Abstract
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.
Highlights
The Prx subfamily enzyme Prx[1], typically called 2-Cys Prx, is among the most highly expressed, soluble proteins in the cell[15], and is characterized by two conserved cysteine residues
The bacterial 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 E. coli thioredoxin reductase (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 Escherichia coli AhpC (EcAhpC) for H2O2 reduction
Summary
The Prx subfamily enzyme Prx[1], typically called 2-Cys Prx, is among the most highly expressed, soluble proteins in the cell[15], and is characterized by two conserved cysteine residues. The detailed structural analysis of bacterial Prx[1], called Alkyl hydroperoxide reductase subunit C (AhpC), reveals two distinct active site conformations linked with their catalytic cycle. The active site is competent for productive substrate binding and is in a so-called fully folded (FF) conformation, where the peroxidatic cysteine, CP47 (according to the Escherichia coli subunit AhpC numbering), is part of the α2 -helix and is located at the bottom of the catalytic cavity, formed by the conserved residues, P40, T44 and R120. The CP and CR are disulphide bonded in the so-called locally unfolded (LU) conformation, where (i) CP of helix α2 is partially unwound, orienting CP towards CR, and (ii) the C-terminal tail unfolds from the active site region and becomes disordered[19,20] (Fig. 1). 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
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