REVERSAL OF ELECTRON TRANSFER IN THE RESPIRATORY CHAIN 1 33 



were made under these experimental conditions, we have no alternative 

 but to conclude that all the components of the respiratory chain itself 

 were not in equilibrium as an electron-transfer system, but the couples 

 of the respiratory chain were interacting individually with the ATP- 

 electron transfer system, presumably because under these particular 

 experimental conditions the latter reaction is much more rapid than that 

 of electron transfer. 



Such an experimental condition calls our attention again to the intense 

 inhibition of electron transfer through the chain attributed to hypothetical 

 " I " compounds. It follows from Figs. 1 1 and 12 that a high concentration 

 of ATP would lead to a concentration of the "I" compounds sufficient 

 to bind the carriers tightly in their inhibited form [5]. Thus the products 

 of the reaction of the ATP-electron transfer activity are concluded to be 

 the inhibited and not the free forms of the carriers, to explain the low 

 values of " phosphate potential " which can cause half-maximal cytochrome 

 oxidation. However, this hypothesis must be considered a tentative one 

 because of our limited experience with this new phenomenon. 



At the present time investigations are under wav to locate "crossover 

 points" for the ATP-electron transfer activity and experiments such as 

 those of Fig. 13 suggest an interaction site between DPNH and flavin. 

 The response of cytochrome h suggests that crossover points mav be found 

 on either side of this component. 



Summary 



These experiments ha\e attempted to elucidate two pathways by 

 which ATP may be used in activating the reversal of electron transfer 

 through components of the respiratory chain. The first pathway investi- 

 gated is a succinate-acti\ ated branch of the respiratory chain which leads 

 to reduction of the majority of mitochondrial pyridine nucleotide, pro- 

 \iding that an energy source such as ATP is available. The specificity of 

 flavin-linked substrates such as succinate has been studied as has the 

 pathway of electron transfer through respiratory carriers. Similarly, the 

 pathway of energy transfer from ATP to DPNH has been shown to involve 

 the transfer system employed in oxidative phosphorylation. 



Of more general concern is the observation that ATP can cause oxida- 

 tion of reduced cytochromes in a magnesium-, phosphate-, and ADP-free 

 system, and in a respiratory chain blocked at the oxygen end by a suitable 

 inhibitor or by the lack of oxygen. This reaction may be observed in spite 

 of the presence of a reducing substrate such as succinate. Three general 

 points that must be borne in mind in carrying out this experiment: (i) 

 that the ATP-electron transfer activity be maximal because of the absence 

 of {a) reaction products such as ADP and phosphate; {b) reagents hydroly- 

 zing high-energy intermediates such as magnesium or uncoupling agents ; 



