On the Overwhelming Complexity of Mechanochemical Disulphide Bond Reduction in Alkaline Solution

The coupling between mechanical stress and the reactivity of disulphide bridges has recently received a great deal of attention due to its broad relevance in biochemistry and materials science. Here, we will highlight the main findings of our computational studies on mechanochemistry of disulphide bridges, which have been carried out in the past few years in the framework of GCS Large Scale Projects. Our investigations have disclosed a very complex mechanistic scenario for the mechanochemistry of disulphides in aqueous alkaline solution. In the low-force regime, external forces play a dual role in the reduction of disulphide bridges via a bimolecular SN2 attack of a hydroxide ion at a sulphur atom. On the one hand, the external tensile force accelerates the reaction by virtue of the mechanical work performed on the system as the reaction proceeds. On the other hand, tensile forces can induce a conformational distortion of the disulphide moiety that drives the system into a spatial arrangement that is less prone to a nucleophilic attack due to steric hindrance. In the high-force regime, in turn, the tensile force gives rise to a competition between bimolecular SN2 and unimolecular C–S bond breaking mechanisms as well as to drastic changes in the free energy landscape of the system as a result of which bimolecular reaction pathways transform into pure bond-breaking processes. Our results not only provide a rationale for the enigmatic outcome of certain single-molecule force spectroscopy experiments but also suggest new experiments to continue unravelling the intricacies of the mechanochemistry of disulphide bridges

Unexpected mechanochemical complexity in the mechanistic scenarios of disulfide bond reduction in alkaline solution

The reduction of disulfides has a broad importance in chemistry, biochemistry and materials science, particularly those methods that use mechanochemical activation. Here we show, using isotensional simulations, that strikingly different mechanisms govern disulfide cleavage depending on the external force. Desolvation and resolvation processes are found to be crucial, as they have a direct impact on activation free energies. The preferred pathway at moderate forces, a bimolecular S(N)2 attack of OH-at sulfur, competes with unimolecular C-S bond rupture at about 2 nN, and the latter even becomes barrierless at greater applied forces. Moreover, our study unveils a surprisingly rich reactivity scenario that also includes the transformation of concerted S(N)2 reactions into pure bond-breaking processes at specific forces. Given that these forces are easily reached in experiments, these insights will fundamentally change our understanding of mechanochemical activation in general, which is now expected to be considerably more intricate than previously thought

The effect of tensile stress on the conformational free energy landscape of disulfide bonds.

Disulfide bridges are no longer considered to merely stabilize protein structure, but are increasingly recognized to play a functional role in many regulatory biomolecular processes. Recent studies have uncovered that the redox activity of native disulfides depends on their C-C-S-S dihedrals, χ2 and χ'2. Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force-clamp spectroscopy and computer simulation. The χ2 and χ'2 angles have been found to change from conformations that are open to nucleophilic attack to sterically hindered, so-called closed states upon exerting tensile stress. In view of the growing evidence of the importance of C-C-S-S dihedrals in tuning the reactivity of disulfides, here we present a systematic study of the conformational diversity of disulfides as a function of tensile stress. With the help of force-clamp metadynamics simulations, we show that tensile stress brings about a large stabilization of the closed conformers, thereby giving rise to drastic changes in the conformational free energy landscape of disulfides. Statistical analysis shows that native TDi, DO and interchain Ig protein disulfides prefer open conformations, whereas the intrachain disulfide bridges in Ig proteins favor closed conformations. Correlating mechanical stress with the distance between the two a-carbons of the disulfide moiety reveals that the strain of intrachain Ig protein disulfides corresponds to a mechanical activation of about 100 pN. Such mechanical activation leads to a severalfold increase of the rate of the elementary redox S(N)2 reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides