220 



Donald L. Keister 



PHOSPHORYLATION COUPLED TO THE REDUCTION OF INDOPHENOLS 



The phosphorylation coupled to the reduction of the indophenol 

 dyes was demonstrated by all three of the above mentioned groups 

 by using very low concentrations of dye and measuring the small 

 amount of ATP formed by the incorporation of high specific 

 activity ^^?i into ATP during short illumination periods. 

 Gromet-Elhanan and Avron^7) using 5 x lO-^M DCI and short illumi- 

 nation periods measured ATP formation at rates up to I63 ymoles/mg 

 chlorophyll/hr. They further demonstrated that the reduction of 

 DCI was stimulated by the inclusion of a phosphate acceptor 

 system (ADP, Mg"*"*" , and Pi) or by ammonium chloride, a well-known 

 uncoupler of photophosphorylation. These criteria place this 

 compound in the same category as ferrlcyanide^y ^ and tpn <>■'■"'' as 

 Hill oxidants. 



Shen et_ al. ^^^ using essentially the same techniques but with 

 higher light intensities demonstrated that rates of ATP formation 

 up to 6^40 ymoles/mg chlorophyll/hr could be obtained with 

 1.28 X 10-5M DCI with an ATP:2e- ratio of 1.0. This is the 

 highest rate yet reported for a non-cyclic phosphorylation. 



Keister^ ^^ first noted that TCI would catalyze ATP formation 

 while studying the photoreduction of cytochrome £ by chloro- 

 plasts^ll''. This is illustrated in Fig. 1. 



o 500 



UJ 



o 



o 

 llJ 

 (c 400 



z 

 o 



i 300 



o 



o 



200 



UJ 



-J 

 o 



2 100 



4 8 12 16 20 24 



TCIP CONCENTRATION M x 10^ 



Fig. 1. The effect of TCI concentration on phosphorylation 

 coupled to cytochrome c reduction. The reaction mixture, 

 pH 7.5, contained 3 ymoles KCN, 3.8 mg cytochrome c, and 

 10 wg chlorophyll in addition to those described in methods. 

 Illumination was for 2 min with 5000 ft. candles of light. 



In this system TCI was reduced photochemically and oxidized by 

 cytochrome £. Thus, the TCI remains primarily in the oxidized 



