132 BRITTON CHANCE 



therefore titrated the oxidation of cytochrome c with ATP and obtained a 

 sigmoid titration curve, presumably due to preferential expenditure of 

 ATP at other couples at low concentrations of ATP. However, the 

 maximum slope of the graph corresponds to roughly 4 ATPs per electron, 

 over twice the observed value of i • 5 ATPs per electron produced in oxida- 

 tive phosphorylation. Our experimental value is surely not a minimum 

 value since some ATP is lost in the simultaneous oxidation of other 

 carriers in addition to cytochrome c and in hydrolysis of intermediates 

 formed from ATP. Thus the efficiency may under appropriate conditions 

 approach the higher values. It is unlikely that the ATP/e value for the 

 reversal of electron transfer would reach the experimentally observed value 

 for oxidative phosphorylation in the forward direction since the efficiency 

 of the latter process is probably less than 100%. In fact an estimate of the 

 efficiency of oxidative phosphorylation can be obtained by the ratio of the 

 two values and on the basis of these preliminary data a value of over 50% 

 is obtained. 



In addition to considerable interest in the stoicheiometric properties 

 of the ATP-electron transfer interaction, the thermodynamic properties 

 are of importance and preliminary titrations of the extent of oxidation of 

 cytochrome c in "phosphate-potential buffers" (ATP/ADP.Pj) have 

 been made. We are for the first time able to study the oxidation levels of 

 cytochromes in the presence of ATP under conditions where electron 

 flow through the respiratory chain is sufficiently small to be negligible. 

 Furthermore, the rate of ATP breakdown due to hydrolysis can be so small 

 that the initial concentration of ATP is practically constant during the 

 measurement of cytochrome concentration. Thus the system can be 

 sufficiently near equilibrium to consider the relationship between phos- 

 phate potential and cytochrome oxidation. Experiments similar to those 

 of Fig. 14 but in the presence of varying concentrations of ATP, ADP and 

 Pj show that the oxidation of cytochrome c is very sensitive to small 

 amounts of phosphate and ADP and a considerable inhibition of the extent 

 of oxidation can be obtained. Actually it is difficult to obtain 50% oxida- 

 tion of cytochrome c under conditions where the ADP and phosphate 

 concentrations are sufficiently high to insure that equilibrium and not 

 stoicheiometric factors are determining. 



Preliminary estimates suggest that the phosphate potential necessary 

 to cause cytochrome oxidation does not correspond to the complete free- 

 energy change from DPNH to oxygen but instead to a value that would 

 be expected for a single redox couple involved in oxidative phosphorylation. 

 This possibility is supported by titration of the respiratory chain in 

 the absence of added succinate where the [ATP /ADP]/ [PJ ratio is about 

 iC* corresponding only to about 15 kcal. Since spectrophotometric observ- 

 ations of pyridine-nucleotide reduction and the oxidation of cytochrome c 



