FUN'CTION OF FLAVOEXZYMES IN ELECTRON TRANSPORT 1 53 



mitochondrial DPN by succinate, earlier described by Chance and 

 Hollunger [9, 10] and by Klingenberg et al. [11, 12]. It was therefore of 

 interest to investigate whether the ATP-induced activation of succinate 

 oxidation in the high-energy phosphate depleted mitochondria was 

 reflected in an increased level of DPNH. 



In this part of our investigation, Dr. Azzone and I had the pri\ilege 

 to benefit by the hospitality and collaboration of Dr. Martin Klingenberg 

 at Marburg. A typical resulc of our experiments, a detailed account of 

 which is being published elsewhere [34], is shown in the lower part of 

 Fig. 8. As can be seen, the ATP-induced stimulation of the aerobic 

 oxidation of succinate in the arsenate-dicoumarol depleted mitochondria 

 was paralleled by an increase of the 340-380 absorption difi^erence, 

 indicative of the reduction of pyridine nucleotides. Similar results were 

 obtained when dicoumarol was replaced by io~^ M dinitrophenol. Dif- 

 ferential analytical data, given at the bottom of the figure, reveal that the 

 increase was due to a major part to the reduction of TPX, and to a minor 

 part to the reduction of DPX. Admittedly, the observed steady-state levels 

 of the reduced pyridine nucleotides were not particularly high, about 10 

 and 30",, of the total contents of DPX and TPX, respectively. On the 

 other hand, the system contained a fully uncoupling concentration of 

 dicoumarol (or dinitrophenol), this causing a maximal flux of electrons 

 towards oxvgen ; the levels of DPXH and TPXH found may thus actually 

 represent the maximal values obtainable in an uncoupled system. 



However, the main importance of these findings in our opinion is the 

 very fact that an ATP-dependent reduction of pyridine nucleotides by 

 succinate could occur at all in the presence of a fully uncoupling concen- 

 tration of dicoumarol or dinitrophenol ; or in other words, that a high- 

 energy intermediate at the level of the respiratory chain could be formed 

 at the expense of ATP in spite of the uncoupled state of the oxidative 

 phosphorvlation svstem. This seems to imply a serious challenge to those 

 proposed schemes of oxidative phosphorylation ([35], [36], [26]; cf. [37] 

 for review) which invoke the participation of non-phosphorylated high- 

 energy intermediates at the level of the electron transport chain, and 

 according to which uncoupling agents act by disconnecting the interaction 

 of this intermediate with inorganic phosphate and ADP. On the other 

 hand, the present finding is in agreement with, and even lends some 

 support to, the hypothesis promulgated by our group [38-40] that phos- 

 phorylated reduced electron carriers are the primary high-energy inter- 

 mediates. According to this hypothesis, uncouplers are \'isualized as acting 

 by preventing inorganic phosphate from becoming activated in connection 

 with the energy-yielding oxidative step, and are thus not expected to 

 interfere with the reversal of this reaction. 



On the basis of the abo\ e findings, the following simple reaction scheme 



