Spinach chloroplasts scavenge hydrogen peroxide on illumination

Intact spinach chloroplasts isolated by the modified silicasol density centrifugation scavenged H₂O₂ on illumination ata rate about 3-fold that of bicarbonate-dependent O₂-evolution.Accompanying the disappearance of 1 mole of H₂O₂ is the evolutionof a half mole of O₂. The photoscavenging of H₂O₂ was inhibitedby 3(3,4-dichloro-phenyl)-l, l-dimethylurea, cyanide and azide.These results indicate that in chloroplasts H₂O₂ is reducedto H₂O by a cyanide and azide-sensitive peroxidase using a photoreductantas an electron donor. (Received July 4, 1980; )     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Resistance of Photosynthesis to Hydrogen Peroxide in Algae

The effects of H₂O₂ on the photosynthetic fixation of CO₂ andon thiol-modulated enzymes involved in the photosynthetic reductionof carbon in algae were studied in a comparison with those inchloroplasts isolated from spinach leaves. In both systems,H₂O₂-scavenging enzymes were inhibited by addition of 0.1 mMNaN₃ 1 h prior to the addition of H₂O₂. A concentration (10^-4M) of H₂O₂ caused strong inhibition of the CO₂ fixation by intactspinach chloroplasts, as observed by Kaiser [(1976) Biochim.Biophys. Acta 440: 476], but not that by Euglena and Chlamydomonascells. The same results were also obtained with cells of thecyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803in the presence of 1 mM hydroxylamine. These results indicatethat algal photosynthesis is rather resistant to H₂O₂. The insusceptibilityto H₂O₂ of thiolmodulated enzymes, namely, fructose-1,6-bisphosphatase,NADP-glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphatekinase, was also observed in the chloroplasts of Euglena andChlamydomonas and in cyanobacterial cells. It seems likely thatthe resistance of photosynthesis to H₂O₂ is due in part to theinsusceptibility of the algal thiol-modulated enzymes to H₂O₂. (Received April 22, 1995; Accepted June 29, 1995)     

Hydroxyl Radicals and a Thylakoid-Bound Endopeptidase are Involved in Light- and Oxygen-Induced Proteolysis in Oat Chloroplasts

The hypothesis that light- and oxygen-induced proteolysis inchloroplasts is mediated by active oxygen species was examined.In order to determine whether or not H₂O₂ and/or ^{dot}OH radicalsare involved in these degradative processes we compared thedegradation of proteins in isolated oat chloroplasts exposedto white light at 80 W m^-2 with that in chloroplasts incubatedin darkness in the absence or presence of H₂O₂ or a ^{dot}OH-generatingsystem composed by ascorbic acid, FeCl₃ and H₂O₂ (Asc-Fe-H₂O₂).Light enhanced the rate of degradation of at least 18 polypeptides,while proteolysis was almost negligible in darkness in the abscenceof additives. H₂O₂ had a very small effect. However, Asc-Fe-H₂O₂-treatedchloroplasts in darkness showed a pattern of protein degradationalmost identical to that observed in the light. A thylakoid-boundendopeptidase (EP), the activity of which increased under photooxidativeenvironmental conditions and treatment with an ^{dot}OH-generatingsystem, was partially purified and characterized as a serinetypeprotease. Treatments with inhibitors of serine-type proteaseprevented both light- and Asc- Fe-H₂O₂-induced proteolysis.EP was more active against both soluble and membranous proteinsthat had been pretreated with Asc-Fe-H₂O₂ than against untreatedproteins. It is proposed that a high dose of light irradiationpromotes proteolysis by increasing the formation of ^{dot}OH,which may modify proteins such that they become more susceptibleto EP-catalyzed hydrolysis. ¹Fisiología Vegetal, Dept. de Biología Vegetal,Universidad de Alcalá de Henares, Present address: 28871Alcalá de Henares (Madrid), España.     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Effect of hydrogen peroxide on the fluorescence and the G550 absorption change of spinach chloroplast fragments

Effects of H₂O₂ on the transient phase of fluorescence and thelight-induced absorption change of C550 in the presence of ferricyanidewere studied in spinach chloroplast fragments at room temperature.In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU),the parameter of the variable fluorescence, work integral, wasincreased by the addition of H₂O₂ and the rate of its recoveryin the dark was decreased. The steady-state fluorescence yieldwas decreased by H₂O₂. Essentially the same results were obtainedin the absence of ferricyanide. In the presence of DCMU, H₂O₂ decreased the steady-state absorptionchange of C550 and inhibited its reoxidation in the dark. Thesame effects were observed when H₂O₂ was added to chloroplastfragments in the presence of DCMU and carbonyl cyanide m-chlorophenylhydrazone.From these data we concluded that the fluorescence quencherQ and C550 are not identical. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13 [EC] ), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19 [EC] )and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11 [EC] ) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)     

Specific inhibition of photosystem II activity in chloroplasts by fumigation of spinach leaves with SO2

The phytotoxic effects of sulfur dioxide (SO₂) were investigatedby fumigating spinach plants with SO₂. Inhibition of 2,6-dichloroindophenol(DCIP) photoreduction was observed in spinach chloroplasts isolatedfrom fumigated leaves. NADP and DCIP photoreductions were inhibitedto a similar extent by fumigation with 2.0 ppm SO₂ but electronflow from reduced DCIP to NADP was not affected. When electronflow from H₂O to NADP was inhibited by 36%, a 39% inhibitionof non-cyclic photophosphorylation was observed. However, phenazinemethosulfate(PMS)-catalyzed cyclic photophosphorylation wasas active as in the control chloroplasts. Moreover, in the presenceof PMS, no significant suppression was observed in the extentof light-induced H⁺ uptake or in the rate of H⁺ efflux in chloroplasts.From these results, it can be concluded that SO₂ inhibits theelectron flow driven by photosystem II when plants have beenfumigated with SO₂. In spinach leaves fumigated with SO₂, the rate of photosyntheticO₂ evolution was reduced under light-limited conditions, whilethe rate of respiratory O₂ uptake changed slightly. (Received February 8, 1979; )     

Photoproduction and Detoxification of Hydroxyl radicals in Chloroplasts and Leaves and Relation to Photoinactivation of Photosystems I and II

The light-dependent production of hydroxyl radicals (HO{dot})by thylakoids, chloroplasts and leaves of Spinacia oleraceawas investigated using dimethylsulfoxide as HO{dot} trappingagent. Maximum rates of HO{dot} production by thylakoids asindicated by the formation of methane sulfinic acid were observedunder aerobic conditions in the absence of added electron acceptors.They were higher than 2 µmol (mg Chl h)^–1. Saturationof HO{dot} production occurred at the low photon flux densityof 100 µmol m^–2 s^–1. Trapping of HO{dot} bydimethylsulfoxide suppressed, but did not eliminate light-dependentinactivation of PSI and II suggesting that HO{dot} formationcontributed to the photosensitivity of isolated thylakoids.DCMU inhibited HO{dot} formation. Importantly, methylviologendecreased HO{dot} formation in the absence, but stimulated itin the presence of Fe³⁺. In intact chloroplasts, HO{dot} formation became appreciableonly after KCN had been added to inhibit effective H₂O₂ scavengingby ascorbate peroxidase. It was stimulated by ferrisulfate,but not by ferricyanide which does not penetrate the chloroplastenvelope. Infiltrated spinach leaves behaved similar in principleto intact chloroplasts in regard to HO{dot} formation but HO{dot}production was very slow if detectable at all by the formationof methylsulfinic acid indicating effective radical detoxification. HO{dot} formation is interpreted to be the result of a Fenton-typereaction which produces HO{dot} in chloroplasts from H₂O₂ andreduced ferredoxin, when O₂ is electron acceptor in the Mehlerreaction and radical detoxification reactions are inhibited. (Received November 13, 1996; Accepted April 23, 1996)     

Carotenoid photobleaching induced by photosystem II action in the membrane fragments of the blue-green alga Anabaena variabilis : Action of O2

Carotenoid photobleaching induced by photosystem II action wasstudied using membrane fragments of the blue-green alga Anabaenavariabilis. Special attention was paid to the action of O₂. Carotenoid photobleaching elicited by carbonyl cyanide m-chlorophenylhydrazone(CCCP) depended on O₂. However, the addition of H₂O₂, sodiumsilicotungstate or potassium ferricyanide (Ferri), an electronacceptor for reaction center II action, removed the O₂-dependency.These results indicate that O₂ acts as the electron acceptorfor this reaction. When both CGCP and Ferri were present, a short illumination(0.25 sec) caused a rapid photobleaching followed by a slowrecovery in the subsequent dark period. The spectrum of theabsorption decrease in the light was identical with that ofthe absorption increase in the subsequent dark, indicating thata reversible process is involved in the carotenoid photobleaching.The size in the dark recovery relative to the light bleachingbecame larger under anaerobic conditions and smaller under higherpartial pressure of O₂. The reuslts were interpreted as indicatingthat O₂ does not function in the primary process including areversible bleaching step, but is involved in the slow and irreversiblebleaching process. (Received April 3, 1978; )     

Attachment of CuZn-Superoxide Dismutase to Thylakoid Membranes at the Site of Superoxide Generation (PSI) in Spinach Chloroplasts: Detection by Immuno-Gold Labeling After Rapid Freezing and Substitution Method

In chloroplasts O^–₂ is photoproduced via the univalentreduction of O₂ in PSI even under conditions that are favorablefor photosynthesis. The photogenerated O^–₂ is disproportionatedto H₂O₂ and O₂ in a reaction that is catalyzed by superoxidedismutase (SOD). The H₂O₂-scavenging ascorbate peroxidase isbound to the thylakoid membranes at or near the PSI reactioncenter [Miyake and Asada (1992) Plant Cell Physiol. 33: 541],and the primary product of oxidation in the peroxidase-catalyzedreaction, the monodehydroascorbate radical, is photoreducedto ascorbate in PSI in a reaction mediated by ferredoxin [Miyakeand Asada (1994) Plant Cell Physiol. 35: 539]. Therefore, SODshould be localized at or near the PSI complex. We report herethe microcompartmentalization of the chloroplastic CuZn-SODon the stromal-faces of thylakoid membranes where the PSI-complexis located. Spinach leaves were fixed and substituted by a rapidfreezing and substitution method that allows visualization ofintact chloroplasts. The embedded sections were immuno labeledwith the antibody against CuZn-SOD by the immunogold method.About 70% of the immunogold particles were found within 5 nmfrom the surface of the stromal-faces of thylakoid membranes.Of these particles, about 40% were found at the ends and marginsof the grana thylakoids and 60% were found on the stromal sideof the stromal thylakoids. From these results, the local concentrationof CuZn-SOD on the stroma-facing surfaces of the thylakoid membraneswas estimated to be about 1 mM. The effect of the microcompartmentalizationof CuZn-SOD on the scavenging of superoxide radicals is discussed. (Received November 25, 1994; Accepted February 23, 1995)     

Drought and Oxidative Load in the Leaves of C3 Plants: a Predominant Role for Photorespiration?

Although active oxygen species are produced at high rates inboth the chloroplasts and peroxisomes of the leaves of C₃ plants,most attention has focused on the potentially damaging consequencesof enhanced chloroplastic production in stress conditions suchas drought. This article attempts to provide quantitative estimatesof the relative contributions of the chloroplast electron transportchain and the glycolate oxidase reaction to the oxidative loadplaced on the photosynthetic leaf cell. Rates of photorespiratoryH₂O₂ production were obtained from photosynthetic and photorespiratoryflux rates, derived from steady-state leaf gas exchange measurementsat varying irradiance and ambient CO₂. Assuming a 10 % allocationof photosynthetic electron flow to the Mehler reaction, photorespiratoryH₂O₂ production would account for about 70 % of total H₂O₂ formedat all irradiances measured. When chloroplastic CO₂ concentrationrates are decreased, photorespiration becomes even more predominantin H₂O₂ generation. At the increased flux through photorespirationobserved at lower ambient CO₂, the Mehler reaction would haveto account for more than 35 % of the total photosynthetic electronflow in order to match the rate of peroxisomal H₂O₂ production.The potential signalling role of H₂O₂ produced in the peroxisomesis emphasized, and it is demonstrated that photorespiratoryH₂O₂ can perturb the redox states of leaf antioxidant pools.We discuss the interactions between oxidants, antioxidants andredox changes leading to modified gene expression, particularlyin relation to drought, and call attention to the potentialsignificance of photorespiratory H₂O₂ in signalling and acclimation.     

Superoxide dismutases from kidney bean leaves

Three isozymes of superoxide dismutase were found in the solubleextract of kidney bean leaves. Two of them, purified to nearhomogeneous states, were inhibited by cyanide and by the antibodyto spinach Cu, Zn-superoxide dismutase. Thus, both isozymeswere the Cu and Zn containing enzyme that has a molecular weightof 32,000 and is composed of two subunits of equal size. Anotherisozyme was insensitive to cyanide and to the antibody, butwas inactivated by acetone or heating. The cyanideinsensitiveisozyme was not inactivated by H₂O₂ showing that this isozymeis Mn-superoxide dismutase. (Received June 13, 1979; )     

Studies on frost hardiness in Chlorella ellipsoidea III. Changes in O2 uptake and evolution during hardening and after freeze-thawing

Chlorella ellipsoidea cells at an intermediate stage in theripening phase of the cell cycle were hardened at 3?C. Oligomycin(OGM) and 3-(3,4-dichiorophenyl)-1,1-dimethylurea (DCMU) addedduring hardening in the light inhibited the development of frosthardiness, suggesting that mitochondria and chloroplasts wereinvolved in the hardening process. The O₂-uptake activity in unhardened cells increased duringhardening in the light while the O²-evolution activity decreased,when these activities were measured at 25?C. The increase inO₂ uptake was suppressed by OGM and DCMU and the decrease inO₂ evolution was stimulated by OGM. While the algal hardinessin the dark was very limited, the addition of glucose duringhardening in the dark caused a remarkable development of frosthardiness. These results suggest that mitochondria and chloroplastsclosely interact at low temperature, and the former plays aprincipal role in the hardening process and the latter servesas substrate-donor in the light. The O₂ evolution in cells which survived freezing was remarkablydecreased by freeze-thawing while the O₂ uptake was hardly affected.The freeze-injured chloroplasts were repaired during the followingincubation. OGM inhibited the repair of freeze-injured chloroplasts.From the results, mitochondria seem to change their membranesinto a structure hardier than chloroplasts, and ATP synthesizedby mitochondria seems to be essential for the repair of freeze-injuredchloroplasts. ¹ Present address: Department of Public Health, Faculty of Medicine,Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka 812, Japan. (Received November 9, 1977; )     

Influence of Organic-Phosphates on 3-Phosphoglycerate Dependent O2 Evolution in C3 and C4 Mesophyll Chloroplasts

In C₄ plants phosphoenolpyruvate (PEP) of the C₄ cycle may betransported on a chloroplast transporter which also transports3-phosphoglycerate (3-PGA) and triosephosphates. In C₃ plantsPEP is not considered to be effectively transported on the chloroplastphosphate translocator. The influences of certain organic phosphates,having a similar structure to either PEP or triose-phosphates,on 3-PGA dependent O₂ evolution by C₄ (Digitaria sanquinalisL. Scop.) and C₃ (Hordeum vulgare L.) mesophyll chloroplastswere investigated. In the C₄ mesophyll chloroplasts phosphoglycolatewas a competitive inhibitor (K_i = 2.1 mM) of 3-PGA dependentO₂ evolution, and was as effective as previously reported forPEP. 2-Phosphoglycerate was also a competitive inhibitor (K_t= 8.6 mM) of O₂ evolution in the C₄ mesophyll chloroplasts with3-PGA as substrate, while phospholactate was a weak inhibitorand glyphosate had no effect. Neither PEP, phosphoglycolatenor 2-phosphoglycerate were effective inhibitors of 3- PGA dependentO₂ evolution in the C₃ chloroplasts. Phosphohydroxypyruvatewas a competitive inhibitor of 3-PGA dependent O₂2 evolutionin both chloroplast types. The selectivity in inhibition ofO₂ evolution with 3-PGA as substrate suggests that the C₄ mesophyllchloroplasts can recognize certain organic phosphates with thephosphate in the C-2 or C-3 position but that the C₄ mesophyllchloroplasts can only effectively recognize certain organicphosphates with the phosphate in the C-3 position. The resultsalso support the view that 3-PGA and PEP are transported onthe same phosphate translocator in C₄ mesophyll chloroplasts. ¹ Current address: Department of Horticulture, 2001 Fyffe Court,The Ohio State University, Columbus, Ohio 43210-1096. (Received March 24, 1987; Accepted April 16, 1987)     

Oxygen- and Light-Induced Proteolysis in Isolated Oat Chloroplasts

The influence of light and O₂ on the degradation of proteinsin isolated oat chloroplasts was studied by SDS-polyacrylamidegel electrophoresis. Proteolysis increased as irradiance andthe concentration of O₂ increased. Dark treatments preventedprotein degradation. Neutral and alkaline aminopeptidase activitiesand neutral endopeptidase activity decreased with the time andwith the increase in the concentration of O₂. However, acidendopeptidase activity increased as the level of O₂ increased,accounting, at least in part, for the proteolysis observed underhigh irradiance and high concentrations of O₂. Acid endopeptidaseactivity was strongly associated with thylakoid membranes. Treatmentwith H₂O₂ increased thylakoid-bound acid endopeptidase activitybut had no effect on the solubilized activity. It is suggestedthat active species of oxygen generated in illuminated chloroplastsmay enhance proteolysis by inducing alterations in membraneswhich, in turn, increase the endopeptidic activity. (Received October 18, 1989; Accepted February 19, 1990)     

Effects of inhibitors of catalase on photosynthesis and on catalase activity in unwashed preparations of intact chloroplasts

The catalase activity of unwashed preparations containing intact spinach (Spinacia oleracea L.) chloroplasts is inhibited both by cyanide and by azide at concentrations which also cause inhibition of photosynthetic CO₂- dependent O₂ evolution.

Aminotriazole can also be used to inhibit this contaminant catalase, and in this case inhibition of catalase can be achieved at aminotriazole concentrations which have little effect on the rate of photosynthetic CO₂ fixation. Aminotriazole may be used as a specific inhibitor of catalase in order to demonstrate inhibition of photosynthesis by added H₂O₂.

It is therefore concluded that inhibition of photosynthesis by cyanide and azide does not necessarily result from inhibition of catalase in the chloroplast preparation, and that intact chloroplasts do not produce inhibitory concentrations of H₂O₂ under the best experimental conditions for CO₂ fixation.

     

Oxidation-reduction reactions between electron transfer components and hydrogen peroxide in photosystem II of chloroplasts

The light-induced oxygen evolution, photoreduction of 2,6-dichlorophenolindophenol (DPIP) and carotenoid photobleaching induced by carbonylcyanide m-chlorophenylhydrazone (CCCP) were investigated withspinach chloroplast fragments in the presence of H₂O₂. Oxygenevolution in the presence of H₂O₂ was not inhibited by CCCPand was only partially inhibited by 5 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) which completely inhibited the Hill reaction with DPIP.The degree of inhibition by DCMU was decreased by a simultaneousaddition of CCCP. Carotenoid photobleaching in the presenceof CCCP was stimulated by H₂O₂. The CCCP-induced carotenoidphotobleaching was completely inhibited by DCMU. However, itwas only partially inhibited by DCMU in the presence of H₂O₂.These data indicate that H₂O₂ donates electrons at a site betweenthe CCCP-sensitive site and the reaction center of photosystemII and is reduced at a site between the DCMU-blocked site andthe reaction center of photosystem II. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

H2O2 destruction by ascorbate-dependent systems from chloroplasts

Washed lamellae from isolated spinach chloroplasts exhibited peroxidative activity with 3,3′-diaminobenzidine or ascorbate as electron donors. By heat treatment or by incubation of the chloroplasts with pronase a heat-labile enzymic activity (system A) and a heat-stable non-enzymic peroxidative activity (system B) could be differentiated.System A is membrane-bound, reacts with 3,3′-diaminobenzidine and with ascorbate as electron donors, shows a sharp pH optimum between 7.5 and 8.0 with both substrates and is inhibited competitively by cyanide.The heat-stable factor can be extracted from the chloroplast lamellae by heat treatment, reacts only with ascorbate as electron donor, shows increasing activity with higher pH values but no optimum and is not inhibited by cyanide.Both peroxidative systems in connection with a relatively high concentration of ascorbate in chloroplasts should represent an important tool for the detoxification of H₂O₂ which is produced in these organelles by photosynthetic O₂ reduction.