1 Department of Chemistry and Biochemistry, Centre for Research in Molecular Modeling (CERMM), Concordia University, 7141 Sherbrooke Street West, Montréal, QC, H4B 1R6, Canada.
2 Quebec Network for Research on Protein Function, Structure, and Engineering (PROTEO), Montréal, Canada.
3 Theoretical Molecular Biophysics Laboratory, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD, 20814, USA.
4 Department of Chemistry and Biochemistry, Centre for Research in Molecular Modeling (CERMM), Concordia University, 7141 Sherbrooke Street West, Montréal, QC, H4B 1R6, Canada. ann.english@concordia.ca.
5 Quebec Network for Research on Protein Function, Structure, and Engineering (PROTEO), Montréal, Canada. ann.english@concordia.ca.
6 Department of Chemistry and Biochemistry, Centre for Research in Molecular Modeling (CERMM), Concordia University, 7141 Sherbrooke Street West, Montréal, QC, H4B 1R6, Canada. gilles.peslherbe@concordia.ca.

Nitroglycerin is a potent vasodilator in clinical use since the late 1800s. It functions as a prodrug that is bioactivated by formation of an enzyme-based thionitrate, E-Cys-NO₂. This intermediate reportedly decomposes to release NO and NO₂^- but their relative yields remain controversial. Hence, we determined barriers for NO and NO₂^- production from the model thionitrate, CH₃SNO₂, using comprehensive high-level quantum chemistry calculations [CCSD(T)//MP2/aug-cc-pVTZ]. We find that the sulfenyl nitrite, CH₃SONO, readily releases NO on (S)O-N bond homolysis but CH₃SONO formation from CH₃SNO₂ either by S-NO₂ bond homolysis or concerted rearrangement faces prohibitively high barriers (?H_calc/?H^‡_calc > 42 kcal/mol). Dramatically lower barriers (?H^‡_calc ~ 17-21 kcal/mol) control NO₂^- release from CH₃SNO₂ by gas-phase hydrolysis or nucleophilic attack by OH^- or CH₃S^- on the sulfur atom within the C-S-NO₂ molecular plane. Moreover, attack by either anion along the S-NO₂ bond results in barrierless NO₂^- release (?H^‡_calc ~ 0 kcal/mol) since a s-hole (i.e., area of positive electrostatic potential) extends from this bond. Consistent with our high-level calculations, ALDH2 and GAPDH, enzymes implicated in nitroglycerin bioactivation via an E-Cys-NO₂ intermediate, catalyze mainly or exclusively NO₂^- release from the prodrug.