Photooxidation of ascorbate as a substitute for oxygen production by illuminated chloroplasts


The ability of freshly prepared spinach or poke weed (Phytolacca americana) chloroplasts to photoreduce triphosphopyridine nucleotide (TPN) under anaerobic conditions decreased in the presence of ascorbate and 2,6-dichlorophenolindophenol (DPIP). On .aging the chloroplasts, however, addition of DPIP and ascorbate was required for a high rate of TPN photoreduction. Qualitatively similar results were obtained for photoreduction of DPN, indigo carmine, riboflavin-5'-phosphate and vitamin K3 . Addition of 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU) at 10-5 M to freshly prepared chloroplasts inhibited the photoreduction of TPN by more than 98 %, and activity was restored to 94 %o f the original value by addition of ascorbate and.DPIF. Hydroxylamine at 10-3 Mand ammonium ion at 0.17 M produced similar effects. Qualitatively similar results were obtained with .these three inhibitors for photoreduction of DPN, indigo , carmine, .riboflavin-5 1 -phosphate, vitamin K3, methyl red and tetrazolium blue. Cesium ion and orthophenanthroline produced inhibitory effects similar to those of DCMU, hydroxylamine and ammonium ion which responded analogously to the DPIP-ascorbate couple. The data substantiate the previously proposed idea that ascorbate is photooxidized in the absence of oxygen by the photochemical oxidizing equivalents produced by illuminated chloroplasts, with the dye DPIP· acting as intermediate electron carrier. With intact .chloroplasts 2 the oxidizing equivalents are utilized preferentially for oxidation of water to produce oxygen. Only after the oxygen-evolution .system becomes .inoperativec1;1.n the ascorbate-DPIP couple compete successfully for the oxidizing equivalents. ExPosure of chloroplasts . to . light for 20 minutes _prior to ,adding .a Hill oxidant resulted in a reduced photoreducin,g capacity in the Hill reaction with TPN, indigo .carmine .and tetrazoli:um blue. However, such a treatment .actually increased the photoreducing _ability of the chloroplasts in the presence of DPIP and .ascorbate. Chloroplasts were inactivated toward the Hill reaction .by a pH of 7.7 at 30°. This inactivation was .readily overcome by addition of ascorbate and DPIP, indicating that .the principal effect of this treatment was to inactivate the oxygen-evolution system. Since indigo carmine photoreduction is stabili.z-ed· much more by the presence of sucrose in the reaction mixture than the photoreduction of DPIP it is indicated that there are components in the electron transport system leading from the site of DPIP photoreduction to that of indigo carmine photoreduction which .are sensitiye to distilled water dilution. The photoreduction of TPN, PPN .and indigo carmine was stimulated by addition of cysteine, whereas reduced glutathione was without effect. Cysteine was unable to overcome the inhibitions of DCMU, hydroxylamine and ammonium ion and no stimulation was observed when cysteine was .added to the inhibited systems containing DPIP and ascorbate even though cysteine was capable of stimulating the uninhibited reaction containing the DI"IP-ascorbate couple. It was there3 fore concluded that cysteine acts by stabilizing or reacting with the oxygen-evolving system. Chloride-deficient chloroplasts .were stimulated by added chloride and cyanide in Hill reaction activities. Reaction .mixtures containing sufficient chloride to provide maximal stimulation were further stimulated by added cyanide, versene and Tris.



College and Department

Chemistry and Biochemistry



Date Submitted


Document Type





Photosynthesis, Hil reaction, Vitamin C, Chloroplasts, Salts



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