Determination of Superoxide Free Radical Ion and Hydrogen Peroxide as Products of Photosynthetic Oxygen Reduction

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H202-dependent oxidation of ascorbate and ascorbate-dependent reduction of H202. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This To date, detailed studies on the mechanism(s) of 02 evolution coupled to H202 reduction have not been presented. Here, we report such studies using ruptured chloroplasts. Reaction 5 is also consistent with catalase activity. Although catalase is predominantly a peroxisomal enzyme (13), residual catalase activity is invariably associated with isolated chloroplast preparations (1). Accordingly, evidence is presented which excludes the possibility that catalase activity is involved in the operation of reaction 5 in ruptured chloroplasts, in the light. MATERIALS AND METHODSChemicals. Chemicals were obtained from the sources described previously (16). H202 and t-butyl hydroperoxide concentrations were determined by titration with standardized KMnO4. The GSH contained 6% GSSG (16).Chloroplast Preparation. Unwashed, sonicated, or osmotically shocked chloroplasts were prepared as described previously (15, 16) from pea seedlings (Pisum sativum cv. Massey Gem). In the studies reported here, the unwashed chloroplasts were repeatedly washed (4 times) with a washing medium (16)

Section: Discussionmentioning

confidence: 76%

“…Pretreatment for 10 min in the dark decreased enzyme activity by 30%, but the activity decreased by 67% in the light. The activity of chloroplasts incubated in the light for 10 (Table V).…”

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Light-Dependent Reduction of Hydrogen Peroxide by Ruptured Pea Chloroplasts

Jablonski

Anderson²

1982

“…Chloroplasts reduce 02 in the light to H202 (2,4). Foyer and Halliwell (5) have proposed a mechanism for the reduction of H202 in chloroplasts involving ascorbate as the reductant.…”

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confidence: 99%

Light-Dependent Reduction of Dehydroascorbate by Ruptured Pea Chloroplasts

Jablonski¹,

Anderson²

1981

Glutathione dehydrogenase (EC 1.8.5.1) was partialHy purified from pea shoots. The pH optimum was 7.6. The Km values for GSH and dehydroascorbate were 4.4 and 0.44 millimolar, respectively. The enzyme was inhibited by iodoacetate and CuS04 but not significantly by ZnCI2 or NaN3. Part of the total enzyme activity was associated with isolated chloroplasts.Illuminated ruptured chloroplasts, in the presence of 50 micromolar NADP(H) and substrate concentrations of GSH or GSSG, catalyzed (dehydroascorbate plus glutathione)-dependent 02 evolution with the concomitant reduction of dehydroascorbate to ascorbate. Oxidation of ascorbate by ascorbate oxidase activity associated with the chloroplasts was relatively insignificant. ZnCI2 inhibited (dehydroascorbate plus glutathione)-dependent 02 evolution but not ascorbate formation. The reaction was attributed to light-dependent reduction of GSSG (involving glutathione reductase) coupled to the reduction of dehydroascorbate (involving glutathione dehydrogenase). Light-dependent reduction of GSSG appears to be the rate-limiting step in this reaction sequence at physiological concentrations of GSH.chloroplasts, especially as light-induced alkalization of chloroplast stroma (10) provides favorable conditions for the nonenzymic reaction.We reported previously (11) that illuminated ruptured chloroplasts catalyze the reduction of GSSG with the concomitant evolution of 02. The properties of these reactions were consistent with the operation of light-coupled GSSG reductase (reactions I and II). Taken in conjunction with the proposal of Foyer and Halliwell (5), this suggests that the reduction of DHA, and ultimately H202, could be light coupled via GSSG reductase. This mechanism predicts that in the presence of catalytic amounts of GSH or GSSG, chloroplasts will exhibit DHA-dependent 02 evolution with the concomitant reduction of DHA to ascorbate. Previously, we reported that ruptured chloroplasts did not support DHA-dependent 02 evolution in the presence of 40 tLM GSSG, 50 /LM NADPH, and 1 mm DHA (11). In this paper we report that illuminated ruptured chloroplasts in the presence of 50 UM NADPH, 0.94 mmDHA, and >0.15 nm GSH oraO.l m GSSG exhibit characteristics consistent with the operation of reactions I to IV and that reaction III involves a chloroplast GSH dehydrogenase.Chloroplasts reduce 02 in the light to H202 (2, 4). Foyer and Halliwell (5) have proposed a mechanism for the reduction of H202 in chloroplasts involving ascorbate as the reductant. Their model also postulates that the DHA3 produced in this reaction is successively reduced by GSH and NADPH, the latter involving GSSG reductase (EC 1.6.4.2) MATERIALS AND METHODS Chemicals. DHA was obtained from Koch-Light, Bucks., England; the purity as estimated by oxidation of GSH in the presence of purified GSH dehydrogenase was 94%. Cyt c (horse heart), ascorbate oxidase (lyophilizate), and GSSG were obtained from Boehringer, Mannheim, Germany. GSH was obtained from Sigma and contained 0.06 mol GSSG/mol GSH by assay ...

“…Suggestions are in the literature (8) for a link between the chloroplast pyridine nucleotide pool and 02. Conceivably, the NADPH formed by either glucose-6-P dehydrogenase or by noncyclic electron transport may be oxidized by superoxide and/or hydroxyl radicals (4) produced by photosynthetic reduction of 02 (9,10).…”

Section: Resultsmentioning

confidence: 99%

Orthophosphate Control of Glucose-6-Phosphate Dehydrogenase Light Modulation in Relation to the Induction Phase of Chloroplast Photosynthesis

Huber

1979

High concentrations of orthophosphate (Pi) inhibited C02-dependent 02 evolution and prevented the inactivation of glucose-6-P dehydrogenase by light in intact spinach and barley chloroplasts. Addition of glycerate-3-P to chloroplasts inhibited by Pi in the light, induced 02 evolution and caused rapid inactivation of glucose-6-P dehydrogenase. The activity of phosphofructokinase detected in chloroplast preparations was not affected by light or by Pi.Dihydroxyacetone-P was a major product of chloroplast photosynthesis when optimum concentrations of Pi were used. Chloroplasts continued to form dihydroxyacetone-P at a slow rate in the presence of Pi at concentrations (2 to 4 millimolar) that gave complete inhibition of C02-dependent 02 evolution. Formation of dihydroxyacetone-P in the presence of 4 millimolar Pi was stimulated by light and either 02 (150 micromolar) or sparker amounts of oxaloacetate.Conditions that favored dihydroxyacetone-P formation (high 02 or low 02 plus oxaloacetate) increased the optimum Pi concentration for C02-dependent 02 evolution and stimulated 02 evolution at high concentrations of Pi. The stimulation of 02 evolution at superoptimal concentrations of Pi by 02 or oxaloacetate was prevented by dithiothreitol.The results suggested that formation of pentose-P pathway intermediates via the oxidative pentose-P pathway may be limited by availability of NADP in the light but may occur at significant rates and thereby contribute to termination of the induction phase of 02 evolution.The induction phase of C02-dependent O2 evolution by isolated chloroplasts is the initial lag observed before photosynthesis reaches maximum rates, and has been postulated to represent the time required for the autocatalytic increase in photosynthetic intermediates in the chloroplast stroma (29). This is supported by the observed reduction in the length of the lag phase by exogenous metabolites (7) that are transported across the chloroplast envelope (12,29) and the increase in the lag phase caused by Pi (6,13).High external Pi also reduces the maximum rate of C02-dependent 02 evolution, presumably by continually draining metabolites from the chloroplasts by operation of the phosphate translocator (11). Furthermore, an increase in triose phosphates and pentose monophosphates before the onset of 02 evolution has been ob-