616 B. Chance 



mitochondria by altering either the type of substrate or the extent of in- 

 hibition of the oxidase. In two cases, double crossover points have been 

 identified under given experimental conditions in guinea pig liver mito- 

 chondria and in intact yeast cells — one between pyridine nucleotide and 

 flavoprotein, and one between cytochromes b and (c + q). 



The crossover theorem does not specify a maximum number of interaction 

 sites, and it is of interest to note that the data of Ramirez on toad heart 

 muscle (Ramirez, 1959) suggest an additional site on the oxygen side of 

 cytochrome a^. This site, observed with considerable experimental difficulty 

 in the intact toad heart muscle, was apparently not present in mitochondria 

 isolated from this tissue. 



It is significant that a crossover point between flavoprotein and cytochrome 

 b has never been observed in any of the wide range of experimental conditions 

 or materials we have studied. This point is particularly relevant to the 

 proposal of Low, Ernster, Lindberg, Grabe and Siekevitz(1958) that an inter- 

 action site exists between these two components. Crossover data provide no 

 evidence in favour of this site (cf. Scheme C, Slater (1958)). 



The room temperature spectroscopic data do not permit us to distinguish 

 cytochromes c and q of the phosphorylating preparation, and we have 

 consistently lumped cytochromes c and q together in our studies so far. 

 Recent developments of the 'frozen steady state'-method now permit us to 

 distinguish a cytochrome Z?-q crossover point. A low temperature difference 

 spectrum clearly shows this crossover point; the absorption band of cyto- 

 chrome b is diminished and that of cytochrome q is increased in material 

 treated with ADP, relative to that without ADP. Thus the inhibitory inter- 

 action is between b and q. This experimental result probably requires a 

 revision of some of the interesting speculations put forward by Slater (1959). 



In summary, the experimental results on the apphcation of the crossover 

 theorem to intact mitochondria clearly identify three interaction sites: 



(1) between pyridine nucleotide and ffavoprotein; 



(2) between cytochromes b and q; and 



(3) between cytochromes c and a. 



These sites have been identified in a wide variety of materials ranging from 

 isolated mitochondria to whole tissues and under a wide variety of experi- 

 mental conditions. This appears to be the only method for locating inter- 

 action sites which can be directly applied to intact mitochondria (Chance, 

 1959; Chance and Conrad, 1959), cells (Chance, 1959c), and tissues (Weber, 

 1957; Ramirez, 1959). 



Representation of Reaction Kinetics 



While it is of interest to propose complex reaction sequences for electron 

 transfer and phosphorylation, they are of little use unless they can be examined 



