II ENERGY-RICH PHOSPHATE BONDS 3 I 



As indicated previously, succinate is oxidized to fumarate and choline is oxidized 

 to betaine aldehyde by flavoproteins rather than by pyridine nucleotide de- 

 hydrogenases. When these substrates are oxidized by liver mitochondria, a P:0 

 ratio of about two is observed (Chance, 1953-1954; Rotschild et al., 1954). Thus, 

 there are two phosphorylation sites between FADHj and oxygen. 



The span between cytochrome c and oxygen was studied by utilizing cyto- 

 chrome c reduced chemically by hydrogen (palladium catalyst) or by ascorbate 

 or adrenalin (Maley and Lardy, 1954). In this case, a P:0 ratio of 0.9 is observed. 

 Since the standard oxidation-reduction potential of cytochrome c is about 0.26 

 and that of cytochrome a is about 0.29, and a difference of 0.25 V is required 

 for one phosphorylation, it can be inferred that the third phosphorylation is 

 localized between cytochrome a and oxygen. 



The rate of aerobic phosphorylation is depressed in rat liver mitochondria by 

 dicumarol without affecting respiration (Martins and Nitz-Litzow, 1953). The 

 uncoupling effect of dicumarol (lo"^ M) may be shown either on the overall 

 span, p-hydroxybutyrate to oxygen, or on the span between hydroxybutyrate and 

 cytochrome c, or cytochrome c and O, (Cooper and Lehninger, 1 956a, 1 956b) . This 

 supports the hypothesis that vitamin K is either an electron carrier in the electron 

 transport chain or is a component of the phosphate transferring enzyme system 

 responsible for the formation of ATP from the primary high energy linkage at 

 the three phosphorylating sites in the respiratory chain. 



Some of the factors which control the rate of oxidative reactions have been 

 demonstrated by Chance (Chance, 1953-1954)- Using intact liver mitochondria, 

 it was shown that the rate of oxygen consumption is low in aerobic resting mito- 

 chondria. When substrate is added in excess, an increased reduction of DPN^ and the 

 cytochrome pigments is observed, although the rate of oxygen consumption remains 

 low. If, however, both ADP and substrate are added to aerobic mitochondria, a rapid 

 increase in the respiratory rate takes place concomitant with a reoxidationof DPNHj 

 and the cytochrome pigments and the phosphorylation of ADP to ATP. As the ADP 

 is phosphorylated to ATP, the rate of respiration falls and an increased reduction of 

 DPN*, flavin, and cytochrome b is again observed. Under conditions where substrate 

 and oxygen are not limiting, the rate of respiration within the mitochondria is 

 therefore dependent upon the availability of ADP and phosphate. 



The requirement for phosphate acceptors in mitochondrial oxidations has 

 also been demonstrated in experimental systems in which glucose and the enzyme 

 hexokinase have been used to regenerate ADP from ATP : 



hexokinase 

 i) ATP + glucose > glucose-6-phosphate + ADP 



Under these conditions, stimulation of oxygen uptake has been noted. The 

 capacity of dinitrophenol to stimulate respiration while completely inhibiting 

 oxidative phosphorylation has been attributed to the fact that "ATPase activity" 

 is stimulated by dinitrophenol. As a result, the ADP and H3PO4 required for 

 maximal respiration is maintained (Chance and W'illiams, 1956). The action of 

 dinitrophenol is depicted schematically in Fig. 13. In Fig. 13, X ~ I represents the 

 labile compound formed during oxidative phosphorylation. 



Literature p. 124 



