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Daniel I. Arnon 



only TPN (E^ = -320 mV, pH 7) but also strongly electronegative 

 redox dyes such as methyl viologen (Eq = -^55 mV, pH 7) , a dye 

 which was shown by Jagendorf and Avron (20) and Hill and Walker 

 (21) to catalyze cyclic photophosphorylation. 



It would follow from these considerations that an accumulation 

 of photoreduced methyl viologen, although never before demonstra- 

 ted, should be possible if its reoxidation by oxygen or by the 

 cyclic electron flow mechanism of chloroplasts is prevented. This 

 was experimentally shown by Mitsui et al. (22). Table 1 shows 

 that illuminated chloroplasts reduced methyl viologen in the 

 presence of cysteine and dichlorophenolindophenol (DPIP) . No 

 reduction of methyl viologen was observed in the dark or after 

 boiling the chloroplasts for 5 min. 



Table 1 

 Photoreduction of Methyl Viologen by Chloroplasts (22) 



Methyl viologen reduced 

 (umoles/hr/mg chl) 



Complete system 5^*2 



DPIP omitted 5*8 



Cysteine omitted 0.2 



Water was not the electron donor since the system was incap- 

 able of evolving oxygen, with or without added CMU. The rate of 

 photoreduction of methyl viologen was greatly decreased when DPIP 

 was omitted from the reaction mixture. Thus, DPIP rather than 

 cysteine appeared to be the effective electron donor. However, 

 the function of cysteine was not limited to donating electrons 

 via DPIP since ascorbate, which can also reduce DPIP, did not re- 

 place cysteine in the same system. Only after a mild heat treat- 

 ment (50° C for 10 min.)> which greatly reduced the total photo- 

 activity of chloroplasts, were chloroplasts able to use the 

 ascorbate-DPIP couple as an electron donor for the photoreduction 

 of methyl viologen (at a low rate). 



The effectiveness of cysteine is explained by its inhibition 

 of the cyclic electron flow by which reduced methyl viologen can 

 be reoxidized by chloroplasts. As shown in Table 2, cysteine 

 strongly inhibited cyclic photophosphorylation catalyzed by 

 methyl viologen, menadione or FMN but not the "shortcut" cyclic 

 photophosphorylation pathway catalyzed by phenazine methosulfate 

 (6). The last observation suggests that the inhibitory effect of 

 cysteine on cyclic photophosphorylation was not related to an 

 activation of ATPase (23) since in that case all photophosphory- 



