THE FUNCTIONAL LINK OF SUCCINIC DEHYDROGENASE 2O3 



inhibit DPN reduction, was derived from the finding (reported by Dr. Ernster) 

 that the energy of ATP could be used for reducing DFX in the presence of 

 dinitrophenol. 



According to the phosphoryl-flavin theory [Low, Siekevitz, Ernster and Lind- 

 berg, Biochirn. biophxs. Acta 29, 392 (1958)] ATP can react with the diaphorase 

 flavin giving rise to a reduced phosphorylated electron carrier : 



2H - + ATP + 2Fe - - + Fp > ADP + zFe " ' - + FpH - P (i ) 



It has been suggested that Reaction (i) is not sensitive to the uncoupling agents, 

 and therefore the energy of ATP can be used, through the intermediate FpH -«- P, 

 for reducing DPN (Reaction (2)): 



FpH ~ P + DPN > Fp + DPXH + Pj + H (2) 



In the presence of dinitrophenol Reaction (2) will take place from right to left 

 without the activation of inorganic phosphate (Reaction (3)): 



DNP 



H+DPXH + Fp >DPX + FpH, (3) 



Thus the sum of Reactions (i), (2) and (3) will account for an ATP-ase activity. 



Chance: I still think there is a discrepancy because it is the rate of reduction 

 of DPX (the second equation) that we observed optically to be inhibited. 



AzzoNE : The fact that the rate of reduction of DPX is lower in the presence 

 than in the absence of dinitrophenol can be explained on kinetic reasoning. 



Once the high energy intermediate postulated in our hypothesis has been formed 

 during succinate oxidation [Azzone, Ernster and Klingenberg, Xattire, Loud. 188, 

 552 (i960)] it can be either utilized for reducing DPX, or reoxidized by the cyto- 

 chrome system. The higher the electron flow toward the oxygen the lower will be 

 the utilization of the intermediate in the backward reaction for reducing DPX. 



Ernster: We do accept the fact that the level and rate of DPX-reduction may 

 be low in the presence of an uncoupling agent which allows full respiration. How- 

 ever, the point we wish to stress is this : Is it at all possible to obtain an ATP- 

 induced DPX-reduction (no matter how little) in the presence of a fully uncoupling 

 concentration of dinitrophenol or dicoumarol ? I think our data clearly show that 

 it is. 



HoLTON : Before the experiments of Chance and Hollunger were published, 

 when one got an activation of oxidation of succinate in mitochondria one normally 

 regarded this as evidence that the mitochondria were breaking up. One knows very 

 well that in intact mitochondria succinate oxidation is rather slow while in mito- 

 chondrial fragments it is extremely rapid, so it is clear that the structural state of 

 the mitochondrion can have some influence on the rate at which it oxidizes suc- 

 cinate cjuite apart from mechanisms of the type postulated by Chance and 

 Hollunger. I wonder whether it might be as well to keep in mind that changes in 

 the rate of oxidation of succinate can be mediated by changes in the structural 

 state of the mitochondrion, that changes in structural state can be brought about 

 by changes in the ATP: ADP ratio and the possibility that these mechanisms of 

 succinate oxidation involving energy requirement and oxidation via DPXH are 

 not the only way of explaining an acceleration of succinate oxidation under any 

 particular experimental conditions. 



