| Abstract
| - The thiol/disulfide oxidoreductases of the thioredoxin family have, in the active site, two cysteines that canbe in a reduced or an oxidized form. One of the cysteines in the reduced state is deprotonated, and it is callednucleophilic cysteine. The pKa of this cysteine is different from that of a normal cysteine and varies widelyamong the different enzymes of this family. However, the factors responsible for the different degrees ofstabilization of nucleophilic cysteine thiolate are not fully understood. Here, we have studied the well-knownhypothesis of proton sharing between the active site thiols by performing a linear transit scan for the transferof the proton between the active site cysteines. We used a two-layered (DFT/MM) ONIOM formalism, withthe active site region treated at the B3LYP/6-31+G(d) level and the remains of the protein treated with theAmber Parm94 force field. The solvation free energy was accounted for with a continuum solvent model, bysolving the Poisson−Boltzmann equation using the program Delphi. We have obtained excellent agreementwith the experimental data available in the literature. Besides refuting the proton sharing hypothesis, ourresults include a value of 14.0 for the pKa of the buried cysteine, a quantity that has not been possible toobtain experimentally but which has been proven to be higher than 11. Additionally, this study also providesdetailed information on the very interesting and so far unknown fact that the contribution of the enzymaticstructure (8.3 kcal/mol) prevails in relation to that of the solvent (0.60 kcal/mol) concerning the differentialstabilization of the active site thiolates.
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