Mechanistic studies of S-nitrosothiol formation by NO [rad]/O 2 and by NO [rad]/methemoglobin

Abstract

The mechanisms of formation of S-nitrosothiols under physiological conditions and, in particular, of generation of SNO-Hb (the hemoglobin form in which the cysteine residues β93 are S-nitrosated) are still not completely understood. In this paper, we investigated whether, in the presence of O 2, NO is more efficient to nitrosate protein-bound thiols such as Cysβ93 or low molecular weight thiols such as glutathione. Our results show that when substoichiometric amounts of NO are mixed slowly with the protein solution, NO , O 2, and possibly NO 2 and/or N 2O 3 accumulate in hydrophobic pockets of hemoglobin. Since the environment of the cysteine residue β93 is rather hydrophobic, these conditions facilitate SNO-Hb production. Moreover, we show that S-nitrosation mediated by reaction of NO with the iron(III) forms of Hb or Mb is significantly more effective when it can take place intramolecularly, as in metHb. Intermolecular reactions lead to lower S-nitrosothiol yields because of the concurring hydrolysis to nitrite.