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.     

Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H₂O₂-dependent oxidation of ascorbate and ascorbate-dependent reduction of H₂O₂. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K_m value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN₃, KCN, and 8-hydroxyquinoline but not ZnCl₂ or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.     

Quantitative relationship between photosystem I electron transport and ATP formation

The Photosystem I-dependent transport of electrons from diaminodurene to methylviologen is linear with reaction time and supports a constant rate of phosphorylation. However, if the diaminodurene is not kept fully reduced by the presence of excess ascorbate, the oxidized diaminodurene accumulates and begins to compete with the methylviologen as the electron acceptor. Thus, although the rate of ATP formation remains unchanged, an increasing proportion of the electron transport becomes cyclic and hence unmeasured. This leads to a rapid increase in the apparent efficiency of phosphorylation which is misleading.In contrast, it is known that the oxidized form of 3,3′-diaminobenzidine polymerizes to form an insoluble substance which should not be available to serve as an electron acceptor. However, 3,3′-diaminobenzidine is not a satisfactory donor of electrons in Photosystem I reactions for two reasons: the rate of electron transport quickly falls with reaction time and the oxidized form of 3,3′-diaminobenzidine seems to be an exceptionally efficient electron acceptor near the beginning of the period of illumination when it is presumably not yet polymerized. Thus in the first 2–3 sec of illumination when the reaction is still rapid much of the electron transport is cyclic and therefore unmeasured, especially in the absence of excess ascorbate. This cycling of electrons, which leads to an inflated apparent efficiency ($\text{P}\text{e}{}_2\text{> 2}$), is particularly pronounced at low donor concentrations.When cyclic electron transport is avoided by the use of ascorbate or by the selection of appropriate reaction times, both diaminodurene and 3,3′-diaminobenzidine support phosphorylation with an efficiency which is approximately half of the efficiency exhibited by the overall Hill reaction. The same is true when 2,5-diaminotoluene, tetrachlorohydroquinone, 4,5-dimethyl-o-phenylenediamine, and reduced 2,6-dichloroindophenol serve as electron donors. With these six substances, the phosphorylation efficiences were 0.57 ± 0.1 molecules of ATP formed for each pair of electrons transferred ($\text{P}\text{e}{}_2$). In the same chloroplasts preparations, the transport of electrons from water to methylviologen-supported phosphorylation with a $\text{P}\text{e}{}_2$ of 1.2.     

DEVELOPMENT AND CYTOCHEMISTRY OF THE THYLAKOIDAL BODY IN TOBACCO CHLOROPLASTS

Developing plastids in young tobacco leaves contain thylakoidal bodies, inclusions bound by a single membrane continuous with stroma lamellae. Both the thylakoidal body and its attached lamellae contain an enzyme that catalyzes an oxidation reaction with 3,3'-diaminobenzidine (DAB). DAB staining of the thylakoidal body and lamellae is not the result of photo-oxidation and is inhibited by potassium cyanide. The thylakoidal body disappears as plastids develop into chloroplasts and, further, the lamellar systems of the mature chloroplasts do not stain with DAB. In developing chloroplasts, it is suggested that the thylakoidal body forms by accumulation of protein which stains with DAB within primary lamellae derived from the inner plastid membrane. The ultrastructural and cytochemical evidence suggests that the thylakoidal body stores protein used later in lamellar formation.     

The roles of weak light,oxygen and dithiothreitol in the photoreactivation of the oxygen evolving center of tris-washed and 2, 6-dichlorophenol indophenol-treated chloroplasts

The inactivated O₂-evolving center of Tris-washed chloroplasts was reactivated by DCPIP-treatment and photoreactivation in the presence of Mn²⁺, Ca²⁺, DTT and weak light. Many electron donors (Asc and reduced DCPIP, etc.) were found to be suitable substitutes for DTT. By studying the anaerobic inhibition of the reactivation, the electron acceptors O₂, NADP⁺, etc. were also found to be essential factors in photoreactivation. Weak light stimulated the chloroplast electron transport from the above-mentioned electron donors to the electron acceptor and effected the photoreactivation. More than 280 electrons were transported to NADP⁺ in the anaerobic photoreactivation of one unit of an O₂-evolving center with 400 Chl. Electron transport in the reactivation was inhibited by omitting DTT or Mn²⁺ ion, and by adding DCMU. The photoreactivated chloroplasts incorporated about 2 Mn by 400 Chl. Omission of DTT in the reactivation caused chloroplasts in the weak light to bind large amounts of excess Mn.Abbreviations Asc ascorbate - Chl chlorophyll - DCPIP 2, 6-dichlorophenol indophenol - DPC diphenyl carbazide - DTT dithiothreitol - Fd ferredoxin - STN a chloroplast preparation medium, containing 0.4 M sucrose, 0.05 M Tris-Cl and 0.01 M NaCl (pH 7.8 and 8.0) - TMPD tetramethyl-p-phenylenediamine     

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H₂O₂ accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl₃ staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H₂O₂ accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl₃ were mainly observed especially around the swelling of thylakoids. H₂O₂ accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H₂O₂ accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H₂O₂ under salinity is responsible for the changes in chloroplast ultrastructure. H₂O₂ accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H₂O₂ and the production is light dependent under salinity.     

Tyrosinase and superoxide dismutase activities of peroxidase in the vacuoles of beet roots

In the absence of H₂O₂, isoforms of vacuolar phenol-dependent peroxidase (PO) in beet (Beta vulgaris) roots oxidized phenolic compounds like tyrosinases. Tyrosinase activity of PO manifested a clearly expressed pH-dependence with the optimum at pH of 8.0–9.0; peroxidase activity was the highest at pH 5.0–7.0. The inhibitory analysis confirmed a specificity of observed reactions. Along with tyrosinase activity, PO manifested SOD-like activity, which was also expressed in the absence of H₂O₂ and depended on some factors, for example, on the way of analyzed sample preparation. This activity appeared at a long-term dialysis and low temperature. SOD-like PO activity was observed in the presence of such substrates as 3,3′-diaminobenzidine and IAA. The results obtained allow a conclusion that vacuolar PO, as well as PO of other localization and origin is a polyfunctional enzyme, which, under definite conditions, can catalyze reactions of oxidase type.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

Efficiency of hydrogen photoproduction by chloroplast-bacterial hydrogenase systems

A comparative study of H₂ photoproduction by chloroplasts and solubilized chlorophyll was performed in the presence of hydrogenase preparations of Clostridium butyricum. The photoproduction of H₂ by chloroplasts in the absence of exogenous electron donors, and with irreversibly oxidized dithiothreitol and cysteine, is thought to be limited by a cyclic transport of electrons wherein methylviologen short-circuits the electron transport in photosystem I. The efficiency of H₂ photoproduction by chloroplasts with ascorbate and NADPH is limited by a back reaction between light-reduced methylviologen and the oxidized electron donors. The use of a combination of electron donors (dithiothreitol and ascorbate), providing anaerobiosis without damage to chloroplasts, makes it possible to avoid consumption of reduced methylviologen for the reduction of oxidized electron donors and to exclude the short-circuiting of electron transfer. Under these conditions, photoproduction of H₂ was observed to occur with a rate of 350 to 400 micromoles H₂ per milligram chlorophyll per hour. In this case, the full electron-transferring capability of photosystem I (measured by irreversible photoreduction of methyl red or O₂) is used to produce H₂.     

Intact chloroplasts display pH 5 optimum of O2-reduction in the absence of methyl viologen: Indirect evidence for a regulatory role of superoxide protonation

The pH-dependence of light-driven O₂-reduction in intact spinach chloroplasts is studied by means of chlorophyll fluorescence quenching analysis and polarographic O₂-uptake measurements. Most experiments are carried out in presence of KCN, which blocks activities of Calvin cycle, ascorbate peroxidase and superoxide dismutase. pH is varied by equilibration with external buffers in presence of nigericin. Vastly different pH-optima for O₂-dependent electron flow are observed in the presence and absence of the redox catalyst methyl viologen. Both fluorescence quenching analysis and O₂-uptake reveal a distinct pH 5 optimum of O₂-reduction in the absence of methyl viologen. In the presence of this catalyst, O₂-reduction is favoured in the alkaline region, with an optimum around pH 8, similar to other types of Hill reaction. It is suggested that in the absence of methyl viologen the extent of irreversibility of O₂-reduction is determined by the rate of superoxide protonation. This implies that O₂-reduction takes place within the aprotic phase of the thylakoid membrane and that superoxide-reoxidation via oxidized PS I donors competes with protonation. Superoxide protonation is proposed to occur at the internal surface of the thylakoid membrane. There is no competition between superoxide reoxidation and protonation when in the presence of methyl viologen the site of O₂-reduction is shifted into the protic stroma phase. In confirmation of this interpretation, fluorescence measurements in the absence of KCN reveal, that non-catalysed O₂-dependent electron flow is unique in beingstimulated by the transthylakoidal pH-gradient. On the basis of these findings a major regulatory role of O₂-dependent electron flow under excess light conditions is postulated.     

Purification and kinetic characterization of the liverwort Pallavicinia lyelli (Hook.) S. Gray. cytosolic ascorbate peroxidase

Ascorbate peroxidase (APX) of the liverwort Pallavicinia lyelli was extracted and purified through ammonium sulfate precipitation, Butyl-Toyopearl, DEAE-Cellulofine and Sephadex G-75 chromatography. The purification factor for APX was 285 with 7.9% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of APX was approximately 28 kDa estimated by SDS-PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 6.0. The enzyme had a temperature optimum at 40 °C and was relatively stable at 60 °C, with 54% loss of activity. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity and the capacity to utilize alternate electron donors. p-chloromercuribenzoate (pCMB), hydroxyurea and salicylic acid (SA) significantly inhibited APX activity. Ascorbate (AsA) and pyrogallol were found to be efficient substrates for Pallavicinia APX, considering the Vmax/Km ratio. We detected the activity of monodehydroascorbate reductase (MDHAR) involved in the regeneration of ascorbate, but failed to detect the dehydroascorbate reductase (DHAR) activity. The data obtained in this study may help to understand desiccation tolerance mechanism in the liverwort.     

Electron flow accompanying the ascorbate peroxidase cycle in maize mesophyll chloroplasts and its cooperation with linear electron flow to NADP+ and cyclic electron flow in thylakoid membrane energization

Potential roles for cyclic and pseudocyclic electron flow in C₄ plants are to provide ATP for the C₄ cycle and, under excess light, to down-regulate PS II activity through membrane energization. Intact mesophyll chloroplasts of maize were used to evaluate forms of electron transport including the Mehler peroxidase reaction (linear electron flow to O₂, formation of H₂O₂ which is reduced by ascorbate, and linear flow linked to reduction of oxidized ascorbate). Addition of H₂O₂ to isolated chloroplasts in the light in the presence of an uncoupler induced Photosystem (PS) II activity, as determined from increases in photochemical quenching of chlorophyll fluorescence (q_p) and the quantum yield of PS II. H₂O₂ also induced dissipation of energy by thylakoid membrane energization and non-photochemical fluorescence quenching (q_n), which was inhibited by addition of an uncoupler. These effects of H₂O₂ on q_p and q_n were inhibited by addition of KCN, an inhibitor of ascorbate peroxidase. The results suggest that H₂O₂ is reduced via ascorbate, and that the oxidized ascorbate is then reduced by linear electron flow contributing to photochemistry and thylakoid membrane energization. Evidence for function of pseudocyclic electron flow via the Mehler peroxidase reaction was obtained with only oxygen as an electron acceptor, as well as in the presence of oxaloacetate a natural electron acceptor in C₄ photosynthesis. KCN decreased q_p and PS II yield in the absence and presence of oxaloacetate and, in the former case, it severely reduced q_{_n}. KCN also decreased pH formation across the thylakoid membrane based on its decrease in the light-induced quenching of 9-aminoacridine fluorescence, particularly in the absence of oxaloacetate. Antimycin A, an inhibitor of cyclic electron flow, also diminished pH formation. These results provide evidence for shared energization of thylakoid membranes by the Mehler peroxidase reaction, cyclic electron flow, and linear electron flow linked to the C₄ pathway.     

Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts

Induction of zeaxanthin formation and the associated nonphotochemical quenching in iodoacetamide-treated, non-CO₂-fixing intact chloroplasts of Lactuca sativa L. cv Romaine is reported. The electron transport needed to generate the required ΔpH for zeaxanthin formation and nonphotochemical quenching are ascribed to the Mehler-ascorbate peroxidase reaction. KCN, an inhibitor of ascorbate peroxidase, significantly affected these activities without affecting linear electron transport to methyl viologen or violaxanthin deepoxidase activity. At 1 millimolar KCN, zeaxanthin formation and ΔpH were inhibited 60 and 55%, respectively, whereas ascorbate peroxidase activity was inhibited almost totally. The KCN-resistant activity, which apparently was due to electron transport mediated by the Mehler reaction alone, however, was insufficient to support a high level of nonphotochemical quenching. We suggest that in vivo, as CO₂ fixation becomes limiting, the Mehler-peroxidase reaction protects photosystem II against the excess light by supporting the electron transport needed for zeaxanthin-dependent nonphotochemical quenching and concomitantly scavenging H₂O₂. Ascorbate is essential for this process to occur.     

Metabolic activities in Cyanophora paradoxa and its cyanelles

Isolated cyanelles of Cyanophora paradoxa perform photosystem I and II dependent Hill reactions. The photosynthetic electron transport of the cyanelles does not show special features uncommon in cyanobacteria or chloroplasts of red algae. A preparation of cyanelles performs photosynthetic O₂-evolution with approximately 1/3 of the rate of intact Cyanophora, in only, however, the first three minutes of the experiment. All attempts to stabilize the CO₂-fixation activity of isolated cyanelles failed. Isolated cyanelles do not perform KCN-sensitive O₂-uptake, indicating that respiratory cytochrome oxidase is lacking in cyanelles. O₂-consumption by crude extracts from Cyanophora is inhibited by KCN when N-tetramethyl-p-phenylenediamine/ascorbate or NADH but not NADPH are supplied as the electron donors in contrast to the situation in cyanobacteria. These findings suggest that cyanelles do not respire. It is concluded that cyanelles are not so much related to cyanobacteria as formerly believed, but share many properties with chloroplasts of eukaryotic cells.Abbreviations Chl chlorophyll - DCPIP dichlorophenol-indophenol - TMPD N-tetramethyl-p-phenylenediamine To whom correspondence should be addressed     

Light-dependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts

A reconstituted spinach chloroplast system containing thylakoids, stroma and 0.1 mM NADPH supported O₂ evolution in the presence of oxidised glutathione (GSSG). The properties of the reaction were consistent with light-coupled GSSG-reductase activity involving H₂O as eventual electron donor. The reconstituted system also supported dehydroascorbate-dependent O₂ evolution in the presence of 0.6 mM reduced glutathione (GSH) and 0.1 mM NADPH with the concomitant production of ascorbate. The GSSG could replace GSH in which case the production of GSH preceded the accumulation of ascorbate. The data are consistent with the light-dependent reduction of dehydroascorbate using H₂O as eventual electron donor via the sequence H₂O→NADP→GSSG→dehydroascorbate. Approximately 30% of the GSH-dehydrogenase activity of spinach leaf protoplasts is localised in chloroplasts: this could not be attributed to contamination of chloroplasts by activity from the extrachloroplast compartment. Washed intact chloroplasts supported the uptake of ascorbate but the uptake mechanism had a very low affinity for ascorbate (K_m approximately 20 mM). The rate of uptake of ascorbate was less than the rate of light-dependent reduction of dehydroascorbate and too slow to account for the rate of H₂O₂ reduction by washed intact chloroplasts.     

Studies on the Energy-coupling Sites of Photophosphorylation: V. Phosphorylation Efficiencies (P/e(2)) Associated with Aerobic Photooxidation of Artificial Electron Donors

The rate of Hill reaction can be measured accurately as O₂ uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O₂ consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O₂ uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O₂ uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O₂) through both photosystems II and I is practically independent of the donor used—catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e₂ ratio is 1.0 ± 0.1. Only ascorbate gives a slightly lower value (P/e₂ = 0.9). (NH₂OH-treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU-insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O₂) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e₂ ratio is 0.6 ± 0.08.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

Summary The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H₂O₂ and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3-diaminobenzidine both in the presence and in the absence of H₂O₂. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3-diaminobenzidine and coniferyl alcohol in the presence of H₂O₂. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.     

Hymenolepis diminuta: Biochemical properties of peroxidase activity in mitochondria

The biochemical properties of a peroxidase previously localized cytochemically in the mitochondria of Hymenolepis diminuta were determined. The method chosen was the o-dianisidine procedure in which the decomposition of hydrogen peroxide has been followed spectrophotometrically. Peroxidase activity was initially demonstrated in the mitochondrial pellet. Subsequently, mitochondrial pellets were sonicated and the membrane and supernatant fractions were tested for peroxidase activity. Enzyme activity was demonstrated in the membrane fraction. The enzyme displayed a pH optimum of 5.0, was ascorbate sensitive, and was inhibited by excess H₂O₂. Neither peroxidase nor catalase were observed in any other fraction of the tapeworm tissue, confirming previous cytochemical investigations.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.