259 



Keelin T. Fry and Anthony San Pietro 



solution equivalent to the volume of the added reactant was forced from the 

 cuvette at each addition. Formation of the ferrous triphenanthrolate complex 

 was determined from the absorbance at 510 mp (E = 11, 100) ^^^' and forma- 

 tion of the ferric complex with Tiron from absorbance at 487 mp ( E = 5, 800 

 as determined at pH 8). The latter extinction coefficient corresponds well 

 with the value of 6, 100 calculated from the data of Yoe and Jones ^'^^' and was 

 used, therefore, to calculate the values presented. 



Control experiments in which ferrous iron was added to the anaerobic 

 reaction mixtures after treatment with PCMS indicated that the ferric iron 

 observed did not arise from oxidation of free ferrous iron in solution. Thus, 

 it would appear from the data in Table 4 that the metal in the native enzyme 

 is in the ferric oxidation state. The color formation observed with OP 

 probably results from reduction by reducing agents present in the protein 

 which are not bound by the mercurial. In this regard it should be noted that 

 the amount of PCMS added was about four times that required to titrate the 

 PPNR. 



PHOTOREDUCTION OF IRON 



(26) 



It has been demonstrated by several investigators that PPNR can be 

 photoreduced in the presence of illuminated chloroplasts U 2, 13, 16, 2 7) j^. 

 was of interest, therefore, to determine whether reduction of the enzyme 

 was accompanied by a concomitant change in the valence state of the non- 

 haem iron. For this purpose reaction mixtures containing PPNR and chloro- 

 plasts were illuminated anaerobically for varying lengths of time. After 

 illumination, the pxtent of photoreduction and the appearance of ferrous iron 

 were determined ^ ^ As shown in Table 5, one- half the iron is in the 

 ferrous state when the protein is completely reduced. 



TABLE 5 



Photoreduction of Iron 



Chance and San Pietro (^'') investigated the photoreduction of PPNR by 

 chloroplasts and showed that the light- dark difference spectrum for PPNR 

 reduction by illuminated chloroplasts corresponded to the difference spec- 

 trum for dithionite- reduced PPNR. The kinetics of reduction of PPNR by 



