Copper Toxicity Affects Photosystem II Electron Transport at the Secondary Quinone Acceptor, Q(B)

The nature of Cu²⁺ inhibition of photosystem II (PSII) photochemistry in pea (Pisum sativum L.) thylakoids was investigated monitoring Hill activity and light emission properties of photosystem II. In Cu²⁺-inhibited thylakoids, diphenyl carbazide addition does not relieve the loss of Hill activity. The maximum yield of fluorescence induction restored by hydroxylamine in Tris-inactivated thylakoids is markedly reduced by Cu²⁺. This suggests that Cu²⁺ does not act on the donor side of PSII but on the reaction center of PSII or on components beyond. Thermoluminescence and delayed luminescence studies show that charge recombination between the positively charged intermediate in water oxidation cycle (S₂) and negatively charged primary quinone acceptor of pSII (Q_A⁻) is largely unaffected by Cu²⁺. The S₂Q_B⁻ charge recombination, however, is drastically inhibited which parallels the loss of Hill activity. This indicates that Cu²⁺ inhibits photosystem II photochemistry primarily affecting the function of the secondary quinone electron acceptor, Q_B. We suggest that Cu²⁺ does not block electron flow between the primary and secondary quinone acceptor but modifies the Q_B site in such a way that it becomes unsuitable for further photosystem II photochemistry.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor QA

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron-quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D| = 0.0286 cm⁻¹, |E| = 0.0044 cm⁻¹ was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T = 100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron-quinone primary acceptor complex.     

Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach

The effect of photosystem II core phosphorylation on the secondary quinone acceptor of photosystem II (Q_B) domain environment was analyzed by comparative herbicide-binding studies with photosystem II preparations from spinach (Spinacia oleracea L.). It was found that phosphorylation reduces the binding affinity for most photosynthetic herbicides. The binding of synthetic quinones and of the electron acceptor 2,6-dichlorophenolindophenol is also reduced by photosystem II phosphorylation. Four photosystem II core populations isolated from membranes showed different extents of phosphorylation as well as different degrees of affinity for photosynthetic herbicides. These findings support the idea that heterogeneity of photosystem II observed in vivo could be, in part, due to phosphorylation.     

Effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on the secondary electron acceptor B of photosystem II

Measurements of chlorophyll fluorescence have been used to monitor electron transport from the primary electron acceptor of photosystem II, Q, to the secondary acceptor, B, in chloroplasts in either the presence or the absence of the plastoquinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Electron transport is markedly slower from Q^? to either B or B^? in the presence of DBMIB. Binary oscillations in the rate of reoxidation of Q^? (equivalent to the reactions Q^?B → QB^? and Q^?B^? → QB^2?) after each of a series of flashes were of a phase opposite to those observed in the absence of DBMIB (J. M. Bowes, and A. R. Crofts, (1980) Biochim. Biophys. Acta590, 573–584). The results confirm that inhibition of electron transport by DBMIB in chloroplasts is not restricted to an inhibition of electron transfer from the plastoquinone pool, but that there is also a specific interaction between the reduced form of the inhibitor and the secondary electron acceptor B. Models are discussed to account for the mechanism of this interaction.     

Sub-microsecond chlorophyll A delayed fluorescence from Photosystem I Magnetic field-induced increase of the emission yield

(1) In photosystem I (PS I) particles in the presence of dithionite and intense background illumination at 290 K, an external magnetic field (0–0.22 T) induced an increase, ΔF, of the low chlorophyll a emission yield, F ($\text{ΔF}\text{F}\text{? 1–1.5\%}$). Half the effect was obtained at about 35–60 mT and saturation occurred for magnetic fields higher than about 0.15 T. In the absence of dithionite, no field-induced increase was observed. Cooling to 77 K decreased ΔF at 685 nm, but not at 735 nm, to zero. Measuring the emission spectra of F and ΔF, using continuous excitation light, at 82, 167 and 278 K indicated that the spectra of F and ΔF have about the same maximum at about 730, 725 and 700 nm, respectively. However, the spectra of ΔF show more long-wavelength emission than the corresponding spectra of F. (2) Only in the presence of dithionite and with (or after) background illumination, was a luminescence (delayed fluorescence) component observed at 735 nm, after a 15 ns laser flash (530 nm), that decayed in about 0.1 μs at room temperature and in approx. 0.2 μs at 77 K. A magnetic field of 0.22 T caused an appreciable increase in luminescence intensity after 250 ns, probably mainly caused by an increase in decay time. The emission spectra of the magnetic field-induced increase of luminescence, ΔL, at 82, 167 and 278 K coincided within experimental error with those of ΔF mentioned above. The temperature dependence of ΔF and ΔL was found to be nearly the same, both at 685 and at 735 nm. (3) Analogously to the proposal concerning the 0.15 μs luminescence in photosystem II (Sonneveld, A., Duysens, L.N.M. and Moerdijk, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5889–5893), we propose that recombination of the oxidized primary donor P-700⁺ and the reduced acceptor A^?, probably A^?₁, of PS I causes the observed fast luminescence. The effect of an external magnetic field on this emission may be explained by the radical pair mechanism. The field-induced increase of the 0.1–0.2 μs luminescence seems to be at least in large part responsible for the observed increase of the total (prompt + delayed) emission measured during continuous illumination in the presence of a magnetic field.     

Luminescence decay kinetics in relation to quenching and stimulation of dark fluorescence from high and low CO2 adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii

Two green algal species, Chlamydomonas reinhardtii and Scenedesmus obliquus, exhibited a relative maximum during the decay of luminescence, when adapted to low CO₂ conditions that was not observed in high CO₂ adapted cells.From the kinetics of transient changes in the level of dark fluorescence, after illumination and parallel to the luminescence maxima, it was concluded that the maximum in Scenedesmus was mainly related to a decrease in nonphotochemical quenching, whereas in Chlamydomonas the maximum was mainly related to a dark reduction of the primary PS II acceptor Q_A.ATP/ADP ratios from low CO₂ adapted Scenedesmus showed transient high levels after a dark/light transition that was not observed in high CO₂ adapted cells. After 30 s of illumination the ATP/ADP ratios however stabilized at the same steady state level as in high CO₂ adapted cells.Dark addition of HCO₃ ^- to low CO₂ adapted cells of Chlamydomonas resulted in a rapid transient quenching of luminescence that was not observed in low CO₂ adapted cells of neither species.It is concluded that the luminescence maxima present in both low CO₂ adapted Scenedesmus and Chlamydomonas reflect adaptation of the cells to low CO₂ conditions. It is further suggested that the difference in mechanistic origin of luminescence maxima in the two species reflects differences in adaptation.Abbreviations ADP adenosine-diphosphate - ATP adenosine-triphosphate - C_i inorganic carbon - F_D dark fluorescence recorded under dark adapted conditions - F₀ fluorescence with all reaction centers open - FV variable fluorescence - PS I photosystem I - PS II photosystem II - Q_A the first quinone acceptor of PS II     

Acetate in mixotrophic growth medium affects photosystem II in Chlamydomonas reinhardtii and protects against photoinhibition

Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris–acetate–phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris–acetate–phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less ¹O₂ than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of ¹O₂ originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (E_m) of the quinone electron acceptors. Thermoluminescence indicated that the E_m of the primary quinone acceptor (Q_A/Q_A⁻) of mixotrophic cells was stabilised while the E_m of the secondary quinone acceptor (Q_B/Q_B⁻) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to ¹O₂ production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of Q_A and Q_B affecting photosystem II charge recombination events and photoinhibition.     

A New Mechanism for Adaptation to Changes in Light Intensity and Quality in the Red Alga Porphyra perforata: III. Fluorescence Transients in the Presence of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea

In the red alga Porphyra perforata, the level of chlorophyll fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased during illumination of the thallus. The results showed that: (a) this decay was related to the photooxidative activity of photosystem I; (b) Q, the primary electron acceptor of photosystem II, became oxidized during the decay of the fluorescence; (c) reagents which inhibit the back reaction of photosystem II inhibited the decay.

From these results, it is suggested that, when conditions in the chloroplasts of this red alga become too oxidative, excess light energy can be converted to heat as a result of an accelerated back reaction of photosystem II. This may be one of the mechanisms by which this alga can cope with the high salt and high light conditions that can occur in its natural habitat.

     

Correlation of the light-induced change of absorbance with ESR signal of photosystem II in presence of silicomolybdate.

The light-induced dark-reversible ESR signal in chloroplast fragments enriched in photosystem II and free from P700 contamination has been observed in the presence of silicomolybdate as an electron acceptor operating directly on the photosystem II primary acceptor. The signal at g = 2.0025 and with line-width ΔHpp = 9G rises and decays in close correlation with the photobleaching band centered at 680 nm and the minor peak at 435 nm.     

Clara cell damage and inhibition of pulmonary mixed-function oxidase activity by naphthalene

The photosystem II electron acceptor 3,6-dichloro-2,5-dimethoxy-p-benzoquinone [DCDMQ] is suggested to replace the second quinone-type two electron acceptor B (or R); the DCDMQ Hill reaction is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is insensitive to dry heptane extraction of thylakoids and other photosystem II inhibitors. Addition of HCO₃^? to CO₂-depleted thylakoids in silicomolybdate, DCDMQ, diaminodurene and ferricyanide Hill reactions brought about 1,3,10 and 10 fold increase in the electron transport rates; these data confirm that HCO₃^? affects both Q^? to B and B^2? to PQ reactions.     

Thermoluminescence and Delayed Luminescence Investigation of the Green Alga,Chlamydobotrys stellata

Thermoluminescence and delayed luminescence investigations of the autotrophically and photoheterotrophically cultivated green alga, Chlamydobotrys stellata, demonstrated that both the thermoluminescence and delayed luminescence yields are much lower in the photoheterotophic algae than in the autotrophic ones due to an efficient luminescence quenching of unknown mechanism. The relative contributions of the so called Q (S₂Q^?_A charge recombination) and B (S₂Q^?_B and S₃Q^?_B charge recombinations) thermoluminescence bands to the glow curve as well as the Q_A(S₂Q^?_B charge recombination) and Q_B (S₂Q^?_B and S₃Q^?_B charge recombinations) delayed luminescence components to the delayed luminescence decay of autotrophically and photoheterotrophically cultivated Chl. stellata were compared using a computer assisted curve resolution method. It was found that, while in the autotrophic cells the area of the B band was considerably larger than of the Q band, in photoheterotrophic cells the Q band was more effectively charged than the B band. In the delayed luminescence decay curves measured in the seconds to minutes time region the amplitude of the Q_A component relative to that of the Q_B component was larger in the photoheterotrophic cells than in the autotrophic ones. These observations demonstrate that, after light-induced charge separation in the photosystem II reaction centers of autotrophic cells, electrons are “quasipermanently” stored mainly in the secondary quinone acceptor pool, Q_B but in the nonquenched photosystem II reaction centers of photoheterotrophic cells the main reservoir of electrons is the primary quinone acceptor, Q_A. This behaviour indicates an inhibition of electron transport in the photoheterotrophic alga at the level of the secondary quinone acceptor, Q_B.     

Iron-induced changes in light harvesting and photochemical energy conversion processes in eukaryotic marine algae

The role of iron in regulating light harvesting and photochemical energy conversion processes was examined in the marine unicellular chlorophyte Dunaliella tertiolecta and the marine diatom Phaeodactylum tricornutum. In both species, iron limitation led to a reduction in cellular chlorophyll concentrations, but an increase in the in vivo, chlorophyll-specific, optical absorption cross-sections. Moreover, the absorption cross-section of photosystem II, a measure of the photon target area of the traps, was higher in iron-limited cells and decreased rapidly following iron addition. Iron-limited cells exhibited reduced variable/maximum fluorescence ratios and a reduced fluorescence per unit absorption at all wave-lengths between 400 and 575 nm. Following iron addition, variable/maximum fluorescence ratios increased rapidly, reaching 90% of the maximum within 18 to 25 h. Thus, although more light was absorbed per unit of chlorophyll, iron limitation reduced the transfer efficiency of excitation energy in photosystem II. The half-time for the oxidation of primary electron acceptor of photosystem II, calculated from the kinetics of decay of variable maximum fluorescence, increased 2-fold under iron limitation. Quantitative analysis of western blots revealed that cytochrome f and subunit IV (the plastoquinone-docking protein) of the cytochrome b₆/f complex were also significantly reduced by lack of iron; recovery from iron limitation was completely inhibited by either cycloheximide or chloramphenicol. The recovery of maximum photosynthetic energy conversion efficiency occurs in three stages: (a) a rapid (3-5 h) increase in electron transfer rates on the acceptor side of photosystem II correlated with de novo synthesis of the cytochrome b₆/f complex; (b) an increase (10-15 h) in the quantum efficiency correlated with an increase in D1 accumulation; and (c) a slow (>18 h) increase in chlorophyll levels accompanied by an increase in the efficiency of energy transfer from the light-harvesting chlorophyll proteins to the reaction centers.     

Properties of photoreductions by photosystem II

Dimethyl-methylenedioxy-p-benzoquinone is found to be an excellent mediator for photoreductions by photosystem II only, i.e. in the presence of dibromothymoquinone (DBMIB) blocking reduction by photosystem I. The new quinone system is highly autoxidizable, thus also supporting pseudocyclic electron flow involving photosystem II only. It is not affected by the uncoupler gramicidin at external pH 8, whereas a phenylenediamine acceptor system is strongly inhibited at this pH. This is taken as further support for the notion that the internal pH affects acceptor systems for photosystem II and that therefore the reduction site of these systems occurs in the internal pH range.     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.     

Analysis of the Heat Stress Induced Quenching of Variable Chlorophyll a Fluorescence in Beet Spinach Leaves: Possible Accumulation of Oxidized Quencher P680+ under High Illumination

Effect of preheating of beet spinach leaves on chlorophyll a fluorescence yield was analyzed with the help of additional high intensity illumination pulses using a pulse modulated fluorometer. Preheating at mildly elevated temperature (35–45°C) causes a shift in the redox state of secondary donor of photosystem II, possibly due to uncoupling of phosphorylation because of thermal induced membrane disorganization and associated alkalinization of intra thylakoid space. Also, at these preheating temperatures, a rise in photosystem I catalyzed electron transfer has been shown to occur. These two effects induce rapid quenching of Chi a fluorescence, which drops even in the presence of actinic light, below the level of initial fluorescence (F_o′ monitored by the weak modulated probing light. Preheating of leaf segments induces an increase in fluorescence in the presence of dluron, which blocks electron flow between two photosystems, and thus this increases in fluorescence yield (F_o′ as monitored by weak modulated light, is not solely due to disorganization of light harvesting Chi-protein complex but also due to a shift in the redox equilibrium of the donor at the oxidizing side of photosystem II resulting in rapid reduction of Q_A the stable primary acceptor of photosystem II. In 50°C preheated DCMU treated samples, the fluorescence yield increases in weak modulated light and it approaches that of maximal steady state (F_max) level. At preheating temperature of 48°–50°C, the inactivation of enzymes in the reducing side of photosystem I, causes an impairment of the reoxidation of QA and under this condition, a strong illumination causes quenching of Chi a fluorescence. This quenching seems to arise because of accumulation of the P680⁺, the oxidized physiological donor of photosystem which is a quencher of Chi a fluorescence. This quenching depended on the pulse intensity and duration which saturates P680⁺ accumulation and is greatly manifested when water oxidation complex is damaged.     

Mechanism of photoinduced electron transfer in photosystem II reaction centers

The shape of the EPR spectrum of the triplet state of photosystem II reaction centers with a singly reduced primary acceptor complex Q_AFe²⁺ was studied. It was shown that the spectroscopic properties do not significantly change when the relaxation of the primary acceptor is accelerated and when the magnetic interaction between the reduced quinone molecule Q_A and the nonheme iron ion Fe²⁺ is disrupted. This observation confirmed the earlier conclusion that the anisotropy of the quantum yield of the triplet state is the main cause of the anomalous shape of the EPR spectrum. A scheme of primary processes in photosystem II that is consistent with the observed properties of the EPR spectrum of the triplet state is discussed.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

Bicarbonate effect on electron flow in a cyanobacteriumSynechocystis PCC 6803

In this communication, evidence is presented from the kinetics of Q_A ^? decay (where Q_A is the first plastoquinone electron acceptor of photosystem II) and oxygen evolution for the requirement of bicarbonate in the electron transport in a cyanobacteriumSynechocystis (Pasteur Culture Collection 6803). A large slowing down of Q_A ^? oxidation, measured from the variable chlorophylla fluorescence after saturating actinic flashes, was observed in the thylakoids ofSynechocystis 6803 depleted of bicarbonate in the presence of 25 mM formate. Qualitatively similar results were obtained with DCMU-treated thylakoids. This shows that bicarbonate depletion inhibits electron transport on the acceptor side of photosystem II between Q_A and the plastoquinone (PQ) pool in cyanobacteria. Addition of 2.5 mM HCO₃ ^? fully reversed the inhibition of electron flow caused by bicarbonate depletion. Two exponential phases of Q_A ^? decay, a fast one and a slow one, were observed with halftimes of approx. 400 μs (fast) and 26 ms (slow) at pH 6.5. At pH 7.5, these phases were approx. 330 μs (fast) and 21 ms (slow), respectively. The amplitude, but not the halftime, of the fast component decreased by about 70% (pH 6.5) or 50% (pH 7.5); this was accompanied by a concomittant increase in the slow phase. Twenty mM bicarbonate stimulated, by a factor of 4, the Hill reaction in bicarbonate-depletedSynechocystis cells. This effect is independent of CO₂ fixation as it was observed even in the presence of an inhibitor DBMIB.     

Changes in [C]Atrazine Binding Associated with the Oxidation-Reduction State of the Secondary Quinone Acceptor of Photosystem II

One hypothesis of triazine-type herbicide action in photosynthetic material is that the herbicide molecule competes with a secondary quinone acceptor, B, for a binding site at the reaction center of photosystem II. The binding affinity of B has been suggested to change with its level of reduction, being most strongly bound in its semiquinone form. To test this hypothesis, [¹⁴C]atrazine binding studies have been carried out under different photochemically induced levels of B reduction in Pisum sativum. It is found that herbicide binding is reduced in continuously illuminated samples compared to dark-adapted samples. Decreased binding of atrazine corresponds to an increase in the semiquinone form of B. With flash excitation, the herbicide binding oscillates with a cycle of two, being low on odd-numbered flashes when the amount of semiquinone form of B is greatest. Treatment with NH₂OH was found to significantly decrease the strength of herbicide binding in the dark as well as stop the ability of p-benzoquinone to oxidize the semiquinone form of B. It is suggested that the mode of action of NH₂OH is disruption of quinones or their environment on both the oxidizing and reducing sides of photosystem II. Herbicide binding was found to be unaltered under conditions when p-benzosemiquinone oxidation of the reduced primary acceptor, Q⁻, is herbicide insensitive; weak herbicide binding cannot explain this herbicide insensitivity. It is concluded that the quinone-herbicide competition theory of herbicide action is correct. Also, since quinones are lipophilic the importance of the lipid composition of the thylakoid membrane in herbicide interactions is stressed.     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.