Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl^? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P⁺-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl^?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl^?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II_f and II_vf. The rate of reduction of P⁺-680 in the Cl^?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z⁺ in the Cl^?-depleted system. Under steady-state conditions, the reduction half-time of Z⁺ in uninhibited systems was about 450 μs, while in the Cl^?-depleted chloroplasts, the reduction of Z⁺ was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P⁺-680 observed following the third flash on excitation of Cl^?-depleted chloroplasts was delayed by two flashed when low concentrations of NH₂OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH₂OH or NH₂NH₂ are uninhibited by Cl^? depletion. Addition of NH₂OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH₂OH oxidation present in Cl^?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl^? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z⁺ is reduced directly by Mn of the complex. In terms of the S-state model, Cl^? depletion appears to inhibit the advancement of the mechanism beyond S₂, but not to inhibit the transitions from S₀ to S₁, or from S₁ to S₂.     

The role of chloride ion in Photosystem II I. Effects of chloride ion on Photosystem II electron transport and on hydroxylamine inhibition

1. Chloroplasts washed with Cl^?-free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl^? is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems.2. Strong Cl^?-dependence of Hill activity was observed invariably at all pH values tested (5.5–8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll).3. In the absence of added Cl^? the functionally Cl^?-depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H₂O₂ at rates which are 4–12 times faster than the rate of the residual Hill reaction.4. The Cl^?-concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl^?ag E · Cl^? (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation.5. The initial phase of the development of inhibition of water oxidation in Cl^?-depleted chloroplasts during the dark incubation with NH₂OH ($\text{1}\text{2}$ H₂SO₄) is 5 times slower when the incubation medium contains Cl^? than when the medium contains NH₂OH alone or NH₂OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O₂ evolution.)6. We conclude that the Cl^?-requiring step is one which is specifically associated with the water-splitting reaction, and suggests that Cl^? probably acts as a cofactor (ligand) of the NH₂OH-sensitive, Mn-containing O₂-evolving enzyme.     

Lactoperoxidase-catalyzed iodination of chloroplast membranes. II. Evidence for surface localization of Photosystem II reaction centers

The enzyme lactoperoxidase was used to specifically iodinate the surface-exposed proteins of chloroplast lamellae. This treatment had two effects on Photosystem II activity. The first, occurring at low levels of iodination, resulted in a partial loss of the ability to reduce 2,6-dichlorophenolindophenol (DCIP), even in the presence of an electron donor for Photosystem II. There was a parallel loss of Photosystem II mediated variable yield fluorescence which could not be restored by dithionite treatment under anaerobic conditions. The same pattern of inhibition was observed in either glutaraldehyde-fixed or unfixed membranes. Analysis of the lifetime of fluorescence indicated that iodination changes the rate of deactivation of the excited state chlorophyll. We have concluded that iodination results in the introduction of iodine into the Photosystem II reaction center pigment-protein complex and thereby introduces a new quenching. The data indicate that the reaction center II is surface exposed.At higher levels of iodination, an inhibition of the electron transport reactions on the oxidizing side of Photosystem II was observed. That portion of the total rate of photoreduction of DCIP which was inhibited by this action could be restored by addition of an electron donor to Photosystem II. Loss of activity of the oxidizing side enzymes also resulted in a light-induced bleaching of chlorophyll a₆₈₀ and carotenoid pigments and a dampening of the sequence of O₂ evolution observed during flash irradiation of treated chloroplasts. All effects on electron transport on the oxidizing side of Photosystem II could be eliminated by glutaraldehyde fixation of the chloroplast lamellae prior to lactoperoxidase treatment. It is concluded that the electron carriers on the oxidizing side of Photosystem II are not surface localized; the functioning of these components is impaired by structural disorganization of the membrane occurring at high levels of iodination.Our data are in agreement with previously published schemes which suggest that Photosystem II mediated electron transport traverses the membrane.     

Differential efficiency of photosynthetic oxygen evolution in flashing light in triazine-resistant and triazine-susceptible biotypes of Senecio vulgaris L.

Photosynthetic oxygen evolution in response to flashing light was studied in triazine-susceptible and triazine-resistant biotypes of Senecio vulgaris L. Studies were conducted to determine if the modification of the herbicide-binding site which confers s-triazine resistance also affects the oxygen-evolving system. Oxygen evolution was measured using a Joliot-type oxygen-specific electrode on broken, stroma-free chloroplasts of both biotypes. We observed abnormal patterns of oxygen evolution in resistant chloroplasts. The S′₁ → S₂ transition is slower while the S₂ decay is faster. The S′₂ → S₃ transition, in contrast, is slightly faster in resistant chloroplasts, while the decay of the S₃ state is the same as in susceptible chloroplasts. These altered kinetics may be due to altered Q → B (B^?) electron flow in resistant chloroplasts. These results are also consistent with the hypothesis that back-reactions from the reducing (acceptor) side of Photosystem II to the oxidizing (donor) side occur with greater frequency in resistant than susceptible chloroplasts. These events are responsible for lower oxygen yield and increased ‘misses’ and ‘double hits,’ resulting in abnormal yield patterns and lower quantum yield of CO₂ fixation in resistant chloroplasts compared to the susceptible ones.     

Cooperativity between Photosystem II centers at the level of primary electron transfer

1. Spinach chloroplasts, but not whole Chlorella cells, show an acceleration of the Photosystem II turnover time when excited by non-saturating flashes (exciting 25 % of centers) or when excited by saturating flashes for 85–95 % inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Following dark adaptation, the turnover is accelerated after a non-saturating flash, preceded by none or several saturating flashes, and primarily after a first saturating flash for 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition. A rapid phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx. 0.75 s) is observed for the deactivation of State S₂ in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.2. These accelerated relaxations suggest that centers of Photosystem II are interconnected at the level of the primary electron transfer and compete for primary oxidizing equivalents in a saturating flash. The model in best agreement with the experimental data consists of a paired interconnection of centers.3. Under the conditions mentioned above, an accelerated turnover may be observed following a flash for centers in S₀, S₁ or S₂ prior to the flash. This acceleration is interpreted in terms of a shift of the rate-limiting steps of Photosystem II turnover from the acceptor to the donor side.     

Photooxidation of ferrocyanide and iodide ions and associated phosphorylation in NH2OH-treated chloroplasts

NH₂OH-treated, non-water oxidizing chloroplasts are shown to be capable of oxidizing ferrocyanide and I^? via Photosystem II at appreciable rates (? 200 μequiv/h per mg chlorophyll). Using methylviologen as electron acceptor, ferrocyanide oxidation can be measured as O₂ uptake, as ferricyanide formation, or as H⁺ consumption (2 Fe²⁺ + 2H⁺ + O₂ → 2 Fe³⁺ + H₂O₂). I^? oxidation can be measured as methylviologen-mediated O₂ uptake, or spectrophotometrically, using ferricyanide as electron acceptor. The oxidation product I₂ is re-reduced, as it is formed, by unknown reducing substances in the reaction system.The rate-saturating concentrations of these donors are very high: 30 mM with ferricyanide and 15 mM with I^?. Relatively lipophilic Photosystem II donors such as catechol, benzidine and p-aminophenol saturate the photooxidation rate at much lower concentrations (< 0.5 mM). It thus seems that the oxidation of hydrophilic reductants such as ferricyanide and I^? is limited by permeability barriers. Very likely the site of Photosystem II oxidation is embedded in the thylakoid membrane or is situated on the inner surface of the membrane.The efficiency of phosphorylation (P/e₂) is 0.5 to 0.6 with ferrocyanide and about 0.5 with I^?. In contrast the P/e₂ ratio is 1.0 to 1.2 when water, catechol, p-aminophenol or benzidine serves as electron donor. These differences imply that only one of two phosphorylation sites operate when ferrocyanide and I^? are oxidized. Ferrocyanide and I^? are also chemically distinct from other Photosystem II donors in that their oxidation does not involve proton release. It is suggested that the mechanism of energy conservation associated with Photosystem II may be only operative when the removal of electrons from the donor results in release of protons (i.e. with water, hydroquinones, phenylamines, etc.).     

Effect of ligands on cytochromed fromAzotobacter vinelandii

Spectra of oxidized and reduced cytochromed in particles ofA. vinelandii were studied in the presence of the ligands CO, azide, and NH₂OH under oxidizing, reducing, and turnover conditions. Under oxidizing conditions, spectral changes were observed on oxidized cytochromed (absorption maximum at 648 nm) in the presence of CO and NH₂OH showing a shift of the maximum to shorter wavelengths (639 and 645 nm, respectively) and a broadening of the half-band width. Under reducing conditions, spectral changes were observed on reduced cytochromed (absorption maximum at 631 nm) in the presence of CO (absorption maximum at 636 nm), NO, NO^– ₂, and NH₂OH (absorption maximum at 642 nm in the presence of dithionite). The spectral changes of cytochromed in the presence of NH₂OH or with dithionite and NO^– ₂ were ascribed to the formation of the NO-cytochromed compound. Under turnover conditions CO, NH₂OH, and azide cause a spectral shift of the absorption maximum of cytochromed from 648 nm to 636, 645, and 655 nm, respectively. With NH₂OH and azide a broadening of the half-band width of 7 and 6 nm, respectively, was also observed. The spectral changes caused by CO and NH₂OH were interpreted as a binding of the ligands to cytochromed changing its conformation from the oxidized state absorbing at 648 nm into a more stable liganded form. Since azide does not affect the spectral bands of oxidized and reduced cytochromed, the spectral change during turnover in the presence of azide were ascribed to a preferential binding of azide to enzymically active conformation of cytochromed (cytochromed _x).     

Cobalt ions inhibit electron-transport activity of Photosystem II without affecting Photosystem I

Investigations on photosynthesis have greatly benefited by the use of specific inhibitors that affect a specific site of inhibition on the electron-transport chain. We show here for the first time that cobalt (Co²⁺) ions can be used specifically to inactivate electron donation to the reaction centre of Photosystem (PS) II without affecting PS I reactions. This conclusion is based on the following observations: (1) addition of exogenous electron donors such as NH₂OH does not relieve Co²⁺-induced inactivation of photoelectron transport or the lowering of steady-state chlorophyll a fluorescence yield; this suggests that the inhibition is beyond the NH₂OH donation site and before the fluorescence quencher Q, i.e., on the reaction centre complex itself. (2) Washing of Co²⁺-pretreated chloroplasts with isolation buffer to remove Co²⁺ does not relieve Co²⁺-induced inhibition of Hill activity, suggesting that the Co²⁺ effect is irreversible. (3) Co²⁺ did not alter the PS I reactions. Thus, Co²⁺-treated chloroplasts can be used to study PS I functions free from PS II reactions in isolated chloroplasts.     

Relative activities of linear and cyclic electron flows during chloroplast CO2-fixation

Evolution of oxygen and turnover of cytochromes b-563 and ? were measured upon illumination of isolated intact spinach chloroplasts with a series of flashes. The flash yield of cytochrome ? oxidation approximated the sum of the yields of cytochrome b-563 reduction and electron transfer through Photosystem II, regardless of whether HCO^?₃, 3-phosphoglycerate or O₂ served as the terminal electron acceptor. No absorbance contribution from cytochrome b-559 was discerned within the time range studied. Some pseudocyclic electron flow occurred when both HCO^?₃ and 3-phosphoglycerate were omitted, and possibly also during induction of photosynthesis; however, the flash yield data suggest that O₂ is not reduced at a significant rate during steady state photosynthesis. The maximum rate of cytochrome ? turnover (1000 μequiv./mg chlorophyll per h) was adequate to support the highest rates of photosynthesis observed in isolated chloroplasts.These results agree with the concept that cytochrome ? is a component both of the linear and cyclic pathways whereas cytochrome b-563 functions only in the cyclic pathway. NH₄Cl decreased the half time of cytochrome b-563 oxidation from 11.6 to 8.2 ms and decreased the half time of cytochrome ? reduction from 7.2 to 2.8 ms. The cyclic and linear pathways thus seem to be jointly regulated by a transthylakoid H⁺ gradient through a common control point on the reducing side of cytochrome ?. Cyclic turnover also increased during the induction phase of photosynthesis, when linear throughput is limited by the rate of utilization of NADPH. The slow rise in the P-518 transient correlated with increased cyclic activity under the above conditions.It is proposed that flexibility in the utilization of linear and cyclic pathways allows the chloroplast to generate ATP and NADPH in ratios appropriate to varying needs.     

Multiple action sites for photosystem II herbicides as revealed by delayed fluorescence

DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) at concentrations higher than 10 M suppresses the second time range delayed fluorescence (DF) of pea chloroplasts, due to inhibition of the oxidizing side of photosystem II (PS II). The inhibition of the reducing side of PS II resulting in the suppression of millisecond DF takes place at much lower (0.01 M) DCMU concentrations. The variation in the herbicide-affinities of the reducing and oxidizing sides of PS II is not the same for DCMU and phenol-type herbicides. The DCMU-affinity of the oxidizing side considerably increases and approximates that of the reducing side upon mild treatment of chloroplasts with oleic acid. Probably this is a result of some changes in the environment of the binding site at the oxidizing side. At DCMU concentrations higher than 1 mM, the chaotropic action of DCMU leads to the generation of millisecond luminescence which is not related to the functioning of the reaction centres.Abbreviations D-1 The 32 kDa herbicide-binding intrinsic polypeptide of PS II, the apoprotein of Q_B - D-2 The 32–34 kDa intrinsic polypeptide of PS II, probably the apoprotein of Z - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DF Delayed fluorescence - Dinoseb 2,4-dinitro-6-sec-butylphenol - DNOC 4,6-dinitro-o-cresol - F_m Maximal fluorescence yield (when all traps are closed) - F_o Constant fluorescence yield (when all traps are open) - PS Photosystem - Q_A and Q_B The primary and secondary plastoquinone acceptors of PS II, correspondingly - Z A plastoquinol electron donor, presumably associated with the D-2 protein     

Interaction of linolenic acid with bound quinone molecules in Photosystem II. Time-resolved optical and electron spin resonance studies

Time-resolved spectroscopic techniques, including optical flash photolysis and electron spin resonance spectroscopy, have been utilized to monitor electron-transport activity in Photosystem II subchloroplast particles. These studies have indicated that in the presence of 100 microM linolenic acid (1) a high initial fluorescence yield (Fi) is observed upon steady-state illumination of the dark-adapted sample; (2) flash-induced absorption transients (t greater than 10 mus) in the region of 820 nm, attributed to P-680+, are first slowed, then abolished; and (3) electron spin resonance Signal IIs and Signal IIf (Z+) are not detectable. Upon reversal of linolenic acid inhibition by washing with bovine serum albumin, optical and electron spin resonance transients originating from the photooxidation of P-680 are restored. Similarly, the variable component of fluorescence is recovered with an accompanying restoration of Signal IIs and Signal IIf. The data indicate that linolenic acid affects two inhibition sites in Photosystem II: one located between pheophytin and QA on the reducing side, and the other between electron donor Z and P-680 on the oxidizing side. Since both sites are associated with bound quinone molecules, we suggest that linolenic acid interacts at the level of quinone binding proteins in Photosystem II.     

Inhibition of photosynthetic electron transport in isolated spinach chloroplasts by two 1,3,4-thiadiazolyl derivatives

Buthidazole (3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2-imidazolidinone) and tebuthiuron (N-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-N,N′-dimethylurea) are two new promising herbicides for selective weed control in corn (Zea mays L.) and sugarcane (Saccharum officinarum L.), respectively. The effects of these two compounds on various photochemical reactions of isolated spinach (Spinacia oleracea L.) chloroplasts were studied at concentrations of 0, 0.05, 0.5, 5, and 500 micromolar. Buthidazole and tebuthiuron at concentrations higher than 0.5 micromolar inhibited uncoupled electron transport from water to ferricyanide or to methyl viologen very strongly. Photosystem II-mediated transfer of electrons from water to oxidized diamonodurene, with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) blocking photosystem I, was inhibited 34 and 37% by buthidazole and tebuthiuron, respectively, at 0.05 micromolar. Inhibition of photosystem I-mediated transfer of electrons from diaminodurene to methyl viologen with 3,4-dichlorophenyl-1,1-dimethylurea (DCMU) blocking photosystem II was insignificant with either herbicide at all concentrations tested. Transfer of electrons from catechol to methyl viologen in hydroxylamine-washed chloroplasts was inhibited 50 and 47% by buthidazole and tebuthiuron, respectively, at 0.5 micromolar. The data indicate that the inhibition of electron transport by both herbicides is primarily at the reducing side of photosystem II. However, since catechol is an electron donor at the oxidizing side of photosystem II, between water and chlorophyll a₆₈₀, and lower inhibition levels were observed in the last study (catechol to methyl viologen), it may be that there is also a small inhibition of the mechanism of water oxidation by both herbicides.     

Sulfide inhibition of photosystem II in cyanobacteria (blue-green algae) and tobacco chloroplasts.

The present study shows that in the presence of 600 nm light, sulfide acts as a specific inhibitor of photosynthetic electron transport between water and Photosystem II in the cyanobacteria Aphanothece halophytica and Synechococcus 6311 as well as in tobacco chloroplasts. In the presence of 600 nm light sulfied affects the fast fluorescence transients as does a low concentration (10 mM) of hydroxylamine; the fluorescence yield decreases in the presence of either chemical and can be restored by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In chloroplasts, however, NH2OH, an electron donor at high concentrations (40 mM), relieves the sulfide effect. In the dark, sulfide affects the cyanobacterial fluorescence transients through decrease of oxygen tension. The fluorescence yield increases in a similar pattern to that observed under nitrogen flushing. Upon omission of sulfide in A. halophytica, the characteristic aerobic fluorescence transients return, consistent with the ease of alternation between oxygenic and sulfide-dependent anoxygenic photosynthesis in many cyanobacteria.     

Flash-induced enhancements in the proton NMR relaxation rate of Photosystem II particles

Flash-induced enhancements in the NMR spin-lattice relaxation rate of solvent protons have been detected in suspensions of Photosystem II particles. The relaxation enhancements are small (less than 1% of background) and have been detected using signal-averaging techniques. The enhancements correlate with the known properties of the S states with respect to (1) decay kinetics, (2) extractants of manganese, (3) sensitivity to atrazine-type inhibition of electron transport, (4) reagents which accelerate S-state decay (ADRY reagents), and (5) the two-flash retardation in the S-state advancement that is produced by low concentrations of NH₂OH. The transient proton relaxation enhancement observed after a single flash arises from a strongly relaxing paramagnetic species that is produced by the S₁ → S₂ transition of the water-oxidizing center. The appearance of such a species on an oxidative transition is suggestive of an Mn(III) → Mn(IV) oxidation.     

Mechanism of bicarbonate action on photosynthetic electron transport in broken chloroplasts

In CO2-depleted chloroplasts electron transport between the Photosystem II electron acceptor Q and plastoquinone is largely suppressed. In the presence of a high concentration of sodium formate (greater than 10 mM), which probably binds to the bicarbonate site, addition of bicarbonate restores the ferricyanide Hill reaction only after incubation in the dark. With lower formate concentrations bicarbonate is able to restore electron transport in the light. The Hill reaction rate in CO2-depleted chloroplasts after bicarbonate addition, divided by the rate in CO2-depleted chloroplasts before bicarbonate addition, shows a sharp optimum at pH 6.5. Furthermore, the rate-limiting step in bicarbonate action is probably diffusion. The results are explained in terms of a hypothetical model: the bicarbonate-binding site is located at the outer side of the thylakoid membrane, but not directly accessible from the "bulk". To reach the site from the bulk, the molecule has to pass a channel with negatively charge groups on its side walls. In the light these groups are more negatively charged than in the dark. Therefore, the formate ion cannot exchange for bicarbonate in the light, and a dark period is necessary to enable exchange of formate for bicarbonate.     

Effects of trypsin upon EPR signals arising from components of the donor side of Photosystem II

EPR signals from components functioning on the electron donor side of Photosystem II (PS II) have been monitored in PS II membranes isolated from spinach chloroplasts after treatment with trypsin at pH 7.5 and pH 6.0. The following information has been obtained. (1) The multiline manganese signal, the g = 4.1 signal and Signal II_slow are lost with trypsin treatment at pH 7.5, but not at pH 6.0. (2) At pH 7.5 the multiline S₂ signal and the g = 4.1 signal are lost with approximately the same dependency on the incubation time with trypsin. At pH 6.0 trypsin treatment is known to block electron transfer between Q_A and Q_B (the first and the second quinone electron acceptors, respectively) allowing only a single turnover to occur. Under these conditions both the g = 4.1 signal and the multiline signal are induced by illumination at 200 K and their amplitudes are almost the same as in untreated samples. These results are interpreted as indicating that the g = 4.1 signal arises from a side path donor or from S₂ itself rather than a carrier functioning between the S states and the reaction center as previously suggested. (3) Cytochrome b-559 is converted to its oxidized low-potential form by trypsin treatment at both values of pH. At pH 6.0 the S-state turnover still occurs indicating that the presence of reduced high-potential cytochrome b-559 is not necessary for this process.     

The effects of carbonic anhydrase inhibitors formate,bicarbonate, acetazolamide,and imidazole on photosystem II in maize chloroplasts

Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N₃^?, I^?, OA_c^?, and Cl^? is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor Q. This inhibitory effect of low levels of HCO₃^? along with the known enhancement by HCO₃^? of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the “bicarbonate-effect” associated with PS II in chloroplasts.     

Oxygen requirement of photosynthetic CO2 assimilation

In the absence of electron acceptors and of oxygen a proton gradient was supported across thylakoid membranes of intact spinach chloroplasts by far-red illumination. It was decreased by red light. Inhibition by red light indicates effective control of cyclic electron flow by Photosystem II. Inhibition was released by oxygen which supported a large proton gradient. Oxygen appeared to act as electron acceptor simultaneously preventing over-reduction of electron carriers of the cyclic electron transport pathway. It thus has an important regulatory function in electron transport. Under anaerobic conditions, the inhibition of electron transport caused by red illumination could also be released and a large proton gradient could be established by oxaloacetate, nitrite and 3-phosphoglycerate, but not by bicarbonate. In the absence of oxygen, ATP levels remained low in chloroplasts illuminated with red light even when bicarbonate was present. They increased when electron acceptors were added which could release the over-reduction of the electron transport chain. Inhibition of electron transport in the presence of bicarbonate was relieved and CO₂-fixation was initiated by oxygen concentrations as low as about 10 μM. Once CO₂ fixation was initiated, very low oxygen levels were sufficient to sustain it. The results support the assumption that pseudocyclic electron transport is necessary to poise the electron transport chain so that a proper balance of linear and cyclic electron transport is established to supply ATP for CO₂ reduction.