PHOTO PHOSPHORYLATION IN FLASHING LIGHT 211 



quantum efficiency to one half. Knowing the maximum amount of de- 

 layed photophosphorylation from a single flash, we postulated two 

 sites of phosphorylation on the electron transport chain in R. rubrum 

 chromatophores as a tentative value (see Discussion), The absolute 

 quantum efficiency measurement of photophosphorylation (24) and 

 valiomycin experiments (25) by Baltscheffsky and others are generally 

 in good agreement with our present studies. 



The operation of the different phosphorylating sites is a function of 

 light intensity. For example, the MPM activation of photophosphoryla- 

 tion is more marked under high light intensities. Likewise, the re- 

 covery of antimycin A or HOQNO inhibition by MPM is greater under 

 higher light intensities. These results are summarized in Fig. 7. 

 Curve A is the ratio of MPM- catalyzed phosphorylation to normal 

 phosphorylation. Curves B and C are ratios of HOQNO- (or antimycin 

 A-) MPM phosphorylation/MFM phosphorylation, respectively. The 

 concentrations of MPM, HOQNO, and antimycin A were identical to 

 those in Fig. 6. These facts suggest that in low light intensity experi- 

 ments the untreated chromatophores are more efficient in the energy 

 utilization than other systems. However, MPM-bypassed electron 

 transfer becomes greater under higher intensities, and the overall 

 rate of phosphorylation can be high in the presence of MPM even if 

 the normal electron transfer is blocked and one of the phosphorylating 

 sites is lost. 



DISCUSSION 



It is concluded that the photochemical and dark processes in photo- 

 phosphorylation can be separated by the technique of flashing illumina- 

 tion. The first photochemical step is rapid and occurs only during il- 

 lumination. The rate of steady state phosphorylation is limited by the 

 rate of dark process, possibly by the rate of electron transfer. The 

 amount of total delayed photophosphorylation is proportional to the 

 amount of light absorbed when flashes are sufficiently short. 



The first step can be the formation and accumulation of oxidized 

 cytochrome (which is rapid and temperature independent) (1-9) and 

 some unknown reduced substance. The reduction of pyridine nucleo- 

 tide is less rapid than cytochrome oxidation (26). The second process 

 (light- independent phase of photophosphorylation) would include the 

 transfer of electrons from the reduced low- redox potential system to 

 the oxidized high- redox potential system (oxidized cytochrome) and 

 the associated reactions which lead to phosphorylation of ADP. MPM 

 and higher temperatures accelerate the decay of delayed photophos- 

 phorylation, whereas HOQNO retards this decay. Under continuous 

 illumination, higher temperatures and MPM raise the level of steady 

 state photophosphorylation; HOQNO lowers the level. These data can 



