PHOTOSYNTHETIC PHOSPHORYLATION AND THE ENERGY CONVERSION PROCESS 363 



A primary phosphorylation reaction, common to all pathways of cyclic 

 photophosphorylation, is en\'isaged as being coupled with the oxidation 

 of the terminal cytochrome in the photosynthetic particle, i.e. the cyto- 

 chrome that reacts with the excited chlorophyll molecule (compare Figs. 

 4 and 5). A second site of phosphorylation in the vitamin K pathway is 

 likely to occur on oxidation of reduced vitamin K (or its analogue) by 

 cytochromes, as was suggested by the model reactions proposed by 

 Wessels [85], Harrison [116] and Clark et al. [117]. In the FMN pathway, 

 an additional phosphorylating site can be readily envisaged in the span 

 between TPN and cytochromes [94]. 



Further evidence, derived from fractionating chloroplasts, will be 

 given in the next Section for the conclusion that cyclic photophosphoryla- 

 tion catalyzed by phenazine methosulphate probably proceeds by way of a 

 "bypass" and is less dependent on enzymic chloroplast constituents than 

 photosynthetic phosphorylation catalyzed by either vitamin K or FMN. 



9. Structural association of chlorophyll with the 

 photophosphorylating system 



In photosynthetic bacteria the photophosphorylating system is 

 structurally bound to chlorophyll in the smallest particles that function as 

 units in the absorption of light energy, the chromatophores. Their 

 analogues in green plants are the grana and it was of interest, therefore, 

 to determine whether in chloroplasts photophosphorylation is indeed 

 localized in the grana. 



Photosynthetic phosphorylation was tirst observed in intact chloro- 

 plasts (Fig. 2) but experiments with disrupted chloroplasts soon demon- 

 strated that structural integrity was not essential for this process. When 

 whole chloroplasts were broken, active photophosphorylation systems 

 were reconstituted by a recombination of chloroplast fractions and added 

 cofactors [38, 39]. This technique proved effective in investigating the 

 mechanism of photophosphorylation but provided no rigid e\idence that 

 the site of photophosphorylation is in the grana. 



Direct evidence for the localization of photosynthetic phosphorylation 

 in grana, freed from other chloroplast fractions, was obtained by Miiller 

 et al. [118] who prepared purified grana by sonication of isolated whole 

 chloroplasts followed by a density gradient centrifugation technique. The 

 purity of the grana obtained by these methods was determined by exami- 

 nation of electron micrographs of freeze-dried and air-dried grana prepara- 

 tions (Figs. II and 12). The freeze-drying technique avoids artifacts 

 resulting from chemical fixation and retains the natural shape of the 

 particles [119]. Cyclic photophosphorvlation bv purified grana is shown in 

 Table IV. 



