Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species
Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species
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DOI：
10.1046/j.1432-1033.2002.03204.x
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发表时间：
2002-10-01
期刊：
影响因子：
--
通讯作者：
Sievers, C
中科院分区：
文献类型：
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作者：
Bunik, VI;Sievers, C
研究了2-含氧酸脱氢酶复合物在催化过程中的自我调节。通过自旋捕集、光泽精荧光和铁细胞色素c还原检测到自由基物种作为催化副产物。将结合的硫辛酸或FAD辅因子转化为非功能性衍生物后的复合物的研究表明，自由基是通过FAD产生的。在氧的存在下，检测到2-含氧酸、CoA依赖性的超氧阴离子自由基的产生。在不存在氧的情况下，通过α-苯基-N -叔丁基硝酮捕获被推断为复合物结合的二氢硫辛酸酯的硫酰基自由基的蛋白质结合的自由基。另一个碳中心自由基在2-酮戊二酸和CoA的复合物的厌氧反应中被5，5 '-二甲基-1-吡咯啉-N-氧化物（DMPO）捕获。自由基物种的产生伴随着酶失活。超氧化物清除剂，超氧化物歧化酶，没有保护酶。然而，一个硫基自由基清除剂，硫氧还蛋白，防止失活。可以得出结论，络合物结合的二氢硫辛酸的硫酰基自由基通过2-含氧酸脱氢酶催化中间体的1 e（-）氧化诱导失活。这种氧化的产物，2-含氧酸底物的DMPO捕获的自由基片段，使复合物的第一组分失活。在不存在末端底物NAD（+）的情况下，失活可防止2-含氧酸转化。自我调节由硫氧还蛋白调节，硫氧还蛋白消除二氢硫辛酸中间体的不利影响，从而刺激复合物产生活性氧。数据表明复合物结合的二氢硫辛酸的双重促氧化作用，通过第一和第三组分酶传播，并由硫氧还蛋白和（NAD（+）+ NADH）库控制。
Self-regulation of the 2-oxo acid dehydrogenase complexes during catalysis was studied. Radical species as side products of catalysis were detected by spin trapping, lucigenin fluorescence and ferricytochrome c reduction. Studies of the complexes after converting the bound lipoate or FAD cofactors to nonfunctional derivatives indicated that radicals are generated via FAD. In the presence of oxygen, the 2-oxo acid, CoA-dependent production of the superoxide anion radical was detected. In the absence of oxygen, a protein-bound radical concluded to be the thiyl radical of the complex-bound dihydrolipoate was trapped by alpha-phenyl-N -tert -butylnitrone. Another, carbon-centered, radical was trapped in anaerobic reaction of the complex with 2-oxoglutarate and CoA by 5,5'-dimethyl-1-pyrroline-N -oxide (DMPO). Generation of radical species was accompanied by the enzyme inactivation. A superoxide scavenger, superoxide dismutase, did not protect the enzyme. However, a thiyl radical scavenger, thioredoxin, prevented the inactivation. It was concluded that the thiyl radical of the complex-bound dihydrolipoate induces the inactivation by 1e(-) oxidation of the 2-oxo acid dehydrogenase catalytic intermediate. A product of this oxidation, the DMPO-trapped radical fragment of the 2-oxo acid substrate, inactivates the first component of the complex. The inactivation prevents transformation of the 2-oxo acids in the absence of terminal substrate, NAD(+) . The self-regulation is modulated by thioredoxin which alleviates the adverse effect of the dihydrolipoate intermediate, thus stimulating production of reactive oxygen species by the complexes. The data point to a dual pro-oxidant action of the complex-bound dihydrolipoate, propagated through the first and third component enzymes and controlled by thioredoxin and the (NAD(+) + NADH) pool.