PHOTOSYNTHETIC PYRIDINE NUCLEOTIDE REDUCTASE. IV FURTHER 

 STUDIES ON THE CHEMICAL PROPERTIES OF THE PROTEIN 



Keelin T. Fry and Anthony San Pietro 



The first demonstration that a soluble factor can be added back to chloro- 

 plasts to reconstitute their over-all electron- transport reaction was reported 

 by Davenport, Hill and Whatley ' ^ Mn 1952. They showed that washed 

 chloroplasts lost their capacity for reducing muscle methaemoglobin in the 

 light. The addition of the soluble fraction restored the activity. They fur- 

 ther demonstrated that the soluble methaemoglobin reducing factor was 

 thermolabile. 



Lang and San Pietro (2, 3) were unaware of the prior work of Davenport 

 et al. (i^when they reported that chloroplasts contain a soluble protein which 

 catalyzed the photochemical reduction of pyridine nucleotides. At that time 

 there wa s no evidence to suggest that the proteins isolated independently by 

 these two groups might be identical. The possible identity of these two pro- 

 teins, namely, photos ynthetic pyridine nucleotide reductase (PPNR) and the 

 methaemoglobin reducing factor, only became apparent when they were 

 available in purified form (^' ^). 



During the course of their studies on the methaemoglobin reducing factor, 

 Davenport and Hill ^^^ observed that the preparation catalyzed the reduction 

 of a number of haem- proteins, including cytochrome c, by illuminated 

 chloroplasts. Moreover, this factor was shown to catalyze the reduction of 

 NADP by illuminated chloroplasts ' ^ 



At the suggestion of Dr. H. E. Davenport, the ability of PPNR to catalyze 

 the reduction of cytochrome c by illuminated chloroplasts was examined and 

 it was found to catalyze this reduction ('*). Thus, the catalytic activities of 

 the se two proteins are identical. 



ABSORPTION SPECTRUM 



The methaemoglobin reducing factor ^^> and PPNR ^^' have been exten- 

 sively purified and shown to be homogeneous, both electrophoretically and 

 in the ultracentrifuge. Both proteins contain two moles of non-haem iron 

 per mole of enzyme and an equivalent amount of "labile sulfur" v9-il}_ ^he 

 absorption spectra of PPNR from spinach and of the methaemoglobin reducing 

 factor from parsley are shown in Figure 1. It is clear that these two spectra 

 are very similar; the major difference between them is the relationship of 

 the absorption in the visible region to that in the ultra-violet region. The 



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