ON THE MECHANISM OF THE DIHYDROTHIOCTYL 

 DEHYDROGENASE REACTION 



Robert L. Searls and D. R. Sanadi 



Gerontology Branch, National Heart Institute, National Institutes of Health, 

 and the Baltimore City Hospitals, Baltimore, Maryland 



The dihydrothioctyl dehydrogenase purified from a-ketoglutaric de- 

 hydrogenase complex of hog heart muscle catalyzes the reversible oxi- 

 dation of DPNH by thioctamide (TSo) or thioctate (lipoate) (Ecjua- 

 tion 1) as well as the oxidation of DPNH by 2,6-dichlorophenol 

 indophenol and ferricyanide (diaphorase reaction, Equation 2) (1). 



DPNH + H+ + TS2 ^ DPN+ + T(SH), (/) 



DPNH + H+ + Dye -^ DPN+ + Dye-Ha (2) 



Recent work (6, 10, 12, 13) has show^n that this flavoprotein is identi 

 cal with Straub's diaphorase (14) . The reduction of thioctate is 

 inhibited by reagents which are known to inhibit vicinal sulfhydryl 

 groups (arsenite and cadmium ions) , while the diaphorase reaction 

 is activated several fold under identical conditions (9, 11). These 

 results indicate that the enzyme reaction involves, besides flavin 

 adenine dinucleotide (FAD), a closely juxtaposed (vicinal) disulf- 

 hydryl group and that this disidfhydryl is functional in the oxidation- 

 reduction ^\■ith thioctate but is not required for the diaphorase re- 

 action. The disulfhydryl on the flavoprotein is not a derivative of 

 thioctic acid (10, 13) . 



Fig. 1 shows that the 530-540 m^u, peak (8, 10, 13) of the flavo- 

 protein reduced with DPNH (curve B) disappears instantaneously 

 upon addition of 4 X 10~^ M arsenite (curve C) , along with further 

 bleaching of the flavin (400-500 m^x) spectrum. When the enzyme 

 is reduced by a-ketoglutarate through the thioctate bound to the 

 a-ketoglutaric dehydrogenase complex (Fig. 2, curve B) , according to 

 equation 3, 



R—CO—COOU + CoA— SH + Flavoprotein ^ 



/?— CO— S— CoA + Flavoprotein— H2 + CO2 (3) 



again the 535 m^ peak is eliminated by the addition of arsenite. At 

 the same time, the flavin spectrum returns to the original oxidized 

 state (curve C) . Similar results were obtained in both experiments 

 by replacing arsenite with 3 X 10"^ ^ Cd++. 



157 



