STUDIES ON PHOTOPHOSPHORYLATION VI. PHOSPHORYLATION COUPLED WITH THE PHOTOREDUCTION OF 2,6-DICHLOROPHENOL INDOPHENOL BY CHLOROPLASTS

Abstract

2,6-Dichlorophenol indophenol (DCPIP) had been distinguished from other Hill oxidants in (1) the rapidness of its reduction and (2) the absence of appreciable ATP formation during its photoreduction. It was generally believed that the electron is transferred to indophenol dyes from some component preceding the coupled phosphorylating step, i.e. the photosynthetic electron transport chain is short-cut by indophenol dyes.With the aid of ~(32)P_i, of high specific activity, we have been able to measure the small amount of ATP formed during short exposures (5-10sec.). It has been found, as reported by other workers, the amount of ATP formed is negligible as compared with the amount of dye reduced when the concentration of DCPIP is high. But as the con- centration of DCPIP is decreased, photophosphorylation becomes more and more appreciable and eventually the P/O value approaches 1 at concentration below 10μg/1.2 ml. (Table 1). It appears, therefore, photoreduction of DCPIP is, like other Hill reactions, coupled with phosphorylation.That the formation of ATP is really coupled with DCPIP reduction rather than with the back oxidation of the reduced DCPIP (oxidative photophosphorylation) can be proved by the following facts: (1) With same concentration of oxidized and reduced DCPIP, the rate of ATP formation is more rapid in the former system (Fig. 1); and (2) while the oxidative photophosphorylation is accelerated upon increasing the concentration of DCPIPH_2, the rate of ATP formation in the DCPIP system is unaffected with added DCPIPH_2 (Table 2).The decrease and finally disappearance of ATP formation at high concentration of DCPIP (Table 1) is due to its own uncoupling effect. This is clearly shown by the decrease of ATP formation when DCPIP is added in darkness to chloroplasts preparation preilluminated with PMS. No such effect is observed with DCPIPH_2 (Table 3). Further experiments show that DCPIP also inhibits the ATP production mediated by K_3Fe(CN)_6 (Table 4).When both DCPIP and K_3Fe(CN)_6 are present, the electrons go to K_3Fe(CN)_6 and not to DCPIP as shown by their behavior toward HOQNO (Fig. 2) (DCPIP here acts only as an uncoupler). The apparent absence of ATP formation with DCPIP and with DCPIP+K_3Fe(CN)_6 has led some investigators to the hypothesis that DCPIP intercepts electron in the photosynthetic chain before the phosphorylating step and K_3Fe(CN)_6.The present investigation shows that DCPIP is yet in no way different from other Hill oxidants in the photosynthetic electron transport and the coupled phosphorylation except that it acts at higher concentrations as an uncoupler as well. The failure to detect ATP formation during its photoreduction by other workers might be due to (1) uncoupling caused by the high concentration of the dye used and (2) the insensitiveness of the methods employed for the determination of ATP.