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F. R. Whatley 



and Vennesland) oxidized f errocytochrome c at a slow rate in the 

 dark in air but not in argon, and that this dark oxidation, 

 which proceeded in accordance with the thermochemical gradient, 

 was completely suppressed by 10"'* M KCN. Ferrocytochrome c was, 

 however, oxidized when the system was illuminated in the pre- 

 sence of KCN-treated digitonin extracts and oxygen was supplied. 

 No photooxidation occurred under an atmosphere of argon or in 

 the dark, as shown in Fig. 1. Nieman and Vennesland concluded 

 from similar evidence that a "cytochrome c photooxidase" had 

 been unmasked in their preparation. However, as will now be 

 reported, triphosphopyridine nucleotide (TPN) can be substituted 

 for oxygen as a terminal electron acceptor in an atmosphere of 

 argon. To accomplish this it was necessary to add both the 

 electron carrier ferredoxin and the enzyme ferredoxin-TPN reduc- 

 tase to transfer the electrons produced by the photoreaction to 

 TPN. An experiment showing the photooxidation of ferrocyto- 

 chrome c by TPN under argon is shown in Fig. 2 and represents 

 another experimental manifestation of Nieman and Vennes land's 

 apparent cytochrome c photooxidase activity. 



Photooxidation of ferrocytochrome c by TPN proceeded against 

 a thermochemical gradient, unlike the reaction with oxygen, and 

 obviously required an input of light energy. It was not imme- 

 diately apparent why the oxidation of ferrocytochrome c by 

 oxygen should also need an input of light. However, our results 

 suggest that the photooxidation of ferrocytochrome c is a mani- 

 festation of the terminal portion of the electron transport 

 chain of noncyclic photophosphorylation in chloroplasts (see 

 ref. 6), in which TPN acts as the physiological electron accept- 

 or, but can be replaced unspecif ically by molecular oxygen. 

 Additional support for the view that the digitonin extracts re- 

 tained the terminal portion of the electron transport chain 

 comes from an experiment, shown in Fig. 3> in which ferrocyto- 

 chrome c was replaced by the ascorbate/dichlorophenol indophenol 

 dye couple. In the light, but not in the dark, TPN became 

 photoreduced at the expense of the oxidation of ascorbate. A 

 preliminary report of these results has appeared elsewhere (7) • 



(2) Cyclic photophosphorylation catalyzed by phenazine metho - 

 sulfate . 



It is generally accepted that PMS catalyzes a truly cyclic 

 electron flow in chloroplasts, accompanied by ATP formation 

 (2-4). The principal evidence to support this conclusion may 

 be summarized: (i) No oxygen exchange was observed to accompany 

 the ATP formation (2,3), (ii) the rates of phosphorylation in 



