VOL. 12 (1953) PHOTOCHEMICAL REDUCTION OF NITRATE 69 



synthetic mechanism, experiments designed to support the "alternate hypothesis" have 

 not been carried out. The following statement of Rabinowitch^^ accurately represented 

 the stage when the experiments discussed below were undertaken : 



"More detailed experiments, with specific inhibitors of tlie type of hydroxylamine, could help 

 to analyze the mechanism of photochemical nitrate reduction and establish its relation to ordinary 

 photosynthesis. Unfortunately, this subject has not received further attention since 1920, although it 

 is certainly -worth renewed study", (p. 540). 



BASIS OF THE EXPERIMENTAL APPROACH 



Instead of following the experimental procedure suggested by Warburg, we have 

 chosen a different one. There is a twofold reason for this. In the first place, it is alto- 

 gether likely that the enzyme system involved in the reduction of nitrate is not present 

 in the chloroplasts or grana, just as that for CO^ assimilation must be deemed missing 

 in these structures .But more important, the study of nitrate reduction was one phase 

 of a more general investigation, concerned with the determination of intermediate 

 products in CO 3 assimilation proper. The approach rests on the following considerations. 



The most general formulation of green plant photosynthesis, in keeping with present 

 knowledge, can be schematically represented by the diagram^' : 



H (JhXT'' 



HOH 



Light 



^&' 



Pigments 



-> 



E" 



^O (R-z/nw) O2 



^E"OH 



We consider the photochemical reaction to be limited to the formation of the entities, 

 E'H and E"OH ; the further transformations represent enzyme-controlled dark reactions. 

 From present information about the effects of light intensity and temperature on the rate 

 of photosynthesis it is reasonable to postulate that at a sufficiently high intensity of 

 illumination the rate of O.^ production is governed by one of the dark reactions. 



In normal photosynthesis the function of B, the hydrogen acceptor participating in 

 the regeneration of E' from E'H, is exhibited by COg or, more probably, by some of its 

 early transformation products, such as carboxylic acids enzymically generated by COg- 

 addition to other compounds. Hence, at high light intensities the rate of photosynthesis 

 will be limited either by an enzyme concerned in the formation and reduction of these 

 substances, or by a component of the enzyme system responsible for the regeneration of 

 E" and the liberation of Og- In the former case it should then become possible to bring 

 into operation additional enzyme systems which can transfer hydrogen from E'H to a 

 different acceptor, B', and therefore to increase the rateof O2 evolution at light intensities 

 greater than that required for saturation of the CO2 assimilating mechanism. 



Since it appeared an almost foregone conclusion that the enzymes involved in 

 nitrate reduction are different from those concerned with the reduction of CO 2, these 

 deductions would imply that the rate of O2 evolution in the presence of both CO 2 and 

 nitrate might be greater than that obtainable in the absence of nitrate. Theoretically, 

 the relation between rate of O2 production, CO 2 assimilation, and light intensity could 

 then be expressed by the curves in Fig. i. 



From this figure it may be concluded that the linear relationship between rate of 

 References p. 73 1 74 ■ 



