223 



Donald L, Keister 



Losada e^ a_l. ^-'-^^ first reported that when indophenols were 

 added to a reaction containing ferricyanide the rate of oxygen 

 evolution was accelerated, whereas the coupled phosphorylation 

 was inhibited. Since they also found that there was no inhibi- 

 tion of ATP formation coupled to TPN reduction by the same 

 concentrations of dye (in fact there was a stimulation), they 

 proposed that the indophenols were the direct electron acceptors, 

 not coupled to phosphorylation, which were reduced at a point in 

 the electron transport chain prior to the site of phosphorylation. 

 In the TPN system the indophenols were rapidly reduced and not 

 reoxidized by TPN (the reduced dyes are not uncouplers). Also, 

 oxygen evolution can be blocked by CMU, and ATP formation and TPN 

 reduction restored by reduced dye and ascorbate ^-'•5) , thus 

 eliminating the requirement for the photooxidation of water. On 

 the basis of these observations they proposed a separation of the 

 photooxidation of water and non-cyclic phosphorylation into two 

 distinct photochemical reactions. Although it appears that these 

 are indeed two separate reactions, the use of the indophenol dyes 

 to separate them was not valid since the above results demon- 

 strated that there was ATP formation coupled to their reduction 

 and that they are potent uncouplers. 



Shen et_ al_. (") have even cast some doubt on whether in a 

 system containing both ferricyanide and indophenol, that the dye 

 is the direct electron acceptor. Taking advantage of the 

 differential inhibition of DCI and ferricyanide reduction by 

 hydroxyquinoline N-oxide^^°) (HOQNO), they demonstrated that in a 

 system containing both ferricyanide and DCI, the degree of 

 inhibition by HOQNO corresponded to the degree of inhibition 

 observed with ferricyanide alone and not to that observed with 

 DCI alone. Thus it appears that in the DCI-ferricyanide system, 

 ferricyanide is the direct electron acceptor and the DCI an 

 inert uncoupler. 



CYCLIC PHOTOPHOSPHORYLATION 



The ability of reduced indophenols to reduce a photochemical 

 oxidant and thus restore the reduction processes in chloroplasts , 

 in which the normal process has been blocked by inhibiting oxygen 

 evolution either with CMU or aging' -^^^ has been established. 

 From these results it could have been postulated that the reduced 

 indophenols should catalyze a cyclic electron transport since 

 they can be both oxidized and reduced by chloroplasts. This was 

 first demonstrated by Trebst and Eck'-^^; who reported that 

 reduced DCI and TCI catalyzed a phosphorylation by chloroplasts 

 that was not inhibited by DCMU. They interpreted this as a 

 cyclic phosphorylation. These results were later confirmed by 

 three laboratories (°~°) . 



Krogmann and Vennesland^^ ) earlier had reported that the 

 indophenol dyes could mediate an oxygen dependent photophosphory- 

 lation and suggested that the phosphorylation occurred upon the 

 photooxidation of the dye and not during the reduction. This 

 suggestion was based upon: 1) reduced dyes catalyzed the ATP 

 formation; 2) the phosphorylation was inhibited by a nitrogen 

 atmosphere; and 3) DCMU inhibition could be reversed by reducing 



