Heme-Thiolate-Mediated Formation of S-Nitrosothiols and NO-Ferroheme: Mechanistic Insights

S-nitrosothiols (RSNO) and NO-ferroheme species have been recognized as important signaling molecules, particularly crucial for NO transport in the vascular system. While it is known that they are concurrently generated from NO and thiols with the assistance of ferric heme complexes, the specific mechanisms of their formation in vivo remain a topic of debate. Here, we describe the formation mechanism of both RSNOs and NO-ferroheme, utilizing a thiolate-bound ferric TMPS porphyrin model system [Fe(TMPS)(SR)], where TMPS stands for meso-tetrakis(sulfonatomesityl)porphyrinato-iron(III), under aqueous, buffered conditions. Spectroscopic studies (Raman, EPR, UV–vis) using biologically relevant thiols (RS), including glutathione, cysteine, and N-acetylcysteine, revealed that heme-thiolate ([Fe³⁺(TMPS)(SR)]) exists in equilibrium with its valence tautomer, thiyl radical-bound ferrous ([Fe²⁺(TMPS)(SR^•)]) species. However, the dominant reaction pathway is not the attack on the thiyl radical but rather the coordination of NO to the iron center, which results in the formation of a ferric nitrosyl species [Fe(TMPS)(SR)(NO)]. In a subsequent, slower step, the transient [Fe(TMPS)(SR)(NO)] converts to a NO-ferroheme through the direct attack of a second NO molecule on the coordinated thiolate, simultaneously releasing S-nitrosothiol. Experimental and theoretical studies showed that the generation of S-nitrosothiols follows a concerted reaction mechanism without the formation of iron coordinated S-nitrosothiol intermediate.