Thioredoxin (Trx) is a small, ubiquitous protein that contains a pair of redox-sensitive cysteine residues within its catalytic domain. This domain comprises the highly conserved sequence motif Trp-Cys-Gly-Pro-Cys-[Lys/Arg] (Holmgren, 1985; Buchanan, 1991; Balmer et al., 2006). The cysteines allow Trx to reduce disulfide bonds on target proteins by catalyzing a dithiol–disulfide exchange reaction, which regulates target activity in several cases. On the basis of the reduction process mediated by Trx studies, the cysteine toward the N-terminal end of the Trx catalytic domain first attacks disulfide bond in a target protein to form an intermolecular disulfide complex, often described as a mixed disulfide intermediate complex (Brandes et al., 1993). Subsequently, the cysteine toward the C-terminal end of the Trx catalytic domain attacks this intermolecular disulfide bond, which results in an oxidized form of Trx and a reduced target.

Trx was first identified as a reducing equivalent donor for the deoxyribonucleotide reductase in E. coli (Laurent et al., 1964). In contrast to bacteria and animals, numerous isoforms of Trx have been identified in plants, although their specific roles are poorly understood (Balmer et al., 2006). Recent proteomics studies revealed that these Trx isoforms could react with a large number of candidate target proteins in vitro (Motohashi et al., 2001; Yano et al., 2001; Balmer et al., 2003; Yamazaki et al., 2004) and in vivo (Hall et al., 2010). For example, approximately 400 proteins have been described as potential targets of the plant Trx system (Montrichard et al., 2009), although only a small portion of these have been confirmed to be bona fide target proteins by biochemical analyses.

With regard to the target specificity and the Trx-mediated reaction process, a number of questions remain unanswered. For example, the mechanism of disulfide bonds recognition on the target proteins by Trx and, during the reaction process, the mechanism by which reduced form of Trx react with the oxidized form of the target protein and release it immediately after the dithiol–disulfide exchange reaction is completed. The target proteins have been categorized into two groups on the basis of their mode of interaction with Trx.

One group is the “switch” type proteins that are predominantly found within the chloroplast such as the chloroplast ATP synthase γ subunit (McKinney et al., 1979; Nalin and Mccarty, 1984), four Calvin cycle enzymes (glyceraldehyde 3-phosphate dehydrogenase, fructose 1,6-bis-phsophatase, sedoheptulose 1,7-bis-phosphatase, and phosphoribulokinase) (Jacquot et al., 1997), and malate dehydrogenase (MDH) (Scheibe and Anderson, 1981). The activities of the target proteins in this group are regulated through the oxidation/reduction of the Trx-targeted disulfide bond located within the molecule. Once reduced by Trx, these target proteins no longer require reducing equivalents provided by Trx.

The second group comprises the “catalytic” types such as peroxiredoxin (Dietz, 2003) and methionine sulfoxide reductase (MSR) (Tarrago et al., 2009). Because “catalytic” type target enzymes use reducing equivalents provided by Trx as an integral part of their catalytic process, they must repeatedly interact with Trx during the catalytic cycle of a target enzyme.

During the dithiol–disulfide exchange reaction, the reduced form of Trx associates with and reduces the oxidized form of the target protein, and then the interchange in redox conditions results in oxidized and reduced forms, respectively. Moreover, when a disulfide bond on the target protein is reduced by the reduced form of Trx, the target protein and Trx immediately dissociate for re-reduction of the oxidized form of Trx by Trx-reductase. This suggests that these proteins must drastically change their affinity for Trx before and after the dithiol–disulfide exchange reaction.

Till date, several attempts regarding the interactions between Trx and the target proteins have been reported. The key residues on the surface of a Trx-f molecule involved in target specificity were revealed by mutation analyses (Geck et al., 1996; Geck and Hartman, 2000). The altered S_0.5 and V_max values for target enzyme activation caused by a Trx mutation indicated the importance of protein–protein interaction in addition to the dithiol–disulfide exchange reaction. In the case of the interaction between MSR and Trx from E. coli, the rate constants for the chemical reaction steps were determined by fluorescence stopped-flow measurements (Antoine et al., 2003; Olry et al., 2004). The authors concluded that the rate limiting step for regeneration of the reduced form of MSR by Trx was the dissociation of oxidized Trx from a reduced MSR complex.

Considering the Trx reduction reaction, there are two interaction modes between Trx and the target protein that depend on their redox states. One is the interaction between the reduced form of Trx and the oxidized form of target protein, and the other is the interaction between the oxidized form of Trx and the reduced form of target protein. Clarifying the difference between these interactions would be helpful for understanding Trx-mediated reduction mechanisms. However, conventional approaches are difficult to use for revealing the changes in affinity between Trx and the target proteins, in particular with regard to the redox states of Trx and its target proteins.

For characterization of these enigmatic but fundamental phenomena that occur within the Trx catalytic domain, we used surface plasmon resonance (SPR) to directly determine the association and dissociation rate constants of Trx to its target proteins. Arabidopsis thaliana cytosolic MDH and chloroplast peroxiredoxin Q (PrxQ) were used as “switch” and “catalytic” type target model proteins, respectively. Cytosolic MDH is a homodimeric enzyme that contains a Cys330–Cys330 intermolecular disulfide bond, and reduction of this disulfide bond by Trx has a strong effect on its activity (Hara et al., 2006). PrxQ is a chloroplast localized Trx-dependent peroxiredoxin, the activity of which is exhibited efficiently by the cytosolic Trx rather than the chloroplast Trx (Rouhier et al., 2004). Moreover, PrxQ is a monomeric protein that contains a Cys45–Cys50 intramolecular disulfide bond. Because MDH and PrxQ each have a disulfide bond, we selected them as target model proteins and Trxh as the reductant for this study.

Several studies attempted to evaluate the affinities of Trx to its target proteins or the reduction efficiencies of the target proteins by determining K_m or k_cat/K_m values obtained from the results of complete reduction of the target proteins using Trx (Collin et al., 2003; Perez-Perez et al., 2009; Maeda et al., 2010). However, a rather enigmatic behavior of Trx remained unresolved; the reduced form of Trx preferentially interacts with the oxidized form of the target protein and the oxidized form of Trx must release the reduced form of the target in order to ensure an efficient reduction cycle. To address this issue, we applied the direct binding measurements to detect the associations and dissociations of Trx with its target proteins.

For this purpose, MDH, PrxQ, and roGFP were selected as model target proteins. Cytosolic MDH is reportedly a target protein for Trxh1 (Hara et al., 2006) whose activity is regulated by its reduction/oxidation. Therefore, we defined this type of target protein as a “switch” type target for Trx. In contrast, PrxQ uses reducing equivalents provided by Trx for catalysis; thus, we defined it as a “catalytic” type target. Although PrxQ is localized in the chloroplasts of higher plants, cytosolic Trx can be an efficient reductant for this peroxiredoxin in vitro, as previously described (Rouhier et al., 2004). In addition, the reduction rate of PrxQ by Trxh1 (0.67 s⁻¹) obtained in this study was comparable to or higher than the reduction rate of PrxQ reduced by chloroplast type Trx (Collin et al., 2004; Perez-Perez et al., 2009). In this study, we selected a combination of PrxQ and Trxh1 for our binding study because PrxQ has only one pair of cysteines in its monomeric molecule and its reaction mode is the simplest among peroxiredoxins.

When the wild type Trxh1 was used as a ligand, no SPR signals were detected with MDH as the analyte. Based on the reaction mechanism of Trx-mediated reduction of a target disulfide bond, the target protein should be immediately released from Trx when the reduction reaction is complete. This implies that the number of bound target proteins on immobilized Trx should be statistically limited by the duration of the reaction period from the formation of a mixed disulfide bond to its reduction. The time during which the target protein remains on immobilized Trx may be too short to allow for detecting the bound molecules on the gold surface of an SPR sensor chip. In addition, when the reduction reaction is completed by the dithiol–disulfide exchange reaction, immobilized Trxh1 adopts its oxidized form and can no longer reduce the target protein because there is no way to simultaneously reduce immobilized Trxh1 alone. Thus, a direct analysis of the binding affinity of Trx to its target protein is difficult.

Therefore, we used the immobilized Trxh1_CS, which had been used for Trx affinity chromatography (Yamazaki et al., 2004), as the ligand in our SPR measurements. Because the Trxh1_CS could efficiently capture the target protein molecules through intermolecular disulfide bond formation, we expected that this mutant Trx could be used for observing the formation of a mixed disulfide intermediate complex, which is the initial contact of Trx and its target protein during the entirety of a reduction reaction. In contrast, a Trxh1_SC mutant, a Trxh1 mutant with a C40S substitution, was not used for these measurements because this mutant could not capture any specific target proteins when used for Trx affinity chromatography (Yamazaki et al., 2004).

As presented in Figure 1, SPR signals for the association of the analyte protein with the ligand were successfully obtained by using the Trxh1_CS as the ligand. In contrast, the Trxh1_CS did not associate with roGFP, which contains a disulfide bond that cannot be reduced by Trx. This indicated that the immobilized Trxh1_CS maintained specificity for the target proteins. In addition, the remarkable differences between the binding time courses for MDH and PrxQ (Figure 1) suggested that the method applied in this study was useful for our purposes.

Because the fast dissociation rate of the analyte from the mixed disulfide intermediate complex by the reverse reaction (k_off−rapid = 0.62 s⁻¹) appear to pose difficulty in accurate determination of the association rate constant when performing SPR analysis using PrxQ, we concluded that the association rate constant for the formation of the mixed disulfide intermediate complex was in the order of 10⁴ M⁻¹ s⁻¹. In addition, the association rate constant for PrxQ was higher than that for MDH by two orders of magnitude. These results were comparable to the reported differences in the reduction rates of PrxQ and MDH by the wild type Trxh1; 0.67 s⁻¹ and 6.5 × 10⁻³ s⁻¹, respectively (Table 1).

Why did Trx (Trxh1_CS) that mimics the reduced form of Trx interact with the oxidized form of MDH but not with the reduced form as presented in Figure 4A? A significant conformational change during the transition between the oxidized form and the reduced form of an MDH dimer has been suggested by size exclusion chromatography and analytical ultracentrifugation (Hara et al., 2006). This large conformational change must be critical for forming an intermolecular disulfide bond between Cys330–Cys330. In addition, the structure or the molecular surface of MDH for its interaction with Trx may only be exposed in its oxidized form but not its reduced form because on the basis of its reported crystal structure, the two Cys330 residues are located at the opposite sides in an MDH dimer molecule. This may be the reason for interaction of the Trxh1_CS with the oxidized form of MDH but not with the reduced form as presented in Figure 4A. In addition, a redox-dependent large conformational change has been reported for the other switch type target protein, HSP33. For HSP33, which is known to be a redox-dependent chaperone that contains four redox responsive cysteines, the domain that contains Trx-targeted disulfide bonds is largely unfolded in the oxidized form, although the reduced form of HSP33 is completely folded and coordinates zinc in this domain (Ilbert et al., 2007).

In contrast, the Trxh1_CS could bind to the reduced form of PrxQ as presented in Figure 4B. On the basis of the reported structures of the reduced and oxidized forms of PrxQ from Aeropyrum pernix (ApPrxQ), a redox-dependent conformational change was clearly observed at the region that contained two active cysteines (Perkins et al., 2012). In contrast, molecular modeling for BCP1, the homolog of PrxQ in Sulfolobus solfataricus, and its reductase, SsPDO (Limauro et al., 2010), revealed that the stable regions such as the end of β3 and α3 were involved in the molecular interaction, although these regions do not change their conformations in ApPrxQ in a redox-dependent manner. These reports suggest that a certain stable region on a PrxQ protein can interact with its partner protein irrespective of their redox states. The reduced form of PrxQ may have a similar interaction region on its molecular surface, and it may have caused the relatively weak association between the Trxh1_CS and the reduced form of PrxQ in our SPR experiments.

In this study, we conclusively confirmed that the redox state of the target protein was the significant determinant for its affinity to Trx. As presented in Figure 4, MDH and PrxQ altered their affinities for Trxh1. As previously noted, the Trxh1_CS cannot associate with the reduced form of MDH. The amount of the reduced form of PrxQ that associated with Trx was definitely less than that of its oxidized form. These results strongly suggest that the interaction between Trx and an oxidized target protein exhibits a higher affinity than that between Trx and the reduced form, a prerequisite for efficient association and subsequent dissociation.

In addition, the Trxh1_SS exhibited a preference for the oxidized target protein, even though its active domain lacks two cysteines (Figure 5). This indicates that the protein–protein interaction defined by the molecular surfaces of Trx and its target proteins is an important determinant for the redox-dependent selectivity of Trx, in addition to the cysteines in the catalytic domain of Trx and the rate of formation of the mixed disulfide intermediate complex. Similarly, the interaction between a Trx mutant that lacked two cysteines and its target ATP synthase complex was previously reported (Stumpp et al., 1999).

Considering together, these data reveal that the efficient reduction of the target proteins by Trx is accomplished by a remarkable change in the affinity between Trx and its target protein before and after the dithiol–disulfide exchange reaction. This selectivity is defined by these protein–protein interactions. The key molecular determinants of Trx that confer these interesting properties need to be elucidated by structural analysis of the Trx–target protein co-complexes.