41 8 BIRGIT VENNESLAND 



Photoreduction of ferricyanide and of trichlorophenol indophenol 



Some of the most interesting results with coupled stoicheiometric 

 photophosphorylation have been obtained in a study of the reduction of 

 ferricyanide in the presence of intact chloroplasts by Jagendorf, et al. at 

 the McCoUum Pratt Institute [26-29]. These investigations started with 

 the original observation of Arnon [8], that the rate of photoreduction of 

 ferricyanide could be increased by addition of ADP and inorganic ortho- 

 phosphate. The Baltimore group showed that with fresh chloroplasts this 

 increase w'as a striking phenomenon. The rate of photoreduction of ferri- 

 cyanide was about 200 /^.moles per mg. chlorophyll per hour, in the 

 absence of the phosphorylation system, and about 800 /xmoles per mg. 

 chlorophyll per hour in the presence of added orthophosphate and ADP. 

 If the chloroplasts were washed in slightly acid isotonic medium, they 

 were " uncoupled ", in the sense that they caused ferricyanide reduction at 

 a maximum rate in the absence of the phosphorylating system. Un- 

 coupling could also be achieved by addition of ammonia, or of a small 

 amount of the dye, trichlorophenol indophenol. Separate examination of 

 the behaviour of the dye showed that it was an excellent Hill reagent and 

 that its rate of photoreduction was always at least as fast or faster than the 

 rate of photoreduction of ferricyanide under any conditions. The photo- 

 reduction of the dye was not associated with coupled phosphorylation, 

 however, and not subject to stimulation by added ADP and orthophosphate 

 to any significant extent. 



All the phenomena described up to this point can be accommodated 

 within a simple mechanistic picture involving only one phosphorylation 

 site, designated as A in Scheme 4. The system behaves as though the dye 

 can by-pass the phosphorylation site, reacting more or less directly with R. 

 Such a picture is supported by the results of Witt et al. [47], who con- 

 cluded that the transfer of electrons to indophenol dyes is a simpler and 

 more rapid process than is the transfer to ferricyanide. Their evidence was 

 based on a study of the kinetics of disappearance of a light-induced 

 absorption increase at 515 m^u in isolated chloroplasts. 



"Oxidative" photophosphorylation 



The behaviour of trichlorophenol indophenol with chloroplasts exhibits 

 an idiosyncrasy which cannot be so easily explained by the assumption of 

 one photophosphorvlation site at A in Scheme 4, unless one makes some 

 fundamental additions to or changes in the Scheme. Although there is no 

 photophosphorylation associated with the reduction of oxidized dye, one 

 can demonstrate a synthesis of ATP from orthophosphate and ADP when 

 the reduced dye is oxidized by molecular oxygen in the presence of 



