426 BIRGIT VENNESLAND 



these findings do not necessarily conflict with Warburg's statement. It is 

 significant, however, that the prolonged incubation at 20° which we 

 employ to remove CO2 results in a loss of the ability of the grana to give net 

 ATP synthesis in any photophosphorylation system. Furthermore, the 

 ferricyanide-dye system employed does not support ATP synthesis, even 

 with fresh grana. Thus, the operation of the CO., sub-cycle shown in 

 Scheme 8 does not appear to require externally added ATP. It is of course 

 possible that high energy phosphate could be transferred more directly, 

 without going through the adenylate system. It seems just as likely that 

 the COo sub-cycle is self-sustaining with regard to high-energy bonds. 

 The oxidation of formate by the Hill reagent involves a sufficient release of 

 free energy to provide for the synthesis of a high-energy phosphate bond, 

 so that no external sources would be required. Scheme 8 was primarily 

 designed to show how Warburg's mechanism for the Hill reaction can be 

 supplemented to account for all the major phenomenology of photophos- 

 phorylation, with only one postulated phosphorylation site similar in its 

 chemical components and properties to the site of mitochondrial phos- 

 phorylation. As should have been apparent in the development of the 

 argument, the data do not compel one particular choice among a variety 

 of possibilities, so that Scheme 8 should be regarded as a flexible working 

 hypothesis only. The reactions diagrammed in this scheme are all presumed 

 to be catalyzed by washed grana. To explain the overall process of photo- 

 synthesis, additional reactions are clearly required. It should be noted that 

 the " formate " of the diagram may be used in part as a source of reducing 

 power, in which event it will be reoxidized to CO2. Though some of the 

 "formate" will probably also be retained as fixed carbon, the occurrence 

 of this extra COa-fixing mechanism does not in any way deny a functional 

 role to the soluble enzymes of the chloroplast which catalyze other COg- 

 fixing reactions. It is reasonably self-evident, however, that if the grana 

 photoreduce COo directly in the manner indicated in Scheme 7, then this 

 reaction must be regarded as the most important " COo-fixing" reaction in 

 nature. It is conceptually incorrect to think of the grana primarily as 

 generators of ATP and reducing power in the form of reduced TPN. The 

 CO2 reduction precedes the reduction of TPN instead of following it [54]. 



The question of a natural cofactor 



One final precaution must be kept in mind. The Hill reagents and 

 phosphorylation cofactors used in our studies with grana are largely 

 artificial. This is true even for the FMN- and menadione-stimulated 

 photophosphorylation systems. Although FMN is certainly present in 

 chloroplasts, the quantities are insufficient to elicit any reasonably rapid 

 rate of photophosphorylation. Menadione does not occur in nature, but 



