Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species

被引:105
作者
Bunik, VI [1 ]
Sievers, C
机构
[1] Moscow MV Lomonosov State Univ, AN Belozersky Inst Physicochem Biol, Moscow 119899, Russia
[2] Univ Tubingen, Inst Physiol Chem, D-7400 Tubingen, Germany
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2002年 / 269卷 / 20期
关键词
dihydrolipoate; 2-oxo acid dehydrogenase complex; reactive oxygen species; thiyl radical; thioredoxin;
D O I
10.1046/j.1432-1033.2002.03204.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:5004 / 5015
页数:12
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