288 



Giorgio Forti, Maria Luisa Bertole and Bruno Parisi 



water (through the photochemical system 2^^^) to the oxidizing agent formed upon 

 absorption of light by system 1. According to our hypothesis, cytochrome f_ is 

 reduced by the electrons coming from light-excited system 1 (chlorophyll a) 

 via PPNR, flavoprotein and TPN. Such an hypothesis is supported by the 

 following evidence: (a) Reduced cyt. f_ is not photooxidized by chloroplasts in 

 the presence of PPMR and TPN, with or without the further addition of purified 

 flavoprotein, in the presence of CMU to prevent photoreduction by water(4). 

 (b) The chloroplasts contain TPNH-cyt. f_ reductase activity (table 1), which 

 is due to a flavoprotein purified together with the TPNH diaphorase-transhydro- 

 genase of Keister et al.('7). The ratio of diaphorase, transhydrogenase and 

 cytochrome f reductase activities of chloroplasts closely resembles the ratio 

 between thes'e activities found with the purified enzyme. Finally, (c), oxidized 

 but not reduced cyt. f_ inhibits the photoreduction of TPN by the system chloro- 

 plasts -PPNR, in spit'e of the fact that the rate of TPN reduction by the same 

 system is much higher than the rate of cyt. f_ reduction (see fig. 1). The in- 

 hibition is largely prevented by the addition of purified flavoprotein (or also 

 by the addition of large amount of chloroplasts, which contain the enzyme), as 

 shown in figure 1. 



This finding can be explained assuming that cyt.£°^ binds to the reduced 

 flavoprotein, which thus would be prevented from reacting with TPN. The 

 following reaction scheme can explain the reported observations: 



(1) 2PPNRH + flavoprotein ^ 2PPKR + fl.pr. H2 



(2) fl.pr. H2 + 2cyt.£0^ ^ fl.pr. + 2 cyt. fj^^^ 



(3) fl.pr. H2 + TPN -^ -fl.pr. + TPNH 



Reactions (3) from right to left and reaction (2) account for the TPNH- 

 cytochrome f reductase of the purified flavoprotein. 



It should be noted that our evidence with extracted cytochrome f_ is not 

 in agreement with currently proposed schemes^^) in which the primary results 

 of photo- act I is the oxidation of bound cytochrome f. Of course, it should not 

 be expected that added cyt. f would take its ordinary place in the chloroplast 

 electron transport chain, since internal, tightly bound cytochrome is already 

 in place. However it is not unreasonable to expect added cyt. f_ to donate or 

 accept electrons from the membrane-bound enzyme; as for instance is the 

 case with cytochrome c added to mitochondria. 



The failure to see photo-oxidation of added cytochrome f_ under any 

 circumstances (including the addition of CMU) might possibly be due to its 

 participation in a cyclic electron flow. If so, however, the steady state level 

 of cytochrome f in the cycle would have to remain very close to 100% reduced; 

 which seems m'ost unlikely. In addition, little or no stimulation of ATP forma- 

 tion has yet been observed due to adding reduced cytochrome f, even though 

 the reduction of the oxidized form does support phosphorylation. 



