m, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin f or m was not observed on affinity chromatography.

Redox control by dithiol-disulfide exchange in plants: I. The chloroplastic systems.

In plants, the photons of light are absorbed at the level of the photosystems in the chloroplasts. The functioning of the photosynthetic electron transfer chain linked to this process is required to generate NADPH and ATP. In addition, the light signal promotes a regulatory cascade, situated in the stroma, that involves ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxins. This redox-based signal transduction chain allows fine regulation of stromal enzymes and tight control of the photosynthetic process. The molecular properties and the functioning of this redox regulatory chain will be described in this review.

Plant thioredoxin gene expression: Control by light, circadian clock, and heavy metals

Thioredoxins (TRX) are small ubiquitous proteins (= 12 kDa) found in all living organisms.l,2 In plants, two isoforms called m and f a re located in the chloroplast and are involved in the control of key carbon fixation enzymes by light. Under illumination the photosynthetic electron transfer chain generates reduced ferredoxin (Fd), which transfers its electrons to many acceptors including TRXs m and f v i a ferredoxin-thioredoxin reductase. In turn, thioredoxins are able to reduce several key enzymes of carbon fixation metabofism such as fructose-l,6-bisphosphatase and NADP malate dehydrogenase, 3,4 which are converted from an inactive to an active form. Another thioredoxin isoform, the h-type, is located in the cytosol and is reduced by NADPH through an NADPH thioredoxin reductase. 5 The exact function of the cytoplasmic isoform(s) is not fully understood in green leaves and algae, z The posttranslational mechanisms involving TRXs have been extensively studied but little is known about the regulation of the expression of the different TRX genes. We have shown that TRX genes are regulated by light, the circadian clock, and heavy metals in the unicellular green alga Chlamydomonas reinhardtii. 6'7 In this article, we describe the experimental procedures used to study their expression.

NMR structures of thioredoxin m from the green alga Chlamydomonas reinhardtii.

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.

Thioredoxin-thioredoxin reductase--a system that has come of age.

Recent publications make it evident that the thioredoxin–thioredoxin reductase system has come of age. The four minireviews presented here attempt to put this field in perspective. The system was first recognized in the early 1960s as the reductant of methionine sulfoxide and PAPS (3′-phosphoadenosine-5′-phosphosulfate) in yeast and of ribonucleotides in Escherichia coli[1–3]. It became clear that fraction B in the E. coli system was a Mr = 12 000 protein having a redox active disulfide; thioredoxin was the name assigned to this protein [4]. It was shown that thioredoxin was reduced by thioredoxin reductase in a NADPH-dependent reaction and that in its dithiol form, thioredoxin served as the reductant of ribonucleotides via a ribonucleotide reductase. It was suggested that thioredoxin was equivalent to enzyme II in the methionine sulfoxide-reducing system and to fraction C in the sulfate-reducing system [4]. Thioredoxin reductase was shown to be a dimeric flavoenzyme containing a redox active disulfide and a FAD in each subunit [3]. The intense study of the various physiological functions of thioredoxin and thioredoxin reductase is the subject of the first Minireview [5]. The thioredoxin–thioredoxin reductase system is very broadly distributed and the two proteins have been isolated from many species. Thioredoxins are similar to one another in structure and the conformation of the single domain is referred to as the thioredoxin fold with the redox-active disulfide forming a protrusion about 35 residues from the N-terminus. Thioredoxin reductases, on the other hand, fall into two classes as first noticed by Holmgren and his colleagues [6]. The low Mr type (Mr = 35 000 per subunit) is typified by the E. coli enzyme. The high Mr type (Mr = 55 000 per subunit) is found in higher eukaryotes and is related in structure and mechanism to glutathione reductase, lipoamide dehydrogenase and other members of the pyridine nucleotide–disulfide oxidoreductase enzyme family [7]. Differences between the mechanisms of the high and low Mr types are covered in the second Minireview [8]. It has become clear only recently that the two proteins comprising the thioredoxin–thioredoxin reductase system are of considerable medical interest as indicators of a wide variety of diseases including rheumatoid arthritis, HIV–AIDS and cancer. Therefore, they are prime prospective drug targets. These fascinating topics are covered in the third Minireview [9]. Several eubacteria contain a single protein that is closely related to the thioredoxin–thioredoxin reductase system. The C-terminal 60% is similar to low Mr thioredoxin reductase, including its redox-active disulfide located just in the pyridine nucleotide binding domain. The N-terminal 40% is a tandem repeat of two thioredoxin-like folds with the redox-active disulfide retained in only one of them. These proteins function as NADH-dependent reductants of peroxyredoxins that in turn reduce alkyl hydroperoxides. The final Minireview will cover this interesting system [10]. The chloroplast thioredoxins f and m, that are reduced by ferredoxin–thioredoxin reductase rather than by thioredoxin reductase are mentioned briefly in the first Minireview [11].

Physiological functions of thioredoxin and thioredoxin reductase.

Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases. Glutaredoxins catalyze glutathione-disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase. Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system. Plants have chloroplast thioredoxins, which via ferredoxin-thioredoxin reductase regulates photosynthetic enzymes by light. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF-kappaB or the Ref-1-dependent AP1 require thioredoxin reduction for DNA binding. The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co-cytokine and chemokine activities. Thioredoxin reductase is a specific dimeric 70-kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol. In contrast, thioredoxin reductases of higher eukaryotes are larger (112-130 kDa), selenium-dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite. All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C-terminal elongation with a cysteine-selenocysteine sequence forming a redox-active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.

A dicistronic construct for the expression of functional spinach chloroplast ferredoxin:thioredoxin reductase in Escherichia coli

Ferredoxin:thioredoxin reductase (FTR) is a heterodimeric FeS containing disulfide reductase involved in the light-dependent activation of photosynthetic enzymes. We have designed a dicistronic construct for the heterologous expression of this nucleus encoded chloroplast protein in Escherichia coli. The coding sequences for the two mature subunits have been inserted in tandem into the expression vector pET-3d. This dicistronic construct is correctly translated yielding soluble, perfectly functional FTR. The recombinant enzyme is composed of both subunits, contains the correctly inserted FeS cluster as evidenced by its spectral properties and is indistinguishable from the enzyme isolated from leaves in its capacity to activate chloroplast fructose-1,6-bisphosphatase, one of the well known light activated enzymes of the Calvin cycle.

Redox signaling in chloroplasts: cleavage of disulfides by an iron-sulfur cluster.

Light generates reducing equivalents in chloroplasts that are used not only for carbon reduction, but also for the regulation of the activity of chloroplast enzymes by reduction of regulatory disulfides via the ferredoxin:thioredoxin reductase (FTR) system. FTR, the key electron/thiol transducer enzyme in this pathway, is unique in that it can reduce disulfides by an iron-sulfur cluster, a property that is explained by the tight contact of its active-site disulfide and the iron-sulfur center. The thin, flat FTR molecule makes the two-electron reduction possible by forming on one side a mixed disulfide with thioredoxin and by providing on the opposite side access to ferredoxin for delivering electrons.

NMR structures of thioredoxinm from the green algaChlamydomonas reinhardtii

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using 15N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids. Proteins 2000;41:334–349. © 2000 Wiley-Liss, Inc.

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes.

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 (Em) 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 >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em 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 Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.