Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of intrinsic radical species.
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.
Bunik
Victoria I
VI
A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia. bunik@genebee.msu.su
Sievers
Christian
C
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002384:Catalysis; D003065:Coenzyme A / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009589:Nitrogen Oxides / Q000737:chemistry; D013113:Spin Labels; D013481:Superoxides / Q000032:analysis / Q000378:metabolism; D008063:Thioctic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D003497:Cyclic N-Oxides; D005609:Free Radicals; D009097:Multienzyme Complexes; D009589:Nitrogen Oxides; D013113:Spin Labels; D009243:NAD; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; C029217:phenyl-N-tert-butylnitrone; D013879:Thioredoxins; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D008063:Thioctic Acid; C015144:dihydrolipoic acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D003065:Coenzyme A