614 B. Chance 



The response of a complete representation of the chemical system operating 

 in accordance with the law of mass action to decreases of flux caused by the 

 simultaneous ten-fold decrease of three of the velocity constants is illustrated 

 in Table 1. It is seen that the crossover points identified by the — , + changes 

 can be moved from the interaction site near the oxygen side of the respiratory 

 chain to an interaction site nearer the substrate side by a decrease in the 

 velocity constant for the reaction with oxygen (k^), stimulating the inhibition 

 of the respiratory chain by the addition of azide (Chance and Wilhams, 

 1956b). Not only can the crossover point be removed by one interaction site, 

 but it can be moved by two interaction sites with a further decrease of the 

 velocity constants. Furthermore, with appropriate conditions, the respiratory 

 chain can show in a single experiment not one, but two of the three possible 

 crossover points, as indicated in the bottom row of Table 1. 



Table 1. Crossover behaviour of electron-transfer system caused by 



decreases of flux in response to changes in interaction at three sites 



(Courtesy Nature, Land.) 



Enzyme concentration 10~*m. Reaction velocity constants are specified in equations or in table, in 



units of 1. X mole~* x sec~*. k^. k^ and k^ are decreased from 0-9 to 009 I. x mole"* x sec~* to 



give the steady-state concentration changes shown in units of 10~* molar. Values for k^ and kg are 



given in the table. 



S RPN =- b ^ c a ^ 03 ^ O2 



This demonstration of the validity of the crossover theorem, although 

 restricted to a sequence of a limited number of components (5 chemical 

 intermediates), has been examined over extreme ranges of velocity constants 

 and no inconsistencies with the theorem have been found. 



The response of the cytochromes of the respiratory chain to the decrease 

 in flux caused by the expenditure of ADP is characteristic of the phosphory- 

 lating system and has shed considerable light upon the location of interaction 

 sites. It is now apparent that, in a wide variety of isolated mitochondria and 

 intact cells and in some tissues, the same response is observed, and that the 

 same sites of interaction can be identified (Table 2). This observation gives 

 a direct answer to the question of whether or not the behaviour of the 

 cytochrome chain is similar in the living cell and in the isolated mitochondrion. 

 Table 2 shows separate identification of three crossover points in isolated 



