Tài liệu Báo cáo Y học: Inactivation of the 2-oxo acid dehydrogenase complexes upon generation of

Inactivation of the 2-oxo acid dehydrogenase complexes

upon generation of intrinsic radical species

Victoria I. Bunik1 and Christian Sievers2

1

A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia; 2

Physiological Chemistry

Institute of Eberhard-Karls-University, Tuebingen, Germany

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 fluor￾escence 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 a-phenyl￾N-tert-butylnitrone. Another, carbon-centered, radical was

trapped in anaerobic reaction of the complex with 2-oxo￾glutarate and CoAby 5,5¢-dimethyl-1-pyrroline-N-oxide

(DMPO). Generation of radical species was accompanied by

the enzyme inactivation. Asuperoxide 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. Apro￾duct 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 alle￾viates 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 thio￾redoxin and the (NAD+ + NADH) pool.

Keywords: dihydrolipoate; 2-oxo acid dehydrogenase com￾plex; reactive oxygen species; thiyl radical; thioredoxin.

The 2-oxo acid dehydrogenase complexes are key mito￾chondrial enzymes functioning at branch points of

metabolism. They catalyze irreversible oxidation of 2-oxo￾acids (pyruvate, 2-oxoglutarate or branched chain 2-oxo￾acids) yielding CO2, acyl-CoAs and NADH via reactions

1–5:

Correspondence to V. Bunik, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.

Tel.: + 7 095 939 14-56, Fax: + 7 095 939 31 81, E-mail: [email protected]

Abbreviations: E1, 2-oxo acid dehydrogenase; E2, dihydrolipoamide acyltransferase; E3, dihydrolipoamide dehydrogenase; DTPA, diethylene￾triaminepentaacetic acid; DMPO, 5,5¢-dimethyl-1-pyrroline-N-oxide; MNP, 2-methyl-2-nitrosopropane; PBN, a-phenyl-N-tert-butylnitrone;

POBN, a-(4-pyridyl-1-oxide)-N-tert-butylnitrone; ROS, reactive oxygen species; SOD, superoxide dismutase; ThDP, thiamin diphosphate.

(Received 31 May 2002, accepted 23 August 2002)

Eur. J. Biochem. 269, 5004–5015 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03204.x