Immunochemical identification of coenzyme Q0-dihydrolipoamide adducts in the E2 components of the α-ketoglutarate and pyruvate dehydrogenase complexes partially explains the cellular toxicity of coenzyme Q0

Abstract

Coenzyme Q0 (Q0), a strong electrophile, is toxic to insulin-producing cells. Q0 was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q0 covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q0, and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide the succinyltransferase, the E2 component of the α-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q0 bonds to the E2 components of the purified KDC and the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q0 was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q0, but oxidizing agents prevented it, suggesting that Q0 bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC Q0 followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q0 bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q 1, and Q2 did not bond to the 50-kDa protein but competed with the bonding of Q0 to this protein. The prevention by Q 1 of the bonding of Q0 to KDC E2, as well as other characteristics of the Q0 effect, are reminiscent of the action of Q0 on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734-25740).