Goldman R, Stoyanovsky D A, Day B W, Kagan V E
Department of Environmental and Occupational Health, University of Pittsburgh, Pennsylvania 15238, USA.
Biochemistry. 1995 Apr 11;34(14):4765-72. doi: 10.1021/bi00014a034.
Thioredoxin is an important cellular redox buffer. In this report, we describe the reaction of thioredoxin with phenoxyl radicals. The vicinal sulfhydryls of the bis(cysteinyl) active site sequence reduced phenoxyl radicals released in horseradish peroxidase-catalyzed oxidation of phenol. Redox cycling of phenol was accompanied by selective oxidation of thioredoxin sulfhydryls to disulfides. HPLC/UV-vis measurements showed that the SH:phenol oxidation ratio was 15:1 under the conditions used. At the end of the reaction, oxidized thioredoxin was quantitatively recovered in the reduced form with dithiothreitol. Oxidation of sulfhydryls to sulfoxy derivatives, oxidation of other amino acid residues, and formation of covalent adducts with phenolic metabolites (quinones) were not detected by LC-MS. While the thiyl radical of glutathione was readily detected with the spin trap 5,5-dimethyl-1-pyrroline N-oxide, no ESR-detectable DMPO-thiyl adducts formed during the oxidation of thioredoxin. Similarly, oxidation of vicinal sulfhydryls of dihydrolipoic acid did not produce DMPO-thiyl spin adducts, indicating that fast intramolecular cyclization to disulfide occurred with thioredoxin. Measurements of the superoxide dismutase-sensitive chemiluminescence response of lucigenin demonstrated that thioredoxin oxidation was accompanied by release of superoxide, most likely via disulfide radical anion-mediated one-electron reduction of oxygen. We propose that formation of disulfides is characteristic of the phenoxyl radical-catalyzed oxidation of vicinal sulfhydryls in both small thiols and disulfide-forming oxidoreductases. Reversibility of the phenoxyl radical-catalyzed modification of thioredoxin may be responsible for its function as an efficient cytosolic antioxidant.
硫氧还蛋白是一种重要的细胞氧化还原缓冲剂。在本报告中，我们描述了硫氧还蛋白与苯氧自由基的反应。双（半胱氨酰）活性位点序列的邻位巯基还原了在辣根过氧化物酶催化苯酚氧化过程中释放的苯氧自由基。苯酚的氧化还原循环伴随着硫氧还蛋白巯基选择性氧化为二硫键。高效液相色谱/紫外可见光谱测量表明，在所使用的条件下，SH:苯酚氧化比为15:1。反应结束时，氧化型硫氧还蛋白用二硫苏糖醇定量回收为还原型。通过液相色谱-质谱未检测到巯基氧化为亚砜衍生物、其他氨基酸残基的氧化以及与酚类代谢物（醌）形成共价加合物。虽然用自旋捕获剂5,5-二甲基-1-吡咯啉N-氧化物很容易检测到谷胱甘肽的硫自由基，但在硫氧还蛋白氧化过程中未形成电子顺磁共振可检测的DMPO-硫自由基加合物。类似地，二氢硫辛酸邻位巯基的氧化也未产生DMPO-硫自由基自旋加合物，表明硫氧还蛋白发生了快速的分子内环化形成二硫键。光泽精的超氧化物歧化酶敏感化学发光响应测量表明，硫氧还蛋白氧化伴随着超氧化物的释放，最有可能是通过二硫自由基阴离子介导的氧的单电子还原。我们提出，二硫键的形成是苯氧自由基催化小硫醇和形成二硫键的氧化还原酶中邻位巯基氧化的特征。苯氧自由基催化的硫氧还蛋白修饰的可逆性可能是其作为高效胞质抗氧化剂发挥功能的原因。
