Exploring the conformational transition between the fully folded and locally unfolded substates of Escherichia coli thiol peroxidase

DIEGO S. VAZQUEZ; ARI ZEIDA; WILLIAM A. AGUDELO SUÁREZ; MÓNICA R. MONTES; GERARDO FERRER-SUETA; JAVIER SANTOS

PHYSICAL CHEMISTRY CHEMICAL PHYSICS

ROYAL SOC CHEMISTRY

Año: 2020 vol. 22 p. 9518 - 9533

1463-9076

Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of differenthydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to asulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CPto form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol anddisulfide behaves as a monomer under near physiological conditions. Although secondary structurerearrangements are present when comparing different redox states of the enzyme, no significantdifferences in unfolding free energies are observed under reducing and oxidizing conditions.A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving apartial unfolding of aH2 and aH3, must occur to enable the formation of the disulfide bond since thecatalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF - LUand LU - FF transitions were studied using conventional molecular dynamics simulations and anenhanced conformational sampling technique for different oxidation and protonation states of the activesite cysteine residues CP and CR. Our results suggest that the FF - LU transition has a higher associatedenergy barrier than the refolding LU - FF process in agreement with the relatively low experimentalturnover number of EcTPx. Furthermore, in silico designed single-point mutants of aH3 enhancedlocally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarilyoptimized to fully speed-up the conformational transition of wild-type EcTPx.