340 M. Morrison and E. Stotz 



and the /-electron carrier compound is the inhibited form of the electron 

 transport system. 



In 'tightly-coupled' mitochondria, oxidation cannot proceed without the 

 presence of phosphate acceptors. This indicates that the component which 

 inhibits oxidation and couples oxidation to phosphorylation, 1, is present in 

 amounts exceeding the electron transport components. 



Cytochrome c is an electron transport system member which is directly 

 involved in the coupling of oxidation and phosphorylation. The compound, 

 /, which inhibits oxidation is present in amounts exceeding the concentration 

 of cytochrome c initially in the mitochondria. The results of the experiments 

 cited above can then be interpreted simply as a logical consequence of the 

 mass law. On addition of cytochrome c, its concentration will equal and then 

 exceed that of the inhibitor, /. Therefore, at high concentrations, more of the 

 cytochrome c can be oxidized and reduced without reacting with the inhibitor. 

 At low concentrations, where the ratio of 7 to cytochrome c is high, there is a 

 great extent of reaction with the inhibitor. Increasing the concentration of 

 mitochondria results in increased ratios of /to cytochrome c, with a resulting 

 increase in the P/O ratio. Conversely, a lower ratio of / to cytochrome c 

 results in a decreased P/O ratio. From these results, it would appear that 

 cytochrome c does interact with the inhibitor, /, 



With this information at hand, the following experiments were performed. 

 'Tightly-coupled' mitochrondria were prepared and incubated with succinate 

 in a nitrogen atmosphere. When the system became sufficiently anaerobic to 

 deoxygenate the haemoglobin present, two volumes of boiling acetone were 

 added. The contents were then placed in a water bath at 60°C for 5 min and 

 the mitochondria were sedimented by centrifuging. The mitochondria were 

 then extracted with salt solutions. The cytochrome c which was extracted was, 

 almost exclusively, in the reduced form. More interesting was the fact that, 

 quantitatively, the amount of cytochrome c extracted under these conditions 

 was a function of the state of oxygenation of the mitochondria. More 



Table 2. Extraction of cytochrome c 



Condition of extraction 



0-5 M NaCl pH 7-0 

 0-5 M NaCl pH 7-0 



(after pretreatment with 



dithionite) 

 0-5 M NaCl pH 7-0 



(after pretreatment with 



ferricyanide) 



Ratio of cytochrome c 



extracted from mitochondria 



anaerobic 



aerobic 



2-0 -1-5 



1-75-1-4 



1-8 -1-4 



