Xanthine oxidase-mediated decomposition of S-nitrosothiols

Abstract

S-Nitrosothiols (RSNO) occur in vivo and have been proposed as nitric oxide (·NO) storage and transport biomolecules. Still, the biochemical mechanisms by which RSNO release ·NO in biological systems are not well defined, and in particular, the interactions between reactive oxygen species and RSNO have not been studied. In this work, we show that xanthine oxidase (XO), in the presence of purine (hypoxanthine, xanthine) or pteridine (lumazine) substrates, induces S-nitrosocysteine (CysNO) and S- nitrosoglutathione (GSNO) decomposition under aerobic conditions. The decomposition of RSNO by XO was inhibitable by copper-zinc superoxide dismutase, in agreement with the participation of superoxide anion (O2/·̄) in the process. However, while superoxide dismutase could totally inhibit aerobic decomposition of GSNO, it was only partially inhibitory for CysNO. Competition experiments indicated that O2/·̄ reacted with GSNO with a rate constant of 1 x 104 M-1·s-1 at pH 7.4 and 25 °C. The decomposition of RSNO was accompanied by peroxynitrite formation as assessed by the oxidation of dihydrorhodamine and of cytochrome c2+. The proposed mechanism involves the O2/·̄-dependent reduction of RSNO to yield ·NO, which in turn reacts fast with a second O2/·̄ molecule to yield peroxynitrite. Under anaerobic conditions, CysNO incubated with xanthine plus XO resulted in CysNO decomposition, ·NO detection, and cysteine and uric acid formation. We found that CysNO is an electron acceptor substrate for XO with a K(m) of 0.7 mM. In agreement with this concept, the enzymatic reduction of CysNO by XO was inhibitable by oxypurinol and diphenyliodonium, inhibitors that interfere with the catalytic cycle at the molybdenum and flavin sites, respectively. In conclusion, XO decomposes RSNO by O2/·̄-dependent and -independent pathways, and in the presence of oxygen it leads to peroxynitrite formation.