Divergent pathways of photosynthetic electron transfer: The autonomous oxygenic and anoxygenic photosystems

The aim of this article is to assemble and integrate, from a personal perspective of a research participant, seldom examined evidence that is incompatible with some basic tenets of photosynthetic electron transport, the cornerstone of which is the Z scheme. The nonconforming evidence pertaining to the mode of ferredoxin reduction and the role of the copper redox protein, plastocyanin, indicates that contrary to the Z scheme ferredoxin is reduced in two experimentally distinguishable ways: oxygenically by PS II (renamed the oxygenic photosystem), without the participation of PS I, and anoxygenically by PS I (renamed the anoxygenic photosystem). It also indicates that plastocyanin is not only, as the Z scheme asserts, the electron donor to the reaction center chlorophyll of PS I (P700) but also to the reaction center chlorophyll of PS II (P680). Other unconventional findings include evidence that the fully functional oxygenic photosystem, when operating separately from the anoxygenic photosystem, reduces plastoquinone to plastoquinol and subsequently oxidizes plastoquinol by two pathways acting in concert: one being the universally recognized DBMIB-sensitive pathway via the Rieske iron-sulfur center of the cytochrome bf complex and the other, a hitherto unrecognized, DBMIB-insensitive electron transport pathway around P680 that centers on cytochrome b-559. These nonconforming findings form the basis of an alternate hypothesis of photosynthetic electron transport that modifies and complements the Z scheme.Abbreviations PS photosystem - PQ oxidized plastoquinone - PQH₂ reduced plastoquinone (plastoquinol) - Q_A and Q_B specialized membrane-bound forms of PQ - PC plastocyanin - Fd ferredoxin - BISC F_AF_B, membrane-bound iron-sulfur centers of PS I - DBM1B 2,5-dibromo-3-methyl-6-isopropyl-n-benzoquinone (dibromothymoquinone) - DNP-INT dinitrophenol ether of iodonitrothymol - NADP⁺ NADPH, oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate - FCCP carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone - CCCP carbonyl cyanide-3-chlorophenylhydrazone - SF 6847 2,6,-di-(t-butyl)-4-(2,2-dicyanovinyl) phenol - diuron (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - DCIP 2,6-dichloro-phenolindophenol - UHDBT 5-(n-undecyl)-6-hydroxy-4-7-dioxobenzothiazole; cytochrome b-559_HP-cytochrome b-559_LP, high- and low potential states of cytochrome b-559 - oxygenic reductions reductions in which water is the electron donor - BBY PS II preparation made according to Berthold et al. (1981) Dedicated to Professor Achim Trebst on his 65th birthday.Based in part on lecture in Advanced Course on Trends in Photosynthesis Research, Palma de Mallorca, Spain, September 18, 1990.     

Enhanced susceptibility of the oxidized and unprotonated forms of high potential cytochrome b-559 toward DCMU

The nature of interaction of cytochrome b-559 high potential (HP) with electron transport on the reducing side of photosystem II was investigated by measuring the susceptibility of cytochrome b-559HP to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under different conditions. Submicromolar DCMU concentrations decreased the rate of absorbance change corresponding to cytochrome b-559HP photoreduction while the amplitude was lowered at higher concentrations (up to 10 M). Appreciable extents of cytochrome b-559HP photoreduction were observed at DCMU concentrations which completely abolished the electron transport from water to methyl viologen under the same experimental conditions. However, the susceptibility of cytochrome b-559HP to DCMU increased with the degree of cytochrome b-559HP oxidation, induced either by ferricyanide or by illumination of low intensity (2 W/m²) of red light in the presence of 2 M carbonyl cyanide-m-chlorophenylhydrazone. Also, the DCMU inhibition was more severe when the pH increased from 6.5 to 8.5, indicating that the unprotonated form of cytochrome b-559HP is more susceptible to DCMU. These results demonstrate that cytochrome b-559HP can accept electrons prior to the Q_B site, probably via Q_A although both Q_A and Q_B can be involved to various extents in this reaction. We suggest that the redox state and the degree of protonation of cytochrome b-559HP alter its interaction with the reducing side of photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - CCCP carbonylcyanide m-chlorophenylhydrazone - FeCN ferricyanide - HP high potential - MV methylviologen CIW-DPB Publication No.1096.     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or Q_B). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680⁺, the yield of its reduction by Q_B or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.     

Kinetics of the photoreduction of cytochrome b-559 by Photosystem II in chloroplasts

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e. hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100±10 ms, compared to 6–10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25–35}\text{ms}$. The addition of uncouplers, which caused a stimulatory effect on ferricyanide reduction under the same experimental conditions, resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.     

Evidence for the Involvement of Loosely Bound Plastosemiquinones in Superoxide Anion Radical Production in Photosystem II

Recent evidence has indicated the presence of novel plastoquinone-binding sites, Q_C and Q_D, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O₂^•−) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O₂^•−, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the Q_B site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the Q_D site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b₅₅₉ (cyt b₅₅₉). Based on these results, we propose that O₂^•− is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the Q_B site and one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_C site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_D site seems to be ambiguous. In spite of the fact that the existence of Q_C and Q_D sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.     

Reconstitution of plastoquinone in the D1/D2/cytochrome b-559 photosystem II reaction centre complex

Reconstitution of plastoquinone in the photosystem II D1/D2/cytochrome b-559 reaction centre complex, in the presence of the detergent Triton X-100, is reported. Illumination of the reconstituted system results in the reduction of cytochrome b-559, the process being partly herbicide-sensitive. In addition, the reconstitution of plastoquinone results in the ability of the isolated reaction centre to catalyse the photoreduction of 2,6-dichlorophenolindophenol in the presence of the exogenous electron donor diphenylcarbazide.     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

Inhibition of plastoquinol oxidation at the cytochrome b6f complex by dinitrophenyl ether of iodonitrothymol (DNP-INT) depends on irradiance and H+ uptake by thylakoid membranes

Inhibitory analysis is a useful tool for studying reactions in the photosynthetic apparatus. After introducing by Aachim Trebst in 1978, dinitrophenylether of iodonitrothymol (DNP-INT), a competitive inhibitor of plastoquinol oxidation at the cytochrome (cyt.) b₆f complex, has been widely applied to study reactions occurring in the plastoquinone pool and the cyt. b₆f complex. Here we examine the inhibitory efficiency of DNP-INT by implementing three approaches to estimate the extent of blockage of electron flow from the plastoquinone pool to photosystem I in isolated thylakoids from spinach (Spinacia oleracea). We confirm that DNP-INT is a potent inhibitor of electron flow to photosystem I and demonstrate that inhibitory action of DNP-INT depends on irradiance and H⁺ uptake by thylakoid membranes. Based on these findings, we infer that affinity of the quinol-oxidizing site of the cyt. b₆f complex to DNP-INT is increased in the light due to hydrogen bonding between DNP-INT molecules and acidic amino acid residue(s), which is (are) protonated in the light.     

A pathway for the reduction of cytochrome b-559 by Photosystem II in chloroplasts

Cytochrome b-559, which is normally reduced in the dark, was oxidized by preillumination in the presence of N-methyl-phenazonium methosulfate with low intensity far-red light. The average half-time for the photoreduction of oxidized cytochrome b-559 by a long actinic flash ranged from 90 to 110 ms. In the presence of 0.25 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea the half-time for the photoreduction increased to 230 ms although the extent of the absorbance increase was unchanged. Under similar conditions inhibition of electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and the increase in the chlorophyll fluorescence show that a large fraction of the Photosystem II reaction centers are blocked. These results are consistent with the concept that electrons are shared between different photosynthetic units by a common pool of plastoquinone and imply that the principle pathway for the reduction of cytochrome b-559 by Photosystem II occurs through plastoquinone. In the presence of the uncoupler gramicidin which stimulates non-cyclic electron transport, the rate of photoreduction of cytochrome b-559 is slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 180}\text{ms}$), from which it is inferred that cytochrome b-559 competes with cytochrome f for electrons out of this pool. Comparison of cytochrome b-559 photoreduction and electron transport rates using untreated and KCN-treated chloroplasts indicate that, under conditions of basal electron transport from water to ferricyanide, approximately one-fifth of the electrons from Photosystem II go through cytochrome b-559 to ferricyanide. Further support for this pathway is provided by a comparison of the effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) on the rates of reduction of cytochrome b-559 and ferricyanide.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome (Cyt b ₅₅₉). At pH 6.5 it was found that Cyt b ₅₅₉ was reversibly photoreduced by red actinic light when Mn²⁺ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b ₅₅₉ was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942–10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b ₅₅₉ is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b ₅₅₉ increases, with increasing pH, the rate of irreversible photo-oxidation of -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

Plastoquinone as a common link between photosynthesis and respiration in a blue-green alga

The role of plastoquinone in a thermophilic blue-green alga, Shynechococcus sp., was studied by measuring reduction kinetics of cytochrome 553 which was oxidized with red flash preferentially exciting photosystem I. Sensitivity of the cytochrome reduction to DBMIB indicates that cytochrome 553 accepts electrons from reduced plastoquinone. Plastoquinone is in turn reduced in cells without electrons from photosystem II, since DCMU, which inhibited methyl viologen photoreduction more strongly than DBMIB, failed to affect the cytochrome reduction. Participation of cyclic electron transport around photosystem I in cytochrome reduction in the presence of DCMU was excluded, because methyl viologen and antimycin A had no effect on the cytochrome kinetics. On the other hand, electron donation from endogenous substrates to plastoquinone was suggested from decreases in rate of the cytochrome reduction by dark starvation of cells and also from restoration of fast reduction kinetics by the addition of exogenous substrates to or by reillumination of starved cells.KCN, which completely suppressed respiratory O₂-uptake, induced a marked acceleration of the cytochrome reduction in starved cells. The poison was less or not effective in stimulating the cytochrome reduction in more extensively starved or reilluminated cells.Results indicate that plastoquinone is functioning not only in the photosynthetic but also in the respiratory electron transport chain, thereby forming a common link between the two energy conservation systems of the blue-green alga.

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Cytochrome b6f complex is required for phosphorylation of light-harvesting chlorophyll a/b complex II in chloroplast photosynthetic membranes

The light-harvesting chlorophyll a/b complex (LHC II) and four photosystem II (PS II) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS II proteins, LHC II is phosphorylated only when the cytochrome b6f complex is active. Two lines of evidence support this conclusion. (1) 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the 2,4-dinitrophenyl ether of iodonitrothymol (DNP-INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC II phosphorylation in spinach thylakoids. (2) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6f complex, phosphorylates the four PS II proteins but fails to phosphorylate LHC II in vivo or in vitro.     

Uncouplers enhance photosynthetic electron transport from water to NADP+ in the presence of plastoquinone inhibitors

Uncouplers have been previously observed to relieve appreciably the inhibition of photosynthetic electron transport from water to NADP⁺ by the plastoquinone analogues, dibromothymoquinone (DBMIB) and dinitrophenyl ether of iodonitrothymol (DNP-INT). These results were now extended by demonstrating that the reversal by uncouplers of DBMIB and DNP-INT inhibition occurred under conditions when the uncouplers did not stimulate or inhibit NADP⁺ reduction in control treatments without the plastoquinone analogues. Since effects of uncouplers on photosynthetic electron transport depend on external pH, we determined for each of the four uncouplers, gramicidin, nigericin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and SF 6847 (a ditertiary phenol derivative) its effect on oxygenic electron transport (H₂O to NADP⁺) over a range of external pH from 6.7 to 8.7. The effect of each uncoupler on counteracting the inhibition of DBMIB and DNP-INT was then measured at its crossover external pH at which the uncoupler had little or no effect on electron transport in the uninhibited controls. Under these controlled conditions, uncouplers increased the rate of plastoquinone-inhibited electron transport, in some cases by almost 300%. To explain these results, a role for plastoquinone in processing protons released by the oxidation of water is postulated.     

Oxygenic photoreduction of ferredoxin independently of the membrane-bound iron-sulfur centers of photosystem I

An investigation of the photoreduction of soluble ferredoxin and membrane-bound Fe-S centers of chloroplasts yielded results that are incompatible with some basic postulates of the now prevalent concept of photosynthetic electron transport (the “Z scheme”). In the Z scheme, plastquinone serves as an essential link in a linear electron transport chain from water via photosystem II to photosystem I and thence to the bound Fe-S centers, soluble ferredoxin and NADP⁺. In this formulation the oxygenic photoreduction of ferredoxin and of the Fe-S centers should have the same sensitivity to the plastoquinone inhibitors, dibromothymoquinone (DBMIB) and dinitrophenol ether of iodonitrothymol (DNP-INT). We found that the photoreduction of ferredoxin and the Fe-S centers exhibited differential sensitivity to these inhibitors. Ferredoxin was fully photoreduced by water at inhibitor concentrations that abolished the photoreduction of the Fe-S centers. These findings suggest that the oxygenic photoreduction of ferredoxin does not involve the participation of the Fe-S centers or other components of photosystem I. Only when an artificial, direct donor to photosystem I is used is the reduction of ferredoxin invariably preceded by the reduction of the Fe-S centers.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain