Exploring the Conformational Transition Between the Fully Folded and Locally Unfolded Substates of the Escherichia coli thiol peroxidase

Abstract

AbstractThiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer in close to physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3 helices, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this crucial process, the mechanism of the FF→LU and the LU→FF transitions were studied using long time scale conventional molecular dynamic simulations and an enhanced conformational sampling technique for different oxidation and protonation states of CP and/or CR. Our results suggest that the FF→LU transition has a higher associated energy barrier than the refolding LU→FF process in agreement with the relatively slow experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of the αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionary optimized to fully speed-up the conformational transition of wild-type EcTPx.