Molecular basis of the mechanism of thiol oxidation by hydrogen peroxide in aqueous solution: challenging the SN2 paradigm

ARI ZEIDA; RYAN BABUSH; MARIANO CAMILO GONZÁLEZ LEBRERO; MADIA TRUJILLO; RAFAEL RADI; DARIO A. ESTRIN

Santiago de Chile

Congreso; ISCB Latin American; 2012

INTERNATIONAL SOCIETY FOR COMPUTATIONAL BIOLOGY

Oxidation of cellular thiol containing compounds such as glutathione and protein Cys residues by reactive oxygen species, is considered to play important roles in a wide array of biological processes including signal transduction, regulation of the activity of enzymes, protein channels, transcription factors and antioxidant responses. Hydrogen peroxide (H2O2) is produced in many cell types as a response to a variety of extracellular stimuli and could work as a ubiquitous intracellular messenger. Particularly, the two-electron oxidation of reactive protein Cys by H2O2 is a key event during redox signaling and regulation. Though this reaction has been productively studied, the exact mechanism remains poorly understood. There is consensus about thiolates being much more prone to oxidation than protonated thiols and that they react with the protonated form of hydroperoxides. The reduction of H2O2 by thiolates in aqueous solution has been described as a bimolecular nucleophilic substitution (SN2) in which the thiolate acts as the nucleophilic moiety generating the breaking of the O-O peroxide bond and displacing the HO- leaving group.5 However, recent theoretical studies have demonstrated that the product of the reaction would be H2O instead of OH-, which is inconsistent with a SN2 mechanism. In this context, Chu and Trout have investigated the reaction between dimethylthiol and H2O2 via DFT calculations, analyzing the effect of the aqueous environment including up to three explicit water molecules. In successive works, using an IMOMO methodology, Cardey and Enescu modeled the oxidation of methanethiolate (CH3S-) and Cys-thiolate (Cys-S-) by H2O2, including solvent effects through a continuum PCM model. The overall oxidation reaction has been reported to occur through: RS- + H2O2 → RSO- + H2O (eq. 1) where RSO- is the deprotonated form of sulfenic acid. Oxidation rate constantsi of the reaction on eq. 1 are in the ~10 M-1s-1 range for low molecular weight thiols in aqueous solution.4 It has been found that rate constants exhibit almost no dependence on the thiol pKa value and all considered low molecular weight thiols presented similar pH independent rate constants. The free energy barrier of the reaction of free Cys with H2O2 was estimated as 15.9 kcal/mol. Remarkably, the rate constants for peroxidatic thiols in Cys-dependent peroxidases are several order of magnitude larger, in the ~104-108 M-1s-1 range. Moreover, very recent works proposed that protein environment could account for a hydrogen network and substrate placing such as to provide an alternative mechanism to the one found in aqueous solution. In this context, a SN2 or other kind of mechanism may explain the higher reaction rates in enzyme-catalyzed reductions of hydrogen peroxide. Clearly the reaction mechanism of this extremely relevant reaction is not still understood from a molecular viewpoint. As a first step towards elucidating this mechanism we present an integrated QM-MM investigation of the oxidation of methanethiolate (CH3S-) by H2O2, embedded in a classical water molecules box, under periodic boundary conditions. The choice of methanethiolate is due to its small size which allows for more efficient sampling in the expensive QM-MM MD simulations. This is justified by the fact that for low molecular weight thiols the reported rate constants of oxidation by H2O2 are very similar, suggesting that the same mechanism is operative. Our model is a realistic representation of the aqueous environment at room temperature. We explore the reaction by means of QM-MM MD simulations using an umbrella sampling scheme. This approach allowed us to obtain thermodynamical information such as the free energy profile in addition to microscopic insight about electronic structure changes throughout the reaction. The results presented in this work suggest that the process cannot be considered as a simple substitution since the product formation involves a hydrogen atom transfer. The data reported herein provides a detailed microscopic view of the thiolate oxidation reaction by H2O2 in water, and is the first step for future studies related to the mechanisms of thiolate oxidation in different protein environments.