Energy Transfer and Conservation in the Respiratory Chain 611 



Intact mitochondria will carry out the reverse of this reaction in a process 

 that is found to be more sensitive to antimycin-A than to cyanide, indicating 

 that electrons pass through some of the carriers partly in the reverse of usual 

 electron transfer. Other studies lead us to propose the following chain 

 (Chance and Hollunger, 1960). 



c 



t 



t 

 succinate -^fpi-- 6 --fp,-^DPNH (14) 



t \ 



antimycin-A Amytal 



The reaction is, however, quite sensitive to uncoupling agents and it is found 

 that 'high-energy' intermediates are used in this reaction. It is very unlikely 

 that ATP or other 'high energy' phosphates can be solely responsible for this 

 reaction; ATP formed in oxidation of other substrates such as glutamate 

 does not cause the reaction to occur and succinate is apparently essential 

 (Chance, 1956, Chance and Hollunger, 1957a, 1960; see Note 6). 



A direct consequence of the finding of reversal of electron transfer in the 

 respiratory chain mediated by 'high-energy' intermediates is that energy 

 accumulated at one site of oxidative phosphorylation can be used to drive 

 reactions in another portion against a thermodynamically-unfavourable 

 gradient. This result vitiates the rigorous application of thermodynamic 

 data to the phosphorylating respiratory chain, and the detailed calculations 

 recently made by Slater (1958) and previously by Chance and Williams 

 (1956a) may well be restricted in situations where phosphorylation is absent. 

 Indeed, phosphorylation efficiencies obtained from portions of the respiratory 

 chain may be quite different from those obtained from the intact chain 

 because energy contributions from, or donations to, other parts of the chain 

 may be inhibited when only part of the chain is activated. 



This observation may also have a considerable influence on our ability to 

 pin-point the exact sites where 'phosphorylation' occurs in the sense that a 

 single electron transfer reaction provides the full value of AF for ATP 

 formation. Instead, we may ultimately rely entirely upon the interaction 

 sites defined by crossover phenomena as the locus of a 'carrier-high energy 

 intermediate' bond of sufficient strength to inhibit electron transfer in the 

 absence of ADP and phosphate. 



Localization of Interaction Sites by Fragmentation and Inhibitor Studies 



In a system of the complexity of the respiratory chain, it is clear that 

 fragmentation cannot be effective in determining the exact sites at which the 

 inhibiting components (I) interact. For example, the use of a solution of 

 cytochrome c in the measurement of phosphorylation between cytochrome c 



