Date of Award

Spring 2017

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Timerghazin, Qadir K.

Second Advisor

Reid, Scott

Third Advisor

Gardinier, James


S-nitrosothiols (RSNOs) have long been proposed as potential sources of the elusive endogenous nitroxyl (HNO) via S-thiolation reaction with thiols. It is however not clear how S-thiolation can compete with the trans-S-nitrosation pathway commonly observed in vitro. Based on the insights into the highly unusual, antagonistic chemical nature of RSNO molecules, we hypothesize that, while difficult in vitro, S-thiolation could easily lend itself to enzymatic catalysis. To explore this possibility, we adopted a bottom-up computational approach that aims to identify possible catalytic mechanisms able to steer the RSNO + thiol reaction toward HNO production. Inspired by recent discovery of small bioactive HSNO molecule and its involvement in HNO production, we first study HSNO molecule and its subsequent isomerization reactions that can possibly lead to other potentially bioactive small molecules or reactive intermediates that can undergo S-thiolation reaction. Then, we mapped out the profile of the uncatalysed S-thiolation reaction that suggests that the most difficult step of S-thiolation is the R’SH to RSNO proton transfer, and that the reaction proceeds through unusual highly polar zwitterionic species R’SS+(R)N(H)O– that further decomposes yielding HNO and disulfide RSSR’. Therefore, facilitating the proton transfer and stabilizing this zwitterionic intermediate could lead to dramatic acceleration of this reaction. Further, we identified the required arrangements of amino acid residues using theozyme (‘theoretical enzyme’) modeling. These calculations showed that several of these putative active site models can drop the energetic barrier for S-thiolation/HNO formation to less than 10 kcal/mol. An extensive search in the RCSB protein data bank yielded over 600 structures that match one of these successful theozyme models. Remarkably, among these proteins we found DJ-1 protein known to be involved in RSNO-related processes. Furthermore, full-scale modeling of S-thiolation between an incoming RSNO and DJ-1 Cys106 using a hybrid quantum mechanics/molecular mechanics approach have shown that the reaction can be indeed efficiently catalyzed by the His126–Glu18 dyad, a prediction whose relevance to the DJ-1/RSNO biochemistry presents an intriguing question to the experimentalists.