603 



Jagendorf and Hind 



In this two stage procedure we are able to discover 

 the effects of varying conditions on each stage 

 separately. One of the most rewarding variables was 

 that of pH (2), since it turned out that much higher 

 yields were obtained on lowering the light stage alone 

 to pH 6. The major effect appeared to be slowing down 

 the dark decay time, to as much as 30 seconds with 

 pyocyanine, or l80 seconds without, ADP, phosphate and 

 magnesium, and the pH 8 optimum, were found to be 

 conditions of the dark phosphorylation steps. As would 

 be expected, calcium (antagonistic with magnesium) and 

 arsenate (competitive with phosphate) inhibit if added 

 to the dark stage alone. Trichlorophenol indophenol dye, 

 recently shown to be an uncoupler of phosphorylation 

 (9,10,11), also inhibits if added only to the dark stage. 

 On the other hand CMU, or uncoupling by removal of 

 cations (12), are effective on the kinetics of the light 

 reaction. 



An important point is that if redox cofactors are 

 to have an effect, they must be present in the light 

 stage. Thus X must be some entity formed subsequent to, 

 rather than before, electron flow reactions mediated by 

 the redox dyes. In addition, we have ruled out X as 

 being a form of pyocyanine itself (2), 



The formation, equilibrium level, and dark decay 

 rate of X are all affected by the nature of the redox 

 cof actor added (3)« Figure 2 shows some kinetic curves 

 for X in light and subsequent darkness. The most rapid 

 rate of formation, the highest equilibrium level, and 

 (to a lesser degree) the fastest decay, are induced by 

 pyocyanine. This correlates well with the position of 

 pyocyanine in supporting the fastest rates of one-step 

 phosphorylation. Ferricyanide in some experiments, but 

 not in others, also provides the high equilibrium level 

 of X . Other cofactors, including FMN, methyl viologen, 

 PPNR^or PPNR plus TPN, usually increase the rate of 

 formation of X , but do not lead to equilibrium levels 

 as high as that seen with pyocyanine. Most striking, 

 at pH 6 the lower yield of X can be achieved entirely 

 without added cofactors. The rate of formation, however, 

 is quite slow, as much as 5 minutes being required to 

 reach the equilibrium point. It is interesting that the 

 steady state level with pyocyanine is generally twice as 

 high as that found with cofactorless, or with other 



