# Structural and Biochemical Characterization of Thioredoxin-2 from _Deinococcus radiotrans_
## 3 Results and Discussion
### Reduction Activity of DrTrx1 and DrTrx2  
To compare the reduction activity of purified DrTrx1 and DrTrx2, we measured the disulfide reduction activity of DrTrx1 and DrTrx2 using an insulin reduction assay. In the presence of DrTrxR/NADPH, both DrTrx1 and DrTrx2 caused a notable decrease in the absorption at 340 nm, indicating that NADPH was consumed during insulin reduction (Figure 2A,B). DrTrx1 showed a relative percentage activity that was 5-fold higher than that of DrTrx2. This result showed that DrTrx1 and DrTrx2 reduce insulin disulfides via the DrTrxR/NADPH regeneration system. Furthermore, the capacity of two Trx proteins to reduce the exposed disulfide bond was explored using DTNB as a generic disulfide substrate. Similarly to the insulin reduction assay, both DrTrx1 and DrTrx2 clearly showed an increase in absorbance at 412 nm, showing that the reduction of DTNB to TNB occurred only in the presence of both DrTrxR and NADPH (Figure 2C,D). The DTNB reduction activity of DrTrx1 was approximately 4-fold higher compared to that of DrTrx2. This result is compatible with a previous report that showed that the catalytic efficiency of Trx1 was three-times higher than that of Trx2 in _B. anthracis_[54]. To further investigate the role of cysteines conserved in the CXXC motif of DrTrx1 and DrTrx2, single amino acid substitution mutants, leading to the conversion of Cys to Ser in the CXXC motif were prepared (C29S and C32S in the active site CXXC motif of DrTrx1, C7S and C27S in the N-terminal zinc-binding motif of DrTrx2, and C64S and C67S in the active site CXXC motif of DrTrx2) (Figure S3). Insulin and DTNB reduction assays were performed under the same conditions utilized for native Trx proteins. DrTrx1C29S, DrTrx1C32S, DrTrx2C64S, and DrTrx2C67S mutants completely lost their insulin or DTNB reduction activity, even under conditions that included DrTrxR and NADPH (Figure 3). However, DrTrx2C7S and DrTrx2C27S mutants still showed reduction activity towards insulin and DTNB, with DrTrx2C27S exhibiting a higher activity than DrTrx2C7S (Figures 3C,D and S4). These results suggest that the binding of zinc ion affects the reduction activity of DrTrx2 by stabilizing the N-terminal domain, which is supposedly involved in Trx-target interactions [23]. Additionally, the ratios of molar concentration between DrTrx2 proteins and zinc ion analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) were 1:1.39, 1:0.63, and 1:0.57 for DrTrx2, DrTrx2C7S, and DrTrx2C27S, respectively (Table S3). Previous reports have indicated that the zinc binding motif in _E. coli_ Trx2 displays an extremely high degree of affinity for zinc ion (\(K_{\alpha}>10^{18}\) M\({}^{-1}\)) compared to other reported zinc binding proteins and that formation of the disulfide bond by this zinc binding do main following H\({}_{2}\)O\({}_{2}\) treatment results in the release of zinc ions and a conformational change [18]. Figure 3: Effects of mutation in the conserved cysteine residues. The reduction activities of DrTrx1, DrTrx2, and its mutant proteins were compared using insulin and DTNB reduction assays coupled to the DrTrxR/NADPH regeneration system. DrTrx1 and its active site CXXC motif mutants (C29S, C32S) were used for insulin (**A**) and DTNB (**B**) reduction assays. DrTrx2 and its zinc-finger domain mutants (C7S, C27S) and active site CXXC motif mutants (C64S, C67S) were used for insulin (**C**) and DTNB (**D**) reduction assays. Relative activities of insulin and DTNB reduction of DrTrx1 and DrTrx2 are depicted as a bar graph, using the absorbance at 15 min (DTNB) and 30 min (insulin), where the activity of native DrTrx1 and DrTrx2 was set to 100%. Error bars indicate the standard deviation for three experimental replicates. Figure 2: Reduction of insulin and DTNB by DrTrx1 and DrTrx2. Reduction of insulin by DrTrx1 (0.1 uM) (**A**) and DrTrx2 (0.5 uM) (**B**) coupled to the DrTrxR/NADPH regeneration system.