BRITTON CHANCE 



enzyme becomes a high-energy compound ('^I) during the 

 oxidative-reduction reaction 



h"-^c"'-l > b'" + c" ^l (11) 



and that further reaction of c" '^ I with the respiratory chain is 

 slow unless transfer of '^ I to another intermediate occurs, 

 whereupon c", the rapidly reacting form, is released. 



c 



II 



I + X ^c" + X~I (12) 



X'^I, of course, can interact with phosphate and ADP to form 

 ATP. 



According to equation (12), the crossover point identifies 

 the component on the oxidation side of the three crossovers 

 mentioned above as sites of oxidative phosphorylation in the 

 intact mitochondrial respiratory chains. The sites are pyridine 

 nucleotide, cytochrome b, and cytochrome c. If the oxidized 

 form of the respiratory enzymes if found to conserve the incre- 

 ment of free energy, the sites would be flavoprotein, cytochrome 

 c, and cytochrome a. 



RAPID REACTION METHODS 



As in the case of the soluble hemoproteins, the rapid kinetic 

 methods have proved to be of considerable value in identifying 

 the sequence of action of the various components of the intact 

 mitochondrial respiratory chain. For a study of this complex 

 system it has been necessary to refine our methods of mathe- 

 matical analysis by the use of an electronic analogue computer 

 and to improve the optical and hydraulic aspects of the rapid- 

 flow method so that satisfactory results could be obtained with 

 intact mitochondria or whole cells. 



THEORETICAL ASPECTS 



On suddenly initiating a change in the steady-state level of 

 one member of the respiratory chain, the time sequence of the 

 response of the other members of the chain should indicate the 

 sequence of chemical reactions. For the sequence of reactions 



324 



