30 INTERMEDIARY METABOLISM AND GROWTH I 



Another interesting question relates to the oxidation of TPNHj. As yet, no 

 decisive demonstration of a coupHng of TPNHj oxidation with phosphorylation 

 has been shown, although such coupling presumably exists. Possibly, phosphory- 

 lation can be obtained with TPNH, oxidation only after electron transfer to DPN"^ 

 via the pyridine nucleotide transhydrogenase (Kaplan et al., ig56b). Although 

 isocitrate plus TPN^ is oxidized by liver mitochondria which have been depleted 

 of pyridine nucleotides, no phosphorylation is observed. However, when supple- 

 mented with TPN^ plus DPN^ they oxidize isocitrate with a P:0 ratio of 2.3. It 

 is possible that the prime function of TPNHj is to serve as a reducing agent, 

 while DPNH2 is primarily oxidized and thereby gives i"ise to utilizable energy in 

 the form of energy-rich phosphate bonds. It has been shown that TPNHj can act 

 as a reducing agent in the conversion of crotonyl-CoA to butyryl-CoA; DPNH2 

 cannot replace TPNH2 in this reaction. According to this hypothesis, pyridine 

 nucleotide transhydrogenase serves as part of a regulatory mechanism for the 

 conservation of oxidative energy. As shown below, the shifting of electrons from 

 DPNH, to the TPN"^ system would result in a lowering of phosphorylation; a 

 shift in the direction of the DPN^ system would increase the level of phosphoryla- 

 tion (Kaplan et al., 1956b) : 



Fast 



Substrate — > DPNH » oxygen 



ATP formed 



Slow 



Substrate -^ TPNH > oxygen 



I No ATP formed 



Reductive synthesis 



The experiments of Lehninger and collaborators, and Maley and Lardy (1954) 

 have done much to clarify the precise localization of the three phosphorylation 

 reactions which occur between DPNH2 and O2, which may be schematically 

 written as follows: 



i) DPNH + H^ + FAD + ADP + Pi ^ DPN^ + FADH. + ATP 



2) 2 cyt. b-Fe^^ + 2 cyt. c-Fe""* + ADP + P; -^ 2 cyt. b-Fe"^^ + 2 cyt. c-Fe^^ + ATP 



3) 2 cyt. a-Fe^^ + 2 ff + >/202 + ADP + Pi -^ 2 cyt. a-Fe*^^ + H2O + ATP 



In the presence of cyanide, which inhibits cytochrome oxidase, the freshly pre- 

 pared mitochondria or the mitochondrial fragments catalyze the reduction of 

 ferricytochrome c at the expense of [3-hydroxybutyrate with a P: O ratio approach- 

 ing two. Since no phosphorylation occurs during the transfer of the electrons 

 and hydrogens from [^-hydroxybutyrate to DPNHj, this implies that there are 

 two phosphorylation sites between DPNH2 and cytochrome c. The phosphoryla- 

 tions are uncoupled by dinitrophenol and gramicidin. Likewise, the oxidation 

 of the substrate by ferricytochrome c and the phosphorylation reactions are com- 

 pletely blocked by antimycin A, an agent which is beheved to act between cyto- 

 chrome b and cytochrome c. With ferricyanide as electron acceptor instead of 

 ferricytochrome c, the P:0 ratio is about one, indicating that one phosphorylation 

 takes place very early in the chain. 



