# Structural and Biochemical Characterization of Thioredoxin-2 from _Deinococcus radiotrans_
## 1 Introduction  
Maintaining a proper redox state in the extracellular or intracellular environments is critical for a variety of cellular processes. The main protein systems that regulate the thiol/disulfide redox balance in the cytoplasm are the thioredoxinoxin (Trx) system, generally composed of thioredoxin reductase (TrxR)/Trx/reduced nicotinamide adenine dinucleotide phosphate (NADPH), and the glutaredoxin (Grx) system, composed of glutathione reductase (GR)/Grx/glutathionee (GSH). The Trx system is a ubiquitous redox system found in all living organisms [1], whereas the Grx system is absent in many prokaryotes, including some anaerobic bacteria [2; 3]. Interestingly, the Trx systems in several anaerobic organisms utilize reduced nicotinamide adenine dinucleotide (NADH) (e.g., _Thermotogamaritiana, Desulfofibrio vulgaris_, and _Thermoplasma acidophilum_[4; 5; 6], reduced coenzyme F\({}_{420}\) (F\({}_{420}\)H\({}_{2}\)) (e.g., _Methanocaldococcus jannaschi_) [7; 8], or reduced ferredoxin (e.g., _Clostridium pasteurianum_) [9] instead of NADPH. Trx is a small protein, of approximately 12 kDa, with a highly conserved CXXC motif in its active site [1; 10; 11; 12], which reduces the disulfide bonds in a wide range of proteins. Trx was first identified in _Escherichia coli_. Two cytoplasmic Trx proteins, Trx1 (EcTrx1) [13] and Trx2 (EcTrx2) [14], are found in _E. coli_. EcTrx1 catalyzes the reduction of ribonucleotide reductase (RNR), methionine sulfoxide reductase (Msr), and 3\({}^{\prime}\)-phosphoadenylylsulfate (PAPS) reductase, which are important for DNA synthesis, protein repair, and sulfur assimilation, respectively [15; 16]. EcTrx2 contains an additional 32 amino acids in its N-terminal region compared to EcTrx1 and shares 28% sequence identity with EcTrx1. EcTrx2 also reduces RNR, the inner membrane protein DsbD [17] and PAPS reductase, similarly to EcTrx1. Although Trx1 is found in all species, some bacteria do not possess Trx2 [15; 18; 19]. Trx is characterized by a common three-dimensional structure, consisting of four \(\alpha\)-helices and five \(\beta\)-sheets [20; 21], wherein, its active site CXXC motif, located after the \(\beta\)2-sheet, forms the N-terminal portion of \(\alpha\)2 [10; 22]. Trx2 contains an additional N-terminal domain, including two CXXC motifs responsible for zinc binding. Based on its structural homology to the zinc-fingers of Npl4 and Vps36, it was proposed that the zinc-finger domain of Trx2 may mediate protein-protein interactions [23]. During the catalytic cycle, the two cysteines in the active site CXXC motif alternately exist in the form of a reduced dithiol or an oxidized disulfide. When Trx reduces a disulfide bridge of its target proteins, it is oxidized and the disulfide in Trx is reduced back to the thiol state by an NADPH-dependent thioredoxin reductase (NTR) [24; 25]. The Trx system is important for cellular defense against oxidative stress, because oxidative stress promotes disulfide bond formation in redox-sensitive proteins, thereby functionally modulating these proteins [26]. Furthermore, Trx directly reduces H\({}_{2}\)O\({}_{2}\) by providing a reduction equivalent to peroxideroxins (Prxs) [27] and functions as a singlet oxygen quencher, a hydroxyl radical scavenger [28], and a hydrogen donor for peroxiderases [29]. Several proteins responsible for oxidative stress response, such as superoxide dismutase, hydrogen peroxidase, ferric uptake regulator, and aconitase, were found to interact with Trx1 in _E. coli_[30]. In addition, alkyl hydroperoxide peroxidase C (AhpC), thiol peroxidase (Tpx), and bacteriferitin comigatory protein (BCP) reportedly also use reduced Trx as an electron donor for the catalytic reduction of substrates [31]. _Deinococcus radiolurans_ is extremely resistant to ionizing radiation (IR), ultraviolet (UV), desiccation, and oxidizing agents [32; 33; 34; 35; 36]. Cellular exposure to IR increases various reactive oxygen species (ROS) and subsequently causes oxidative damage to DNA, lipids, and proteins [37; 38]. _D.