20 INTERMEDIARY METABOLISM AND GROWTH I 



much lower rate only the reduction of cytochrome c by DPNH2 or the oxidation of reduced 

 cytochrome c by molecular oxygen (reaction i) (Green et al., 1954; Mackler et al., 1954; 

 Green and Beinert, 1955). 



i) DPNH2 + V2O2 -* DPN^ + H2O 



Reaction i) is completely blocked by low levels of antimycin A. When this purified 

 particulate system was treated with desoxycholate, the capacity to catalyze the overall 

 oxidation of DPNH2 by molecular oxygen was greatly reduced whereas the capacity to 

 catalyze the oxidation of DPNH2 by cytochrome c, (cytochrome reductase activity) or 

 the oxidation of reduced cytochrome c by molecular oxygen (cytochrome oxidase activity) 

 increased 25 fold (reactions 2 and 3) : 



2) DPNH2 + 2 cyt. c-Fe^^"^ -> DPN"^ + 2 cyt. c-Fe^"^ + 2W 



3) 2 cyt. c-Fe"* + 2H* + 1/2O2 -^ 2 cyt. c-Fe^^"^ + H2O 



The desoxycholate treated enzyme, though devoid of succinic dehydrogenase or the 

 oxidizing enzymes of the citric acid cycle showed absorption peaks when in the reduced 

 state at 5250 A, 5550 A, and 6070 A. A broad bifurcated Soret band at 4300 A and 4450 A 

 was also observed. 



These observations have been interpreted as follows (Green and Beinert, 1955). DPNH2 

 oxidase exists as an enzymatic unit in which electrons are transferred from the primary 

 prosthetic group to the final electron acceptor (molecular oxygen) by way of a series of 

 intermediary electron carriers such as heme groups and metals. This unit has the character 

 of an impenetrable "closed" unit in the sense that electrons can only enter by way of 

 DPNH2 and leave by way of oxygen. There is thus little possibility of interaction with 

 electron acceptors like cytochrome c or dyes. When the unit is exposed to desoxycholate, 

 the conducting circuit is interrupted in the sense that a barrier is raised to the flow of 

 electrons at some point, possibly by the loss of some component. Such an "opened" 

 DPNH2 oxidase unit can now interact with acceptors like cytochrome c, which then 

 undergoes a cycle of reduction by DPNH2 in the reducing moiety of the unit and of 

 oxidation by molecular oxygen in the oxidizing moiety. In other words, the opened 

 DPNH2 oxidase behaves as if it were a complex of two enzymes, DPNH2-cytochrome c 

 reductase and cytochrome oxidase which are no longer in electronic communication with 

 each other. According to this interpretation, both DPNH2 cytochrome c reductase and 

 reduced cytochrome c oxidase are, in this case at least, artifactual. The opening of the 

 DPNH oxidase unit has created an artificial situation in which exogenous cytochrome c 

 is closing the opened circuit again. It should be pointed out that the highly purified 

 particulate DPNH2 oxidase contains no detectable cytochrome c and that cytochrome c 

 is not liberated in consequence of the treatment with desoxycholate. 



By treating the particulate DPNH2 oxidase with trypsin in the presence of cholate, and 

 subsequent ammonium sulfate fractionation, two heme types having diflferent catalytic 

 activities can be obtained. One contains a green heme group and copper and manifests 

 cytochrome oxidase activity ; the other contains a red heme group and non-heme iron and 

 manifests DPNH2 dehydrogenating activity with dyes as electron acceptors. Cytochrome 

 reductase activity is lost during the treatment necessary for separation. 



A DPNH2 oxidizing enzyme system which does reduce cytochrome c has been isolated 

 in highly purified form from heart muscle (Mahler and Elowe, 1953, 1954). This enzyme 

 system contains flavin and non-heme iron, in a ratio of 4 : i but no heme. Both the iron 

 and the flavin can be reduced by the substrate and oxidized by the acceptor. Prolonged 

 dialysis against 8' nydroxy-quinoline results in a marked lowering of the non-heme iron of 

 the ferroflavoprotein enzyme and concomitantly, the capacity of the enzyme to interact 

 with cytochrome c is lost. On the other hand, the enzyme with reduced iron content is 

 still capable of reacting with dyes such as 2,6-dichlorophenolindophenol (diaphorase 

 activity). Preparations of Straub's* diaphorase are also very low in iron content. Full 

 enzyme activity can be restored by the addition of ferric iron at a concentration of 5- io~''Af : 



