Oxidative stress plays a key role for the development of cardiovascular, metabolic and neurodegenerative disease. This concept was never widely proven by the therapeutic use of classical antioxidants in large scale clinical trials. However, we know from numerous (pre)clinical studies that the formation of reactive oxygen and nitrogen species (RONS) as well as stable oxidative stress markers are a hallmark of most forms of diseases and higher oxidative stress markers are frequently associated with higher disease risk and mortality. Besides the detrimental role of RONS, they are also involved in cellular functions via redox signalling. We have previously identified an efficient mechanism of S‐nitrosation by low levels of nitric oxide and superoxide (3:1 ratio) with potential formation of N2O2 [Daiber A, Schildknecht S, Müller J, Kamuf J, Bachschmid MM, Ullrich V. Chemical model systems for cellular nitros(yl)ation reactions. Free Radic Biol Med. 2009 Aug 15;47(4):458–67.]. Here we, elucidated whether S‐nitrosation in the presence of higher nitric oxide than superoxide concentrations (as observed under hypoxic conditions) could prevent sulfoxidation and thereby oxidative inactivation of enzymes in the presence of higher superoxide than nitric oxide concentrations (as observed during reoxygenation). We found that increasing concentrations of xanthine oxidase/hypoxanthine caused conversion of S‐nitrosoglutathione (GSNO) to reduced glutathione (GSH) up to a certain concentration of xanthine oxidase, indicating that superoxide can induce denitrosation of GSNO. This finding was unexpected since in the presence of excess superoxide one would not expect regeneration of reduced GSH from GSNO. This was even more surprising since we observed substantial dihydrorhodamine oxidation that was prevented by uric acid and tyrosine nitration of albumin during denitrosation of GSNO by superoxide, all of which points to intermediary formation of peroxynitrite. In summary, we propose that S‐nitrosation of (mitochondrial) proteins during ischemia represents a protective mechanism to prevent irreversible overoxidation of thiols during the reperfusion phase and to re‐establish reduced thiol state in (mitochondrial) key enzymes of energy metabolism and cell survival. Wide‐spread mitochondrial protein S‐nitrosation may represent a central feature of the protective preconditioning effects of nitric oxide.Support or Funding InformationThe present work was supported by the European Cooperation in Science and Technology (COST Action BM1203/EU‐ROS and COST Action CA16225/EU‐CARDIOPROTECTION).