FUNCTION OF FLAVOENZYMES IN ELECTRON TRANSPORT 1 65 



case, it is indicative of a complex pattern of compartmentation of energy- 

 transfer routes within the mitochondria. Similar indications have recently 

 been obtained in our laboratory along other lines of approach [52, ^t,]. 



CONCLUSIONS 



The main conclusions of the second section of this paper may be 

 summarized as follows : 



1. In rat liver mitochondrial preparations depleted of high-energy 

 phosphate by preincubation with arsenate and dicoumarol or dinitrophenol, 

 the oxidation of succinate is greatly stimulated by ATP. Parallel to the 

 respiratory stimulation, the mitochondrial pyridine nucleotides become 

 reduced to a slight but significant extent. It is concluded that the ATP- 

 induced activation of succinate oxidation and the ATP-induced reduction 

 of DPN by succinate involve a common high-energy intermediate, which 

 consists of a phosphorylated, reduced electron carrier, and whose formation 

 at the expense of ATP and succinate is not inhibited by uncoupling con- 

 centrations of dinitrophenol and dicoumarol. The reduction of DPN by 

 succinate is thought to involve a re\ersal of the DPN-flavin-linked oxida- 

 tive phosphorylation, and its extent in a respiring system is consequently 

 a resultant of the rate at which electrons derived from succinate enter the 

 respiratory chain and the rate at which these electrons are transferred from 

 their site of entrance towards oxygen. 



2. Intact liver mitochondria incubated under aerobic conditions in the 

 absence of phosphate acceptor catalyze a reduction of acetoacetate to ^- 

 hydroxybutyrate, coupled to the oxidation of succinate to fumarate. The 

 reaction, which provides conclusive evidence for a substantial transfer of 

 hydrogen from succinate to mitochondrial DPN, is completely inhibited 

 by amytal, as well as by low concentrations of dinitrophenol, addition of 

 terminal phosphate acceptor, or omission of Mg^ *. The phosphate 

 acceptor effect is removed by oligomycin A, which presumablv acts by 

 blocking the transfer of phosphate between the primary high-energy 

 intermediates and ADP. In the Mg+ ^-deficient system acetoacetate 

 reduction is restored by ATP and by sodium fluoride. The succinate- 

 linked acetoacetate reduction is also suppressed by antimycin A, cyanide, 

 or in the absence of oxygen ; under these conditions, the reduction is not 

 restored by added ATP. Ferricyanide, in the presence of cyanide, allows 

 phosphorylation coupled to succinate oxidation, and also restores aceto- 

 acetate reduction. Under appropriate conditions, both the coupled phos- 

 phorylation and the acetoacetate reduction of the ferricyanide svstem are 

 insensitive to antimycin A. Respiration with succinate as substrate in the 

 absence of phosphate acceptor is stimulated by acetoacetate and the stimu- 

 lation corresponds to 0-5 /^atom oxygen per /xmole acetoacetate reduced. 



