Natural bond orbital (NBO) analysis (Figure , SI) provided insights into the electronic structure differences between the two peptides. For oxytocin, overlap between the lone pair of the peptide nitrogen atom and the antibonding sigma orbital of the newly formed C-O bond stabilizes the TI by approximately 11.2 kcal mol -1 . In angiotentin IV, this stabilization is more pronounced at 18.9 kcal mol -1 . The NBO analysis also revealed a unique stabilizing interaction (~7 kcal mol -1 ) in oxytocin between the hybridizing nitrogen lone pair and the antibonding   of the disulfide S-S' bond. This interaction was observed exclusively in the TI state of oxytocin. Both the DFT/MM optimized geometries and DFTB3/MM umbrella sampling snapshots showed a close contact between the scissile nitrogen and the sulfur atom of the disulfide bond in the oxytocin tetrahedral intermediate. This interaction was visualized using NCI isosurfaces between Npep and S (Figure and Figure S9, SI). A similar type of interaction has previously been found in other proteins where it was proposed that the engagement of the S-S bonds in a close contact with a carbonyl O atom increases the susceptibility for their reduction. These findings suggest that the mechsnism of hydrolysis in IRAP likely involves nitrogen inversion, which is stabilized by a hydrogen bond with Ala429 from the GAMEN loop. The unique S•••N interaction observed in oxytocin may contribute to its enhanced reactivity compared to angiotensin IV. The enhanced stability of the TS1 and TI with the substrate can be attributed to a favourable interaction between the sp 2 -hybridized nitrogen and the sulfur atom of the disulfide bond. However, elevated overall barriers for inhibitor cleavage are elucidated by multiple factors: stabilization of the angiotensin IV E-R state via hydrogen bonding with the GAMEN loop, destabilization of the E-I complex due to steric clash between the bulky valine side chain and the hybridizing nitrogen and significant peptide strain, manifested through deviation of the valine C-Cα-Cβ angle and the N-terminus ψ dihedral from the ideal conformation (see Figure and Table in the SI).