Trujillo M, Alvarez M N, Peluffo G, Freeman B A, Radi R
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.
J Biol Chem. 1998 Apr 3;273(14):7828-34. doi: 10.1074/jbc.273.14.7828.
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 (O-2) 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 O-2 reacted with GSNO with a rate constant of 1 x 10(4) M-1.s-1 at pH 7.4 and 25 degreesC. 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 O-2-dependent reduction of RSNO to yield .NO, which in turn reacts fast with a second O-2 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 Km 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 O-2-dependent and -independent pathways, and in the presence of oxygen it leads to peroxynitrite formation.
S-亚硝基硫醇（RSNO）存在于体内，并被认为是一氧化氮（·NO）的储存和运输生物分子。然而，RSNO在生物系统中释放·NO的生化机制尚未明确，特别是活性氧与RSNO之间的相互作用尚未得到研究。在这项工作中，我们表明，在嘌呤（次黄嘌呤、黄嘌呤）或蝶啶（鲁马嗪）底物存在的情况下，黄嘌呤氧化酶（XO）在有氧条件下会诱导S-亚硝基半胱氨酸（CysNO）和S-亚硝基谷胱甘肽（GSNO）分解。XO对RSNO的分解可被铜锌超氧化物歧化酶抑制，这与超氧阴离子（O₂⁻）参与该过程一致。然而，虽然超氧化物歧化酶可以完全抑制GSNO的有氧分解，但对CysNO只有部分抑制作用。竞争实验表明，在pH 7.4和25℃条件下，O₂⁻与GSNO反应的速率常数为1×10⁴ M⁻¹·s⁻¹。通过二氢罗丹明和细胞色素c²⁺的氧化评估，RSNO的分解伴随着过氧亚硝酸根的形成。提出的机制涉及O₂⁻依赖的RSNO还原以产生·NO，而·NO又会与第二个O₂分子快速反应生成过氧亚硝酸根。在厌氧条件下，将CysNO与黄嘌呤加XO一起孵育会导致CysNO分解、检测到·NO，并形成半胱氨酸和尿酸。我们发现CysNO是XO的电子受体底物，Km为0.7 mM。与此概念一致 的是，XO对CysNO的酶促还原可被氧嘌呤醇和二苯基碘鎓抑制，这两种抑制剂分别在钼和黄素位点干扰催化循环。总之，XO通过O₂⁻依赖和非依赖途径分解RSNO，并且在有氧存在的情况下会导致过氧亚硝酸根的形成。
