Kinetic study of oxygen evolution parameters in tris-washed,reactivated chloroplasts

Tris-washed chloroplasts which have lost the ability to evolve oxygen can be reactivated by the procedure of Yamashita. T., Tsuji, J. and Tomita, G. ((1971)Plant Cell Physiol. 12, 117–126) [7] to give 100% of the rate of control chloroplasts in continuous illumination. Furthermore, in flashing light the reactivated chloroplasts exhibit oxygen-yield oscillations of period four that are characteristic of the control. Similar kinetic parameters for intermediate steps in the water-splitting process are observed for the two preparations. We conclude that the reactivation procedure restores the native oxygen evolution mechanism to Tris-washed chloroplasts.A relatively rapid and reversible (0.5 s decay) light-induced component of EPR Signal II is observed upon inhibition of O₂ evolution by Tris washing (Babcock G. T. and Sauer, K. (1975) Biochim. Biophys. Acta 376, 315–328) [10]. Reactivated chloroplasts are similar to untreated chloroplasts in that this Signal II transient is not observed. Manganese, which is released by Tris treatment to the interior of the thylakoid membrane in an EPR-detectable state, is returned to an EPR-undetectable state by reactivation. The reactivation procedure does not require light to restore O₂ evolution and EDTA has no effect on the extent of reactivation. These results are discussed in terms of possible mechanisms for manganese incorporation into photosynthetic membranes.     

Kinetic study of oxygen evolution parameters in Triswashed, reactivated chloroplasts.

Tris-washed chloroplasts which have lost the ability to evolve oxygen can be reactivated by the procedure of Yamashita T., Tsuji, J. and Tomita G. (1971) Plant Cell Physiol. 12, 117-126) [7] to give 100 percent of the rate of control chloroplasts in continuous illumination. Furthermore, in flashing light the reactivated chloroplasts exhibit oxygen-yield oscillations of period four that are characteristic of the control. Similar kinetic parameters for intermediate steps in the water-splitting process are observed for the two preparations. We conclude that the reactivation procedure restores the native oxygen evolution mechanism to Tris-washed chloroplasts. A relatively rapid and reversible (0.5 s decay) light-induced component of EPR Signal II is observed upon inhibition of O2 evolution by Tris washing (Babcock G. T. and Sauer, K. (1975) Biochim. Biophys. Acta 376, 315-328) [10]. Reactivated chloroplasts are similar to untreated chloroplasts in that this Signal IItransient is not observed. Manganese, which is released by Tris treatment to the interior of the thylakoid membrane in an EPR-detectable state, is returned to an EPR-undetectable state by reactivation. The reactivation procedure does not require light to restore O2 evolution and EDTA has no effect on the extent of reactivation. These results are discussed in terms of possible mechanisms for manganese incorporation into photosynthetic membranes.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. I. Kinetic analysis for untreated chloroplasts

An analysis of electron paramagnetic resonance Signal II in spinach chloroplasts has been made using both continuous and flashing light techniques. In order to perform the experiments we developed a method which allows us to obtain fresh, untreated chloroplasts with low dark levels of Signal II. Under these conditions a single 10-μs flash is sufficient to generate greater than 80% of the possible light-induced increase in Signal II spin concentration. The risetime for this flash-induced increase in Signal II is approx. 1 s. The close association of Signal II with Photo-system II is confirmed by the observations that red light is more effective than is far red light in generating Signal II, and that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) does not inhibit the formation of the radical. Single flash saturation curves for the flash-induced increase in Signal I and Signal II indicate that the quantum efficiency for Signal II formation is close to that for Signal I. While one or two flashes (spaced 10 ms apart) are quite efficient in generating Signal II, three or four flashes are much less effective. However, if this spacing is decreased to 100 μs, three or four flashes become as efficient as one or two flashes. From observations of a deficiency of O₂ evolved during the initial flashes of dark-adapted chloroplasts, we conclude that the species which gives rise to Signal II is able to compete with water for oxidizing equivalents generated by Photosystem II. On the basis of these results we postulate a model in which Signal II arises from an oxidized radical which is produced by a slow electron transfer to the specific states S₂ and S₃ on the water side of Photo-system II.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. II. Alternative spectral forms and inhibitor effects on kinetics of Signal II in flashing light

Linewidth and hyperfine structure measurements of the EPR spectrum of Signal II in spinach chloroplasts show that the signal reflects two alternative states. One state is characterized by a 16-G linewidth and four partially resolved hyperfine components. The other state has 19 G linewidth and five partially resolved hyperfine components. It is possible to interconvert these two states by changing the ionic strength of the chloroplast suspension. Both states of Signal II show similar light-induced increases in dark-adapted chloroplasts and respond to 10-μs white light flashes with identical kinetics.

In chloroplasts at room temperature, Signal II dark decays to 50% of its total light-induced level in about 1 h. Single flashes increase the spin concentration in these aged chloroplasts but with decreased effectiveness compared with fresh, dark-adapted chloroplasts. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) decreases the decay time of Signal II from hours to seconds without appreciably altering the level of Signal II formed in saturating continuous light. However, both the formation time constant and the extent of Signal II increase stimulated by a single saturating flash are decreased in CCCP-treated chloroplasts.

These results are interpreted in terms of the model, proposed in the preceding paper, in which Signal II is generated by oxidation-reduction reactions on the water side of Photosystem II.     

A flash photolysis ESR study of photosystem II signal IIvf, the physiological donor to P-680+.

In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (g approximately 2.0045, deltaHpp approximately 19G). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with "red" illumination (lambda less than 680 nm) and occurs stoichiometrically in a 1:1 ratio with P-700+. The Photosystem II transient is formed in less than 100 mus and decays via first-order kinetics with a halftime of 400-900 mus. Additionally, the t1/2 for radical decay is temperature independent between 20 and 4 degrees C; however, below 4 degrees C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal-mol-1. Inhibition of electron transport through Photosystem II by o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1-5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this "quinoidal" type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, P-680+.     

Herbicide-induced oxidative stress in photosystem II.

Some herbicides act by binding to the exchangeable quinone site in the photosystem II (PSII) reaction centre, thus blocking electron transfer. In this article, it is hypothesized that the plant is killed by light-induced oxidative stress initiated by damage caused by formation of singlet oxygen in the reaction centre itself. This occurs when light-induced charge pairs in herbicide-inhibited PSII decay by a charge recombination route involving the formation of a chlorophyll triplet state that is able to activate oxygen. The binding of phenolic herbicides favours this pathway, thus increasing the efficiency of photodamage in this class of herbicides.     

Primary reactions of Photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts

The effects of lowering the pH on Photosystem II have been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts.At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese.The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H. J., Pulles, M. P. J., Haveman, J. and den Haan, G. A. (1976) Biochim. Biophys. Acta 423, 217–226).     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

EPR properties of immobilized quinone cation radicals and the molecular origin of Signal II in spinach chloroplasts

In Photosystem II, Z reduces P-680⁺ and gives rise to a characteristic EPR signal, termed II_vf in oxygen-evolving chloroplasts and II_f in non-oxygen-evolving chloroplasts. Previous model compound studies of Signal II have centered on the immobilized anionic and neutral forms of semiquinone radicals. These radicals, however, exhibit an essentially structureless band shape in constrast to the partially resolved hyperfine pattern observed for Signal II. In the experiments reported here, we show that some cationic semiquinone radicals (e.g., 2-methyl-5-isopropylhydroquinone cation radical) exhibit band shape and micro-wave power saturation characteristics upon immobilization which are similar to Signal II. Examination of a series of quinone cation radicals shows that a Signal-II-like spectrum is observed when significant unpaired spin density occurs at a ring carbon to which a methyl group is bound. Whether this will occur for a specific quinone depends on the extent to which the peripheral substituent pattern favors a contribution from the antisymmetric benzenoid molecular orbital to the ground state of the radical. For the 2-methyl-5-isopropylhydroquinone cation radical, for example, a 26% contribution of this orbital is estimated. A plastoquinone cation radical in which the electron-donating ability of the quinol-OH groups has been decreased is compatible with antisymmetric orbital stabilization and, therefore, is identified as the Z⁺ species. Hydrogen bonding of the quinol oxygen to hydrogen-donating amino acid residues in vivo plus an out-of-plane geometry for the quinol-OH groups is proposed to stabilize the antisymmetric orbital. The partially resolved structure of Signal II indicates that the antisymmetric orbital is the major contributor to the ground state; the principal hyperfine splitting in the spectrum arises from the 2-CH₃ group of the plastoquinone cation radical. The estimated electrode potential of the Z⁺ radical is in close agreement with the in vitro electrode potential of quinone cation radicals.     

EPR characteristics of oxygen-evoloving Photosytem II particles from Phormidium laminosum

The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome b-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described.The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated algal membranes for the study of Photosystem II with a functional water oxidation system.     

On the Molecular Identity of ESR Signal II Observed in Photosynthetic Systems: The Effect of Heptane Extraction and Reconstitution With Plastoquinone and Deuterated Plastoquinone

Speculation as to the identity of Signal II, the light-induced, broad, slow decaying electron spin resonance signal with hyperfine structure observed in photosynthetic materials, has tended to center on the semiquinone of plastoquinone. Experiments reported here were designed to give direct evidence bearing on that speculation. Heptane extraction of lipids from lyophilized spinach and tobacco chloroplast fragments reduced the amplitude of Signal II and increased the ratio of Signal I:Signal II. Reconstitution of the system by the addition of plastoquinone partially restored Signal II as well as the ratio of Signal I:Signal II to its pre-extraction condition. Addition of totally deuterated plastoquinone to extracted chloroplasts in which considerable Signal II had survived heptane extraction resulted in a spectrum which showed the characteristic hallmarks of Signal II observed in totally deuterated organisms. These results establish that a free radical immediately derived from plastoquinone contributes to Signal II. The data taken by themselves are consistent with plastochromanoxyl as well as plastosemiquinone free radicals giving rise to Signal II. Other contributors to Signal II are not ruled out.     

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.Abbreviations Chl Chlorophyll - EDTA Ethylenediamine Tetraacetic Acid - FeCN Potassium Ferricyanide - Hepes N-2-Hydroxyethylpiperazine-N¹-2-ethanesulfonic acid - pBQ Para-benzoquinone - PSII photosystem II     

The existence of a high photochemical turnover rate at the reaction centers of System II in Tris-washed chloroplasts

In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of $\text{≈ 1 μs}$. It has been found that X 320 is reduced by a flash in $\text{? 1 μs}$. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.     

Reversible and irreversible effects of alkaline pH on Photosystem II electron-transfer reactions

Incubation of highly active, O2-evolving PS II preparations at alkaline pH inhibits donor side electron-transfer reactions in two distinct fashions, one reversible the other irreversible. In both cases, O2 evolution is inhibited, with concomitant loss of the light-induced multiline and g = 4.1 EPR signals and an increased steady-state level of EPR Signal II induced by continuous illumination. However, the inhibition that is observed between pH 7.0 and 8.0 is readily reversible by resuspension at low pH, while above pH 8.0 the effect is irreversible. In addition, under repetitive flash conditions the ms decay kinetics remains largely unchanged at pH less than or equal to 8.0 but shows about a 2-fold increase in amplitude and is slowed at pH above 8.0. The irreversible component of inhibition most likely can be attributed to the loss of Mn and the 16, 24 and 33 kDa proteins. The reversible component may be mediated by displacement of Cl- from an anion-binding site by OH- or by titration of ionizable groups on the protein(s) associated with water-splitting. We propose that the reversible inhibition blocks electron transfer between the O2-evolving complex and an intermediate which serves as the direct donor to Signal II, while the irreversible inhibition blocks the reduction of Signal II by this intermediate donor species.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport.

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO2-dependent oxygen evolution by isolated pea chloroplasts. Both alpha, beta- and beta, gamma-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO2-dependent oxygen evolution. The concentration of adenylylimidodiphosphate required for 50% inhibition of CO2-dependent oxygen evolution was 50 micronM. 2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation. 3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [14C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator. 4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO2 fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: Quantum Accumulation in Photosynthetic Oxygen Evolution

Three independent methods have been used to determine the size of the quantum accumulation unit in green plant photosynthesis. This unit is defined as that group of pigment molecules within which quantal absorption acts must take place leading to the evolution of a single O₂ molecule. All three methods take advantage of the nonlinearity of oxygen yield with light dose at very low dosages. The experimental values of this unit size, based on an assumed model for the charge cooperation in O₂ evolution, ranging from 800 to 1600, suggest that there is either limited energy transfer between energy-trapping units or chemical cooperation among oxygen precursors formed in several neighboring energy-trapping units. Widely diffusible essential precursors to molecular oxygen are ruled out by these results. Inhibition studies show that O₂ evolution is blocked when 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) is added to chloroplasts after two preliminary flashes and before a third flash which would have yielded O₂ in the absence of DCMU. This experiment is interpreted as evidence that the site of DCMU inhibition is on the oxidizing side of system II. Pretreatment of chloroplasts with large concentrations of Tris, previously believed to destroy O₂ evolution by blocking an essential reaction in the electron chain between water and system II, may be alternately interpreted as promoting the dark reversal of the system II light-induced electron transfer.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Some Chemicals on the Oxygen Evolution and Oxygen Uptake in Isolated Wheat Chloroplasts Irradiated without the Addition of an Oxidant

The oxygen exchange, obtained when isolated chloroplasts of Triticum aestivum, wheat, are irradiated without the addition of a Hill oxidant has been investigated using an oxygen electrode. Ascorbate, catalase, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone(DBMIB), diethyldithio-carbamate (DEDT), dichlorophenylmethylurea (DCMU), and potassium cyanide were added to the Chloroplasts in order to investigate the oxygen exchange. At least two oxygen uptake reactions, one sensitive to catalase and one catalase-insensitive, appeared upon irradiation. Hydrogen peroxide was the product of the oxygen uptake in the former process, and water was the reductant. The formation of hydrogen peroxide was probably associated with photosystem I. The other oxygen consuming reaction was found to be insensitive to both catalase and potassium cyanide. After the chloroplasts had been treated with DCMU, it was possible to show that the catalase-insensitive oxygen uptake was localized in photosystem I, and that a cyclic electron transport system or some endogenous reductant (-s) acted in the oxygen uptake. Addition of ascorbate or DEDT to the chloroplasts led to an enhanced oxygen uptake in 710 nm light. This was probably due to the effect of these compounds on the superoxide radical ion formed in photosystem I. The stimulated oxygen uptake was only weakly affected by catalase, indicating that hydrogen peroxide was not a product of this oxygen uptake. Addition of DEDT and potassium cyanide inhibited (strongly respectively weakly) the oxygen uptake when photosystem II was functioning. The effect of these compounds was probably due to an inhibition of the electron transport at the plastocyanin. DBMIB inhibited the oxygen uptake reactions and the cooperation between the two photosystems. The cooperation between the photosystems was also studied in DCMU-treated chloroplasts. The reactions in photosystem II, measured as oxygen evolution, were more inhibited than the coupling between the photosystems. The oxygen “gush” appearing upon irradiation in light of 650 nm was not affected by a DBMIB-treatment, showing that the oxygen evolution was due to the reduction of plastoquinone. The reoxidation in the dark of the plastoquinone pool was stimulated by DBMIB and potassium cyanide indicating that an oxygen uptake could be associated with plastoquinone. The sites of interaction of oxygen with the electron transport pathways in chloroplasts, and the different reductants for the oxygen consuming reactions are discussed.