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Giorgio Forti, Maria Luisa Bertole and Bruno Parisi 



all or almost all the cyt. _f has been reduced, TPN reduction begins. Under 

 this aspect, cyt. f behaves like ferricyanide(6). pig. 1 also shows that the 

 addition of the purified chloroplast flavoprotein (prepared according to Keister 

 et al.C'') prevents the inhibition of TPK reduction by oxidized cyt. f. These 

 observations indicate that cyt. £ photoreduction by chloroplasts is competitive 

 with the reduction of TPN. In other words, the same photochemical system 

 (chlorophyll a, or system 1 of Duysens et al.^^)) ig providing electrons for 

 the reduction of both TPN and cytochrome f, in contrast with the widely ac- 

 cepted scheme according to which cytochrome f_ is reduced by the accessory 

 pigment system and oxidized by chlorophyll a. More direct evidence was 

 found to support this hypothesis. The chloroplasts are able to reduce cyto- 

 chrome f in the dark, utilizing TPNH as the specific electron donor. The 

 ratio of This enzymatic activity to diaphorase and transhydrogenase activity of 

 chloroplasts is shown in table 1. Under the conditions indicated in table 1, the 

 rate of non-enzymatic reduction of cyt.f_°^ by TPNH is approximately 3% of 

 the enzymatic reaction. It can be seen from the data reported that the ratios 

 of the three activities are comparable for chloroplasts and for the purified 

 enzyme. Furthermore, the observed rates of TPNH-cytochrome f_ reductase 

 activity of spinach chloroplasts are adequate to account for the observed rates 

 of cytochrome photoreduction, ranging around 40-50 |jmoles per milligram of 

 chlorophyll per hour, under light saturation conditions. Higher rates of photo- 

 reduction, up to 120 jimoles/mg Chi. per hour, have been observed with 

 Tetragonia chloroplasts. 



TABLE 1 

 Enzymic Activities of Chloroplasts and of Purified Flavoprotein 



Spinach chloroplasts Purified flavoprotein 

 Activity yequivalents/mg chl. yequivalents/mg protein 



h. min. 



TPNH — .►Fe(CN)g 



790 48.00(152) 



TPNH .► DPN 8.04 0.93(290) 



TPNH — ^cyt.fox 40.00 3.00 



Conditions: Tris buffer 0.05 M, pH 8.0; TPNH lO'^M; 



Ferricyanide 5 x lO'^M DPN lO'^M. Cytochrome f^^lO-^M. 

 The number between brackets indicates specific activities 

 as defined by Keister et al.C^). 



TPNH-CYTOCHROME f REDUCTASE OF SPINACH 



As indicated in table 1, the purified diaphorase-transhydrogenase^'^) has 

 a strong cytochrome f reductase activity. Table 2 summarizes the different 



