re\t:rsal of electron transfer in the respiratory chain 123 



Experiments of the type indicated by Figs. 2 and 3 suggest an essential 

 role for succinate in particular, and for a flavin-linked substrate in general, 

 in reduction of a considerable portion of mitochondrial pyridine nucleo- 

 tide. However, such experiments do not plainly separate the electron- 

 transfer and energy requirements for the reaction. 



An energy requirement for this reaction is apparent from two stand- 

 points. First, there is at least a 300-mv. unfavourable potential difference 

 between the succinate-fumarate and the DPN-DPNH couples and second, 

 the sensitivity of the reaction to uncoupling agents suggests that the 

 energy requirement was met by internal high-energy intermediates of 

 oxidative phosphorylation (Fig. 5). In more recent experiments at the 

 Johnson Foundation, preparations of pigeon heart mitochondria [14] have 

 been studied in which succinate causes no appreciable pyridine-nucleotide 

 reduction in the absence of ATP and, more important, ATP causes no 

 appreciable pyridine-nucleotide reduction in the absence of succinate. 



Succinate + X~I + DPN - — - fumarate + DPNH + H* + X +1 

 Fig. 5. 



Figures 6 and 7 illustrate the properties of succinate-linked pvridine- 

 nucleotide reduction in pigeon-heart mitochondria where the electron- 

 donor and energy-donor requirements are separable. In Fig. 6 pigeon- 

 heart mitochondria are suspended in an aerobic medium containing o • 27 M 

 mannitol, 0-03 M sucrose, 0-02 m "tris" buffer, pH 7-4, free of added 

 magnesium and phosphate.* Under these conditions succinate addition 

 causes no downward deflection of the spectrophotometric trace; there is 

 no measurable pyridine-nucleotide reduction. However, upon addition of 

 100 /LtM ATP there is an abrupt downward deflection of the trace, indicating 

 reduction of DPN. This reaction continues for about 2 min. In a similar 

 experiment under the same conditions mitochondria are pretreated with 

 78 /LiM ATP (Fig. 7). There is only a very small downward deflection of the 

 trace amounting to about 4-',, of the total pyridine nucleotide. However, 

 upon addition of succinate there is an initial rapid reduction of DPN 

 which then proceeds at a slower rate. 



These experimental results can now be considered against the back- 

 ground of the various hypotheses that have been considered. It is apparent 

 that the "switch" hypothesis (Fig. i) is inapplicable to these experimental 

 conditions since succinate alone causes no measurable pvridine-nucleotide 

 reduction. 



An hypothesis suggesting that onlv succinate is required for pyridine- 

 nucleotide reduction is apparently inconsistent with these data, as is one 



* Thanks are due to Mr. K. Kaminker and Miss H. Diefenbach for pigeon- 

 heart preparations and to Dr. U. Fugmann for "digitonin" preparations. 



