Transition steps in peroxide reduction and a molecular switch for peroxide robustness of prokaryotic peroxiredoxins.

In addition to their antioxidant function, the eukaryotic peroxiredoxins (Prxs) facilitate peroxide-mediated signaling by undergoing controlled inactivation by peroxide-driven over-oxidation. In general, the bacterial enzyme lacks this controlled inactivation mechanism, making it more resistant to high H2O2 concentrations. During peroxide reduction, the active site alternates between reduced, fully folded (FF), and oxidized, locally unfolded (LU) conformations. Here we present novel insights into the divergence of bacterial and human Prxs in robustness and sensitivity to inactivation, respectively. Structural details provide new insights into sub-steps during the catalysis of peroxide reduction, enabling the transition from an FF to a LU conformation. Complementary to mutational and enzymatic results, these data unravel the essential role of the C-terminal tail of bacterial Prxs to act as a molecular switch, mediating the transition from an FF to a LU state. In addition, we propose that the C-terminal tail has influence on the propensity of the disulphide bond formation, indicating that as a consequence on the robustness and sensitivity to over-oxidation. Finally, a physical linkage between the catalytic site, the C-terminal tail and the oligomer interface is described.

As shown in Fig. 1, the detailed structural analysis of bacterial Prx1, called Alkyl hydroperoxide reductase subunit C (AhpC), reveals two distinct active site conformations linked with their catalytic cycle. In the reduced state, the active site is competent for productive substrate binding and is in a so-called fully folded (FF) conformation, where the peroxidatic cysteine, C P 47 (according to the Escherichia coli subunit AhpC numbering), is part of the α 2-helix and is located at the bottom of the catalytic cavity, formed by the conserved residues, P40, T44 and R120. In parallel, the resolving cysteine (C R 166), placed at the C-terminal tail of an adjacent subunit, is folded across the active site, placing C P and C R 14 Å away and in opposite orientation 18 (Fig. 1). In the oxidized state, the C P and C R are disulphide bonded in the so-called locally unfolded (LU) conformation, where (i) C P of helix α 2 is partially unwound, orienting C P towards C R , and (ii) the C-terminal tail unfolds from the active site region and becomes disordered 19,20 (Fig. 1). Additionally, the redox-state dependent active site conformations are proposed to modulate the quaternary structure of Prxs, whereas the reduced FF active site favors the decamer formation and the oxidized LU active site weakens the decamer into a dimer 19 . Our recent studies on the Escherichia coli AhpC (EcAhpC) explored the different functional role of its C-terminal tail, as influenced by the redox-state of the enzyme. In the oxidized state, the C-terminal tail of EcAhpC, which includes amino acids 172 to 187, is essential for regeneration by its specific Alkyl hydroperoxide reductase subunit F (AhpF) (Fig. 1). At the same time, the C-terminal tail is essential for the formation of a stable doughnut-shaped decamer under reduced conditions [21][22][23] .
Based on the elucidation of distinct redox-state-linked active-site conformations, it has been proposed that during the formation of the C P -SOH intermediate, the active site should move out of the FF conformation to facilitate the formation of the disulphide bond with C R 16 . Nevertheless, a detailed picture of the structural transition(s) between the FF and LU state during the catalysis is missing. In contrast, the structural studies on eukaryotic Prxs revealed that the FF active site conformation persists during and after oxidation of C P to C P -SOH (Fig. 1), which eventually promotes the over-oxidation of C P -SOH into C P -SO 2 H/C P -SO 3 H, and finally inactivates the enzyme 24,25 . The presence of two conserved sequence features in human and other eukaryotic Prxs, the so-called GGLG motif and the extended C-terminal helix containing the YF motif, are thought to be essential for regulating H 2 O 2 mediated signal transduction 5,18 . However, the greater importance of the C-terminal helix for sensitivity to over-oxidation has been confirmed 25,26 . The C-terminal helix with its YF motif folds across the active site, thereby delaying the conformational change from FF to LU, and favoring over-oxidation 15 (Fig. 1). It has been implicated that the disulphide formation or over-oxidation depends on the rate of FF to LU transitions. According to the proposed floodgate model, over-oxidation of Prxs facilitates the local accumulation of intracellular H 2 O 2 required for signaling events 18 . Finally, the enzyme sulfiredoxin (Srx) catalyzes the repair of over-oxidized Prxs, to restore their peroxidase activity 27 .
In order to (i) gain deep insight into the delicate balance between the FF and LU active site during the catalytic cycle, (ii) to explore the underpinning role of the C-terminal tail in active site conformation, and (iii) to understand the features making the bacterial and human Prxs more robust and sensitive to inactivation, respectively, a combination of genetic engineering, enzyme kinetics and crystallography were used to establish the values The basic functional dimeric unit of 2-Cys Prxs and their active site region is shown. During peroxide reduction, the reduced C P assumes the FF conformation and reacts with H 2 O 2 to form a C P -SOH intermediate. This local unfolding of the active site enables the formation of a disulphide bond with the C R located in the C-terminal tail of adjacent subunit. AhpC is then reduced by AhpF to its FF conformation for future catalytic cycles, with AhpF being oxidized in this process. AhpF is regenerated with the utilization of NADH molecules for further catalytic cycles. In comparison, human Prx is stabilized in the FF active site conformation even after the formation of the intermediate C P -SOH form, which eventually promotes over-oxidation.
Scientific RepoRts | 6:37610 | DOI: 10.1038/srep37610 of kinetic parameters and sensitivity for inactivation using the well-established E. coli AhpC and its mutants. By generating and determining the structure of a chimeric EcAhpC 1-186 -YFSKHN, which includes the extended C-terminal helix with the YF motif of human Prx2, we deduced for the first time the intermediate active-site conformations, lying between the FF and LU conformations. Furthermore, the studies reveal that the C-terminal tail acts as a molecular switch to mediate the structural transition between the FF and LU state. Finally, we propose the detailed conformational transition states that accompany the peroxide reduction and over-oxidation cycle, providing novel insight into the evolutionary divergence of the resistant and sensitive Prxs catalysts of bacteria and human, respectively.

Sensitivity to oxidative inactivation of a novel chimeric EcAhpC 1-186 -YFSKHN protein. The bac-
terial Prx is less sensitive to inactivation by hyper-oxidation, whereas the human Prx is sensitive towards inactivation with its C-terminal helix (YFSKHN-helix), including the conserved residues YF, playing a vital role 18 . The question arises, whether the apparent sensitivity to over-oxidation exerted by the C-terminal helix of human Prx can be transformed into Prxs of prokaryotes. We used the mechanistically well understood E. coli as a prototype to construct a chimeric Prx, EcAhpC 1-186 -YFSKHN, composed of EcAhpC (residues 1-186) and the C-terminal YFSKHN-segment of human Prx 2, to be compared with wild-type (WT) EcAhpC ( Fig. 2A). The E. coli TrxR-Trx system, the common reductase system to characterize Prxs, was used in the peroxidase assay, in which NADPHoxidation by TrxR provides the electrons via Trx to EcAhpC for H 2 O 2 reduction.
As shown in Fig. 2A, WT EcAhpC reacts with 1 mM H 2 O 2 and actively consumes NAPDH. Increasing the H 2 O 2 concentration to 10 and 20 mM caused an activity drop of ~12% and ~18%, respectively. The inhibitory effect of H 2 O 2 to EcAhpC increased significantly at a concentration of 30 mM, resulting in an enzyme activity reduction of ~44%. In comparison, an increase of 10 mM H 2 O 2 decreased the chimeric EcAhpC 1-186 -YFSKHN enzyme activity by already ~33% (Fig. 2B,C), followed by a further drastic decrease of ~50% in the presence of 20 mM H 2 O 2 , and finally to a ~64% drop with 30 mM H 2 O 2 (Fig. 2C). The data indicate that WT EcAhpC is resistant to inactivation up to 20 mM H 2 O 2 , as it shows little variation for peroxidase activity. In contrast, the peroxidase activity of chimeric EcAhpC 1-186 -YFSKHN is affected at H 2 O 2 concentrations above 1 mM, revealing that the transformation of the human C-terminal YFSKHN-helix inside the bacterial EcAhpC increased its sensitivity to over-oxidation.

Crystallographic structure of oxidized and decameric-shaped EcAhpC 1-186 -YFSKHN.
To understand the structural effect of the human YFSKHN-helix in general, in particular inside the chimeric EcAhpC 1-186 -YFSKHN, as well as to identify the individual amino acids essential for sensitivity to oxidative inactivation of Prxs, the crystal structure of oxidized EcAhpC 1-186 -YFSKHN has been solved at 2.7 Å resolution. The asymmetric unit contains one decamer composed of five catalytic dimers (α 2 ) 5 with each subunit containing one catalytic active peroxidatic and one resolving cysteine (Fig. 3A,B). The subunit consists of a central seven-stranded β -sheet, flanked at one side by four and at the other side by two α -helices (Fig. 3C). Each subunit has a dimer and oligomer interface. The dimer interface is mainly stabilized by salt bridge and hydrogen bond interactions, while the oligomer interface is mainly stabilized by hydrophobic interactions. Superposition of the recently resolved oxidized EcAhpC structure 20 with the corresponding dimer of the oxidized EcAhpC 1-186 -YFSKHN one resulted in an r.m.s.d. value of 0.4 Å. The intermolecular disulphide bond observed between C P 47 and C R 166′ in the dimer interface ( Fig. 3B,C) confirms the oxidized state of the EcAhpC 1-186 -YFSKHN structure. The peroxidatic cysteine (C P 47), located in the first turn of helix α 2, adopts an LU conformation in the oxidized structure (Fig. 3B,C and Supplementary Fig. S1A). In most of the disulphide bonded active sites of the decameric and oxidized EcAhpC 1-186 -YFSKHN, the C-terminal arm beyond the resolving cysteine (C R 166) is disordered, indicating the high flexibility of the C-terminus.
However, the structure in chain (H) could be resolved until residue 177, showing amino acids 167 to 177 oriented away from the active site and stacked against the neighboring symmetry molecule. Similarly, the C-termini of chains A, G and J are also stabilized by the packing interactions with the symmetry related molecules ( Supplementary Fig. S1B,C). The average main chain B-factors analysis revealed three dynamic regions of oxidized EcAhpC 1-186 -YFSKHN: (i) the C P 47 containing α 2-helix, (ii) helix α 5 that contains residues S86 and T88, and (iii) the C-terminal tail region (Fig. 3D). The sulfate ions observed in the interface region of the structure, emphasis their role in stabilizing the oxidized AhpC in a decameric form in solution 23 .
The active site conformation in reduced EcAhpC 1-186 -YFSKHN. To shine a light on the redox modulated structural alterations in the active site, the crystallographic structure of reduced EcAhpC 1-186 -YFSKHN was determined to 3.1 Å resolution (Fig. 4A). Except for the C-terminus, clear electron density was observed for all the residues and the resolving cysteine (chains C, E, F, H and J), which is located about 10 Å away from the peroxidatic one (Fig. 4B). No disulphide bond between C P 47 and C R 166′ was observed in any of the active site interfaces of the decameric EcAhpC 1-186 -YFSKHN, confirming that the structure fairly represents the reduced form of EcAhpC 1-186 -YFSKHN. In the reduced state, the typical FF active site is arranged in a way that the C P 47 positions in the first turn of helix α 2 and C R 166′ , containing the C-terminal arm, is folded across the dimer interface to come in proximity to the first turn of helix α 2 of another subunit. This conformational alteration brings the C P and C R ' to face opposite directions and to become separated by about 10 Å.
Interestingly, two different active site conformations were observed in the reduced EcAhpC 1-186 -YFSKHN structure ( Supplementary Fig. S2A), where the residues in the active site region are clearly defined in the electron density map except for the side chains of F45 and V46 (Fig. 4C,D). The first conformation (chains B-J) reveals that the peroxidatic C P 47 adopts helical conformation, bringing the C P 47 in a narrow solvent accessible pocket, which is surrounded by the highly conserved active site residues P40, T44 and R120 (Fig. 5A). In the second active site conformation (chain A), the first turn of α 2-helix is partially unfolded to place C P 47 in the loop conformation (Fig. 5A). However, no significant influence of crystal packing on the conformations of C P 47 was observed in the reduced EcAhpC 1-186 -YFSKHN structure ( Supplementary Fig. S2B,C). A comparison of these two reduced active sites with the oxidized LU state of EcAhpC 1-186 -YFSKHN reflects a significant difference in the loop region (T44-P48) (Fig. 5B), where the FF active site residues of the first active site of reduced EcAhpC 1-186 -YFSKHN are rotated, to expose C P 47 towards the C R 166′ , and finally forming a disulphide bond in the oxidized structure.
WT While in the second active site of reduced EcAhpC 1-186 -YFSKHN, the unfolded C P 47 adopts a lower magnitude of rotation, thereby preventing its full exposure towards C R 166′ to form a disulphide (Fig. 5B).
FF to LU transition state like active site conformation. To gain insight into (i) the structural transition pathway between the FF and LU state, and (ii) the role of the C-terminal tail on the active site conformation, the two presented distinct active sites of reduced EcAhpC 1-186 -YFSKHN were compared with a typical reduced FF and oxidized LU conformation, represented by the crystallographic structure of the Salmonella typhimurium AhpC 28 (PDB ID: 4MA9) and EcAhpC 1-186 -YFSKHN (see above), respectively. For clarity, we termed the first and second active sites of the reduced EcAhpC 1-186 -YFSKHN as FF like and LU like , respectively, to denote the folded and unfolded C P 47 in the active site environments (Fig. 6A). Firstly, the reduced EcAhpC 1-186 -YFSKHN and the S. typhimurium AhpC structure (PDB ID: 4MA9) share an overall similarity indicated by an r.m.s.d. of 0.5 Å for the superposition of the catalytic dimers. When zoomed into the catalytic relevant α 2-helix, the FF and FF like active sites differ significantly (Fig. 6A). While the residues F45 and V46 are placed in the starting position of helix α 2 inside the FF state, the α 2-helix becomes unwound in the FF like conformation and the main chain Cα atoms F45 and V46 shifted by about 0.9 Å. Similarly, in the FF like conformation the main chain Cα atoms of C P 47 and P48 moved about 0.6 Å and 0.5 Å, respectively, compared to their corresponding positions in the FF state (Fig. 6A). Furthermore, no obvious difference in the main chain conformations of the loop amino acids P40, T44 and R120 were observed between the two conformations.
The comparison of the FF (PDB ID: 4MA9) and LU like active site (see above) shows, significant structural variation in the first turn of helix α 2 and the loop region (Fig. 6B,C). Due to the partial unfolding of the α 2-helix, the main chain Cα atoms of V44, F45, V46, C47 and P48 of the FF active site moved by 0.4, 1.8, 1.9, 5.1 and 1.1 Å, respectively, to achieve the LU like state positions. As shown in Fig. 6B,C, the same residues moved during the transition from the FF to the LU state but with a higher magnitude of 1.4, 3.8, 3.8, 5.9 and 1.7 Å, respectively. Because of the magnitude of displacement, the data indicate that the FF like and LU like state, adopt intermediate conformations, lying between the catalytic FF and LU conformation. In addition, significant differences in the backbone torsion angles were determined between the FF, LU like and LU active sites (Supplementary Table S1). Taken together, it can be proposed that the FF like and LU like active sites mimic the initial and the intermediate phase of the structural deformation that occurs during the redox-modulated FF to LU conformational switch.
Oligomer interface region. The oligomeric behavior of Prxs is proposed to be redox modulated 29 . Under reduced conditions, the FF active site buttresses the oligomer interface, and favors decameric ring formation. Whereas under oxidized conditions, the LU active site mediates the restructuring of the oligomer interface region, resulting into lower order oligomers 19 . A structural view to the oligomeric interface in Fig. 7 highlights differences of the distinct active site loop conformations observed between FF, FF like , LU like and LU, where the C P -containing loop residues of the LU conformation are pulled away from the oligomer interface. This facilitates the destabilization of the oligomer interface through structural rearrangements of F43 and F45, which participate in the interface region 19,29 . The rest of the interface region residues remain unchanged in the crystal structures.
Effect of the C-terminal tail on substrate-binding and catalytic efficiency. By genetically engineering and enzymatically characterizing the EcAhpC mutants AhpC 1-172 , AhpC I187G and AhpC S86A,T88A , the questions were addressed, whether the folding of the C-terminal tail across the active site region as well as the dynamic region of helix α 5 with residues S86 and T88 (Fig. 8A), influences the enzyme kinetics.

The role of the C-terminus of bacterial AhpC in H 2 O 2 robustness.
Revealing that the transformation of the human C-terminal YFSKHN-helix inside the bacterial EcAhpC increased its sensitivity to over-oxidation, leads now to the question of whether the C-terminus of bacterial 2-Cys Prxs keeps the enzyme more robust and enabling the prokaryotes to survive under higher peroxide concentrations. To test this, the ability of the two C-terminal variants, EcAhpC 1-172 and EcAhpC I187G, to inactivation was studied. As shown in Fig. 9A (Figs 2C and 9A,B). The inhibitory effect of H 2 O 2 in EcAhpC 1-172 and EcAhpC I187G increased significantly at a concentration of 30 mM, resulting in an enzyme reduction of ~64% and ~62%, respectively. These values are comparable to the once of the chimeric EcAhpC 1-186 -YFSKHN, which were determined to be ~33% at a concentration of 10 mM H 2 O 2 followed by a further drastic decrease of 50% in the presence of 20 mM H 2 O 2 , and finally to a ~64% drop with 30 mM H 2 O 2 (Fig. 2B). The results demonstrate that both EcAhpC 1-172 and EcAhpC I187G are sensitized to inactivation in a magnitude similar to that of EcAhpC 1-186 -YFSKHN, and finally, that the C-terminus, in particular residue I187, is essential for the robustness at high H 2 O 2 concentrations (Supplementary Fig. S3). The sensitivity of EcAhpC 1-172 and EcAhpC I187G is surprising, since the destabilized active site, indicated by the increased Km value, is supposed to expose the C P for disulphide formation with C R ' 30 . We propose, that the EcAhpC 1-172,   The decamer building interface region between two subunits are shown. The significant structural difference in the interface region is observed only in the residues that form the C P -loop. Compared with "FF" (green) C P -loop structure, the "FF like " (blue), "LU like " (magenta) and "LU" (cyan) is shifted away (solid arrow) from the interface region and this facilitates the destabilization of the oligomeric interface through F43 and F45 (dotted arrow). The complementary interface region formed by the neighboring subunit is shown with light colors shade. and EcAhpC I187G mutants might affect the propensity of the disulphide formation ( Supplementary Fig. S3) which would indicate, that the C-terminus plays a role in aiding the disulphide bond formation.

Discussion
Evolutionary adaptation to differing environmental conditions was fundamental to the formation of the different kingdoms of life. Eukaryotic 2-Cys Prxs show a high sensitivity to peroxide inactivation by over-oxidation of the peroxidatic cysteine, C p , to sulphinic acid (C p -SO 2 H). Due to this adaptation, the enzyme keeps resting levels  of H 2 O 2 low, while permitting higher levels during signal transduction 5,18 . The eukaryotic GGLG motif and its extended C-terminal helix, including the YF motif (residues YFSKHN), are responsible for regulating H 2 O 2 mediated signal transduction 18 . The chimeric EcAhpC 1-186 -YFSKHN described here demonstrates, that transformation of the human C-terminal YFSKHN-helix into the related bacterial AhpC confers sensitivity to over-oxidation on the chimeric enzyme. As for human Prx2 (Fig. 1), the engineered YFSKHN-helix inside the chimeric EcAhpC 1-186 -YFSKHN falls across the active site of the chimeric enzyme, thereby delaying the conformational change from fully folded (FF) to locally unfolded (LU), thereby favoring over-oxidation.
The crystallographic structures of the oxidized and reduced chimeric EcAhpC 1-186 -YFSKHN also provide new insights into the sub-steps during catalysis of peroxide reduction, characterized by the initial formation of a C P -SOH intermediate that is generally unstable and prone to further oxidation. This would indicate that the protective mechanism is likely to oppose over-oxidation. The local unfolding (LU) of the active site is such a protection mechanism, but the precise nature of the structural alterations that accompany LU was not known, due to the lack of structural details of this important intermediate C P -SOH state in the bacterial 2-Cys Prx family member AhpC. Although the existence of a C P -SO 2 H state in the FF active site was demonstrated in human Prx 24 , but did not provide the needed insights for FF to LU transition. As demonstrated by the reduced EcAhpC 1-186 -YFSKHN structure (Fig. 10A-I, -II), a shift of the peroxidatic cysteine, C P 47, was observed combined with a first turn of the α 2-helix, which is now called FF like active site. This novel stage is proposed to mimic the early stage of conformational changes around the active site environment due to the oxidation of C P (Fig. 6A and Supplementary Fig. S4). The reduced C P adopts FF conformation and is essential to activate and stabilize the C P -thiolate for peroxidation. (II) The oxidative modification of C P to C P -SOH leads to localized structural alterations around the conserved active site pocket as seen from the "FF like " conformation of the reduced EcAhpC 1-186 -YFSKHN. (III) The "LU like " conformation in the reduce EcAhpC 1-186 -YFSKHN structure, reveals a severe clash between the active site residues with the C-terminal tail. The entire C-terminal arm (aa 166-187) is needed to undergo alterations and to enable the disulphide formation between C R ' and C P (solid arrow). (IV) The disulphide bonded active site is stabilized in the LU conformation, wherein the C-terminal tail is disordered (denoted by asterisk). (V) The C-terminal tail of AhpC binds to its reducing partner, the N-terminal domain (NTD) of AhpF, to regenerate for another catalytic cycle. (VI) In human Prxs, the extra C-terminal YFSKHN-helix is folded across the active site, thereby delaying the FF to LU transition during the intermediate C P -SOH state. In such case, the C P -SOH form can further react with peroxide and leads to enzyme inactivation (VII). (B) The cartoon representation shows that the C P and the first turn of the α 2-helix is stabilized by the weak interactions with the I187 and C-terminal tail, which is hold by residues S86 and T88. Disruption of weak interactions of the C-terminal tail, like between I187 and the α 2-helix or between I187 with S86A and T88A, leads to unfolding of the C-terminal tail from the active site region.
Scientific RepoRts | 6:37610 | DOI: 10.1038/srep37610 The critical step, which decides about inactivation or continues resolving and recycling of AhpC, is combined with the newly described LU like conformation in the reduced EcAhpC 1-186 -YFSKHN structure (Figs 6B and 10A-II). This structurally trapped conformation reveals a severe clash between the active site residues C P 47 and F45 with the C-terminal tail, causing the C-terminal tail residues L183′ , V184′ and I187′ , to move away in order to accommodate the preceding structural alteration in the FF active site due to oxidative modification of the peroxidatic cysteine, C P . This dynamic behavior of C-terminal tail residues is supported by the FF like structure (Fig. 6A), demonstrating that alterations in the C P and the first turn of the α 2-helix caused by oxidation can disrupt interactions of the C-terminal tail, like between L183′ , I187′ and the C P -loop residues or between I187 with S86A and T88A, the C-terminal tail unfolds partially and moves away from the active site (Fig. 10B). Therefore, alterations of the C-terminal tail confer instability on the FF active site, which in turn increases the rapid shift from the FF to the LU state. These structural rearrangements explain the significant increase of the binding constants observed for the EcAhpC I187G and EcAhpC S86A,T88A mutants (Table 1). Therefore, we propose that the side chain interactions of I187 with the C P -loop as well as with amino acids S86A and T88A affect binding of peroxide in the catalytic site.
The comparison of the presented LU like and oxidized structure of EcAhpC 1-186 -YFSKHN also provides novel insights into the resolution of the C P -SOH form. As shown in Fig. 10A-III, the transfer from a LU like to a LU state requires the entire C-terminal arm, including residues 166 to 187, to undergo a structural rearrangement that brings the resolving cysteine, C R 166′ , into proximity to finally resolve the C P -SOH form. Unfolding of the C-terminal tail might aid disulphide formation by favorably orienting both C P and C R ' . During the LU conformation, the C P and C R ' disulphide bonded active site becomes stabilized (Fig. 10A-IV), which weakens the oligomer building interface 19 . In this oxidized state, the C-terminal tail is highly disordered and not visible in the EcAhpC 1-186 -YFSKHN structure (Fig. 3C), but essential for binding with its reducing partner, the N-terminal domain (NTD) of AhpF (Fig. 10A-V). In this essential regenerative step of the catalytic cycle, the C-terminus of EcAhpC wraps around the NTD and slows the dissociation rate for an efficient electron transfer process 22 . Once the disulphide bond is reduced by the NTD of AhpF, the active site is present in equilibrium between the FF and LU state. In this way, the folding of the C-terminal tail of AhpC back into the active site region and the formation of a stable decamer interface maintain the conformational integrity of the FF active site (Fig. 10A-I). Such a FF active-site pocket is essential to activate and stabilize the C P -thiolate for peroxidation 16 .
Establishing the delicate balance between FF and LU conformation and identifying the critical C-terminal tail (amino acids 172 to 187 according to the EcAhpC) and residues (S86, T88) are important as they provide insights not only into the catalytic cycle of Prxs, but also the divergence of the bacterial and human 2-Cys Prxs in robustness and sensitivity to inactivation, respectively. In the sensitive human and more generally eukaryotic Prxs, the two important segments identified here, namely the C-terminal tail and residues S86 and T88, of the less sensitive bacterial Prxs are structurally replaced by the conserved eukaryotic GGLG motif and the YF motif inside the extra C-terminal helix (YFSKHN -helix). Although both the eukaryotic as well as the bacterial enzyme reveal similar catalytic efficiency, the structurally distinct motifs/segments make the eukaryotic enzyme susceptible to over-oxidation 31 . As revealed in Fig. 10A-VI, the eukaryotic C-terminal YFSKHN -helix is complemented by the GGLG motif, forming a stable structural segment across the active site to securely pack the peroxidatic cysteine, C P , in the FF conformation, even after the oxidative modification of the C P to the sulfenic acid form 24,25 . Based on the proposed model of bacterial AhpC, the C-terminal tail is resistant to unfolding from the active site due to the oxidation induced structural perturbation, thereby delaying the unfolding step needed to switch from the FF active site to the LU conformation. Moreover, the resolution of C P -SOH depends on the availability of the resolving cysteine, C R ' , which is also mediated by the unfolding of the C-terminal tail. This perspective is reflected in the rate of disulphide formation during catalysis, which in case of the bacterial AhpC is significantly higher (75 s −1 ) 32 , when compared to the low rate of 1.7 s −1 determined for human Prx2 33 . Taken together, natural selection for the stable C-terminal tail over a stable FF active site reveals the essential role of the C-terminal tail to act as a molecular switch that mediates the structural transition between the FF and LU state during the catalytic cycle.
The oxidized EcAhpC 1-186 -YFSKHN structure in Fig. 3C,D reveals that the active site loop-helix-motif and the C-terminal tail region adopt structural alteration, bringing C P and C R ' to the disulphide bonded LU state. This structural feature is important, since the redox-linked active site conformations are proposed to regulate oligomerization. This regulative mechanism proposes that the disulphide bonded LU state destabilizes the decamer while the C P and C R ' reduced FF conformation favors decamer formation 30 . With the studies presented here, we delineate the linkage between the active site conformation and the C-terminal tail residues. Our earlier studies demonstrated that the EcAhpC 1-172 , EcAhpC 1-182 , and EcAhpC I187G mutants prevented decamer formation in solution 21 , leading to the conclusion that the destabilized active site, caused by these mutants, might confer instability to the oligomer building interface (Fig. 7). Taken together, we propose a physical linkage can be established between the three major regions, namely the C P containing active site, the C-terminal tail and the oligomer interface. In the reduced state, this physical linkage enables the folded C-terminal tail and stable oligomer interface to facilitate formation of the FF active site and vice versa. However, in the oxidized state, these three structural regions are destabilized 19,30 .
In summary, a combination of complementary approaches of genetic engineering, protein chemistry, enzymatic assays, and structural biology has provided new insights into the unique aspect of divergence between the bacterial and human 2-Cys Prxs in robustness and sensitivity to inactivation, respectively. Structural details of the oxidized and reduced chimeric EcAhpC 1-186 -YFSKHN provide novel insights into sub-steps during the catalysis of peroxide reduction, enabling the transition from a fully folded to a locally unfolded conformation. Together with mutational and enzymatic studies these data unravel the fundamental role of the C-terminal tail as a molecular switch that mediates the structural transition between the FF and LU state during the catalytic cycle. Finally, a physical linkage between the C P -containing active site, the C-terminal tail and the oligomer interface was established.
Enzymatic characterization using a peroxidase assay. Peroxide-dependent activity of the various forms of purified recombinant EcAhpC proteins was measured by coupling its activity with NADPH-oxidation (ɛ 280 = 6220 M −1 s −1 ) catalyzed by EcTrxR and EcTrx. The peroxidase activity was carried out at 25 °C by monitoring the decrease in NADPH-absorbance at 340 nm for 120 sec using a stopped-flow spectrophotometer SX20 (Applied Photophysics, UK). The reaction mixture containing 100 μ M NADPH, 50 mM HEPES buffer pH 7.0, 100 mM of ammonium sulfate, 0.5 mM EDTA, 0.25 μ M of EcTrxR, 4 μ M of EcTrx, and 4 μ M of EcAhpC were mixed with varying concentrations of hydrogen peroxide (250 nM-100 μ M) to initiate NADPH-oxidation. The rate of NADPH-oxidation was calculated by a least square fit to the linear portion of the curve. NADPH consumption measured in the absence of peroxiredoxin was taken as a control. The background rate was subtracted from the experimental rate to determine the activity due to EcAhpC. All rates reported here are the average of three independent experiments. Inactivation assay. Peroxide-dependent overoxidation of EcAhpC, -AhpC 1-172 , -AhpC 1187G and the chimeric EcAhpC 1-186 -YFSKHN were measured similar to the above mentioned condition, using hydrogen peroxide concentrations of 1-30 mM. NADPH-oxidation was monitored at 340 nm at 25 °C for 210 sec. Background NADPH-oxidation observed for EcTrx and EcTrxR in the absence of Prx, which is significant at higher H 2 O 2 concentrations, was subtracted to estimate the activity due to Prx. The rate of reaction for each hydrogen peroxide concentration was calculated by fitting the linear portion of the curve. The rate observed for the lowest concentration of hydrogen peroxide is taken as 100% activity, to calculate the remaining enzyme activity in percentage at each concentration of hydrogen peroxide.
Peroxide reduction assay using SDS-PAGE. This assay is based on the observations that the reduced and oxidized 2-Cys Prxs run as a monomer and dimer, respectively, in a non-reducing SDS-PAGE 33 . Prior to each experiment, WT EcAhpC, EcAhpC 1-172 and EcAhpC 1187G were reduced with 20 mM dithiothreitol (DTT) in 50 mM phosphate buffer, pH 7.4 containing 1 mM diethylenetriaminepentaacetic acid for 1 h. Reduced proteins were separated from excess of DTT using a PD-10 desalting column (GE Healthcare). The different forms of EcAhpC (30 μ M) were incubated with varying concentrations of H 2 O 2 for 5 min. The reaction was stopped by adding 50 mMN-ethyl maleimide in a sample buffer (4% SDS, 10% glycerol and 62.5 mM Tris-HCl, pH 6.8) and analyzed by a non-reducing 17% SDS-gel.
Crystallization of EcAhpC 1-186 -YFSKHN. Chimeric EcAhpC 1-186 -YFSKHN was concentrated to 8 mg/ml in buffer containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, using a Millipore spin concentrator with a molecular-mass cutoff of 10 kDa. An initial crystallization attempt was carried out using the recent protocol of EcAhpC (1.8 M ammonium sulfate, 100 mM MES (2-(N-morpholino) ethanesulfonic acid), pH 6.5 and 5% Dioxane) 20 , and the hanging-drop vapour diffusion method in 24-well VDX plates with sealant at 291 K. Diffraction quality crystals were obtained in the optimized condition of 1.6 M ammonium sulfate, 100 mM MES pH 6.5, 5% Dioxane and a protein concentration of 4.5 mg/ml, yielding rod shaped crystals of 0.3 mm × 0.2 mm × 0.1 mm. Reduced EcAhpC 1-186 -YFSKHN crystals were grown by soaking the oxidized crystals with 1 mM Tris(2-carboxyethyl) phosphine (TCEP) for 1-3 min. There was no cracking or disintegration of crystals observed during the soaking.
Data collection and structure determination. Crystals of EcAhpC 1-186 -YFSKHN were quick-soaked in a cryoprotectant solution containing 25% glycerol in the mother liquid and flash-cooled in liquid nitrogen at 100 K. A single wavelength dataset for both the oxidized and reduced EcAhpC 1-186 -YFSKHN were collected at 140 K, beamline 13B1 of the National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan) using a ADSC Quantum 315 CCD detector. The diffraction data were indexed, integrated and scaled using Mosflm 36 and HKL2000 suite 37 . Data collection and processing statistics for oxidized and reduced EcAhpC 1-186 -YFSKHN are summarized in Table 2. Oxidized EcAhpC 1-186 -YFSKHN crystals belong to the monoclinic space group P2 1 with the unit cell parameters a = 99.47 Å, b = 134.73 Å, c = 107.53 Å and β = 111.06°. The unit cell parameters of reduced EcAhpC 1-186 -YFSKHN are similar to that of the oxidized one ( Table 2). The asymmetric unit contains 10 molecules with the solvent content of about 60%. The structure of oxidized EcAhpC 1-186 -YFSKHN was solved using the crystallographic structure of oxidized EcAhpC (PDB ID: 4O5R) 20 as model for molecular replacement by the program PHASER 38 . The reduced form of EcAhpC 1-186 -YFSKHN was solved using the reduced structure of Salmonella typhimurium AhpC (PDB ID: 1N8J) 18 . Refinement 39 was done until convergence and the geometry of the final model was validated with MolProbity 40 . The figures were generated using PyMOL 41 and structural comparison analysis was carried out by SUPERPOSE 42 as included in CCP4 suite 43 .