ENZYME-SUBSTRATE COMPOUNDS 



over tenfold by the reversal of some inhibitory process in the 

 respiratory chain (45), and the localization of the sites of action 

 of the inhibitor should be demonstrable in terms of the crossover 

 point for spectroscopic changes in the respiratory pigments 

 caused by ADP addition. 



In rat liver preparations, it is found that the addition of 

 ADP causes an oxidation of the steady state of cytochromes c 

 and b, flavoprotein, and DPNH and a reduction of cytochromes 

 a and 03, the crossover point being between cytochromes c and 

 a (28). The action spectrum for ADP is illustrated by Figure 4. 

 With other substrates, mitochondria from other sources, the 

 crossover point can be moved along the respiratory chain so that 

 it lies between cytochromes c and b. In the presence of appro- 

 priate concentrations of inhibitors of cytochrome a^ such as 

 azide, only DPNH is oxidized when ADP is added. 



These crossover points identify pairs of components that can 

 be involved in oxidative phosphorylation. For example, in the 

 oxidation of cytochrome b" hy c'" 



b" -{- c'" > b'" + c" (10) 



a portion of the free-energy change can be conserved and later 

 utilized to add high-energy phosphate to ADP. The crossover 

 theorem does not identify which one of the reaction products 

 acquires this portion of the free-energy change. Current the- 

 ories of oxidative phosphorylation (63a) suggest, by analogy 

 with glyceraldehyde-3 phosphate dehydrogenase, that the 

 oxidation product is involved (for a recent summary, see Lehn- 

 inger (46)), but thermodynamics would make no distinction 

 between the two pathways for energy transfer. Our experi- 

 mental data suggest that the reduced form is involved. We 

 find that DPNH exists in an inhibited form in mitochondria 

 lacking ADP and therefore propose that it is the reduced form 

 of the reaction products that are involved in the free-energy 

 conservation. In order to translate this view into chemical 

 terminology, it is assumed that an unknown ligand (I) of the 



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