A high-potential redox component located within cyanobacterial Photosystem II

The calcium-dependent oxygen evolution activity of preparations of Phormidium luridum shows a marked selectivity in favor of ferricyanide over benzoquinone as Hill oxidant. In addition, the rate of oxygen evolution increases with increasing solution redox potential over the range +350 to +550 mV vs. the standard hydrogen electrode. These properties pertain to both 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive and -insensitive fractions of the total oxygen evolution activity. Neither changes in solution potential nor use of oxidants other than ferricyanide obviate the need for added Ca²⁺.

To explain these observations, two models are proposed, each of which invokes the existence of a redox component located within Photosystem II and having a midpoint potential greater than +450 mV. In one model, the postulated species is a donor which competes with water for oxidizing equivalents generated by System II. In the other model, the 450 mV species is a high-potential primary acceptor of System II electrons.     

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.     

A reaction-induced FT-IR study of cyanobacterial photosystem I.

In oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB. A difference Fourier transform infrared spectrum, associated with P(700)(+)-minus-P(700), can be acquired using PSI from the cyanobacterium Synechocystis sp. PCC 6803. This spectrum reflects contributions from oxidation-sensitive modes of chlorophyll, as well as from oxidation-induced structural changes in amino acid residues and the peptide backbone. Oxidation-induced structural changes may play a role in the facilitation and control of electron-transfer reactions involving the primary donor. In this paper, we report that photooxidation of P(700) in cyanobacterial PSI perturbs a cysteine residue. At 264 and 80 K, a downshift of a SH stretching vibration from 2560 to 2551 cm(-1) is observed. Such a downshift is consistent with an increase in hydrogen bonding, with a change in C-S-H conformation, or with an electric field effect. Deuterium exchange experiments were also performed. While the perturbed cysteine is in a protein region that is resistant to exchange, other (2)H-sensitive vibrational chl and amino acid bands were observed. From the (2)H exchange experiments, we conclude that photooxidation of P(700) perturbs internal or bound water molecules in PSI and that the P(700)(+)-minus-P(700) spectrum is (2)H exchange-sensitive. The results are consistent with structural complexity in the PSI primary donor, as previously suggested [Kim, S., and Barry, B. A. (2000) J. Am. Chem. Soc. 122, 4980-4981]. Possible explanations, including a partial enolization of P(700)(+), are discussed.     

Cation-induced,inhibitor-resistant photosystem II reactions in cyanobacterial membranes

Ferricyanide-supported oxygen evolution in sonic vesicles from the cyanobacterium $\text{Spirulina}\text{platensis}$ is only partially sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and addition of cations to inhibited membranes stimulates the rate of oxygen evolution. The order of cation effectiveness (M³⁺ > M²⁺ > M⁺) suggests that this stimulation is due at least in part to surface charge screening effects which permit freer access of anionic ferricyanide to the vesicle membrane surface; La³⁺, Ca²⁺, and K⁺ are most effective in this regard. Ferricyanide photoreduction is completely sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and neither mono- nor divalent cations affect this inhibition. Addition of La³⁺, on the other hand, causes a nearly complete restoration of ferricyanide-supported oxygen evolution. We conclude that the membrane surfaces of these vesicles are uniquely different from those o higher plants; sites of ferricyanide reduction associated with the interphotosystem chain are surface localized, and the primary acceptor region of photosystem II is susceptible to a trivalent cation-specific reaction in which ferricyanide may directly oxidize the primary acceptor.     

Structurally conserved channels in cyanobacterial and plant photosystem II

In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn₄Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn₄Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel). Membrane-extrinsic protein subunits PsbU and PsbV in cyanobacterial PSII are replaced with PsbP and PsbQ in plant PSII. However, these four proteins have no structural similarity. It remains unknown whether the corresponding channels also exist in plant PSII, because water molecules are not identified in the plant PSII cryo-electron microscopy (cryo-EM) structure. Using the cyanobacterial and plant PSII structures, we analyzed the channels that proceed from the Mn₄Ca cluster. The cyanobacterial O4-PsbU and O1-PsbU/V channels were structurally conserved as the channel that proceeds along PsbP toward the protein bulk surface in the plant PSII (O4-PsbP and O1-PsbP channels, respectively). Calculated protonation states indicated that in contrast to the original geometry of the plant cryo-EM structure, protonated PsbP-Lys166 may form a salt-bridge with ionized D1-Glu329 and protonated PsbP-Lys173 may form a salt-bridge with ionized PsbQ-Asp28 near the O1-PsbP channel. The existence of these channels might explain the molecular mechanism of how PsbP can interact with the Mn₄Ca cluster.     

Proteins of the cyanobacterial photosystem I.

Cyanobacterial photosystem (PS) I is remarkably similar to its counterpart in the chloroplast of plants and algae. Therefore, it has served as a prototype for the type I reaction centers of photosynthesis. Cyanobacterial PS I contains 11-12 proteins. Some of the cyanobacterial proteins are modified post-translationally. Reverse genetics has been used to generate subunit-deficient cyanobacterial mutants, phenotypes of which have revealed the functions of the missing proteins. The cyanobacterial PS I proteins bind cofactors, provide docking sites for electron transfer proteins, participate in tertiary and quaternary organization of the complex and protect the electron transfer centers. Many of these mutants are now being used in sophisticated structure-function analyses. Yet, the roles of some proteins of the cyanobacterial PS I are unknown. It is necessary to examine functions of these proteins on a global scale of cell physiology, biogenesis and evolution.     

Characterization of photosystem II in salt-stressed cyanobacterial Spirulina platensis cells

PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0-0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20-50 ms. Thermoluminescence measurements revealed that S(2)Q(A)(-) and S(2)Q(B)(-) recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the Q(B) niche at the acceptor side and an increase in the stability of the S(2) state at the donor side, which is associated with a dissociation of the PsbO protein.     

Structural investigation of PsbO from plant and cyanobacterial photosystem II

The manganese-stabilizing protein PsbO is associated with the luminal side of thylakoids close to the redox-active Mn₄Ca cluster at the catalytically active site of photosystem II (PSII). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn₄Ca cluster against photoinhibition. Using small-angle X-ray scattering, we investigated the low-resolution structure of wild-type spinach PsbO and that of chimeric spinach PsbO fused with maltose-binding protein. Small-angle X-ray scattering data revealed that both proteins are monomeric in solution, and that plant and cyanobacterial PsbO have similar structures. We show a highly efficient expression system that allows recombinant production of the active wild type and the chimeric PsbO from spinach and cyanobacteria, with yields compatible with biophysical and structural studies. The binding of spinach PsbO fused with maltose-binding protein to PSII depleted of extrinsic subunits (PSII-ΔpsbO,P,Q) was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The reconstituted PSII was shown to have similar oxygen evolution rates as obtained with wild-type spinach PsbO.     

Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.

Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.     

Structural basis of cyanobacterial photosystem II Inhibition by the herbicide terbutryn

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q_B site in the D1 subunit and thus block the electron transfer from Q_A to Q_B. Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q_B site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q_A, which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334–342). This discovery is discussed in the context of proton transfer to the lumen.     

Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane

Cyanobacterial cells have two autonomous internal membrane systems, plasma membrane and thylakoid membrane. In these oxygenic photosynthetic organisms the assembly of the large membrane protein complex photosystem II (PSII) is an intricate process that requires the recruitment of numerous protein subunits and cofactors involved in excitation and electron transfer processes. Precise control of this assembly process is necessary because electron transfer reactions in partially assembled PSII can lead to oxidative damage and degradation of the protein complex. In this communication we demonstrate that the activation of PSII electron transfer reactions in the cyanobacterium Synechocystis sp. PCC 6803 takes place sequentially. In this organism partially assembled PSII complexes can be detected in the plasma membrane. We have determined that such PSII complexes can undergo light-induced charge separation and contain a functional electron acceptor side but not an assembled donor side. In contrast, PSII complexes in thylakoid membrane are fully assembled and capable of multiple turnovers. We conclude that PSII reaction center cores assembled in the plasma membrane are photochemically competent and can catalyze single turnovers. We propose that upon transfer of such PSII core complexes to the thylakoid membrane, additional proteins are incorporated followed by binding and activation of various donor side cofactors. Such a stepwise process protects cyanobacterial cells from potentially harmful consequences of performing water oxidation in a partially assembled PSII complex before it reaches its final destination in the thylakoid membrane.     

Regulation of cyanobacterial photosynthesis determined from variable fluorescence yields of photosystem II

Abstract The cyanobacteria Fremyella diplosiphon 7601 and Synechocystis 6701 were grown in continuous cultures with monochromatic red light (680 nm). The distribution of light energy over photosystem I and II was determined from changes in PS II fluorescence at 685 nm. In both organisms, wavelengths absorbed primarily by chlorophyll a caused the high fluorescent state of PS II (State 1), while wavelengths absorbed by the phycobilisome led to low PS II fluorescence (State 2). Superimposing continuous light 2 on the excitation light yielded State 2 fluorescence patterns for Synechocystis 6701, while F. diplosiphon 7601 showed fluorescence patterns similar to state 1 → 2 transitions and changes in fluorescence yield were related to the intensity of the background light. Some ecological implications of energy (re)distribution in cyanobacterial photosynthesis are discussed.     

Beta-carotene redox reactions in photosystem II: electron transfer pathway

A carotenoid (Car), a chlorophyll (Chl(Z)), and cytochrome b(559) (Cyt b(559)) are able to donate electrons with a low quantum yield to the photooxidized chlorophyll, P680(+), when photosystem II (PSII) is illuminated at low temperatures. Three pathways for electron transfer from Cyt b(559) to P680(+) are considered: (a) the "linear pathway" in which Cyt b(559) donates via Chl(Z) to Car, (b) the "branched pathway" in which Cyt b(559) donates via Car and where Chl(Z) is also able to donate to Car, and (c) the "parallel pathway" where Cyt b(559) donates to P680 without intermediate electron carriers and electron donation from Chl(Z) and Car occurs by a competing pathway. Experiments were performed using EPR and spectrophotometry in an attempt to distinguish among these pathways, and the following observations were made. (1) Using PSII with an intact Mn cluster in which Cyt b(559) was preoxidized, Car oxidation was dominant upon illumination at < or =20 K, while electron donation from Chl dominated at >120 K. (2) When Cyt b(559) was prereduced, its light-induced oxidation occurred at < or =20 K in what appeared to be all of the centers and without the formation of a detectable Car(+) intermediate. The small and variable quantity of Car(+) photoinduced in these experiments can be attributed to the residual centers in which Cyt b(559) remained oxidized prior to illumination. (3) The relative rates for irreversible electron donation from Cyt b(559) and Car were determined indirectly at 20 K by monitoring the flash-induced loss of charge separation (i.e., the accumulation of Cyt b(559)(+)Q(A)(-) or Car(+)Q(A)(-)). Similar yields per flash were observed (13% for Cyt b(559) and 8% for Car), indicating similar donation rates. The slightly lower yield with Car as a donor is attributed at least in part to slow charge recombination occurring from the Car(+)Q(A)(-) radical pair in a fraction of centers. (4) Light-induced oxidation of Cyt b(559) and Car at 20 K was monitored directly by EPR, and the rates were found to be indistinguishable. The parallel pathway predicts that when both Cyt b(559) and Car are prereduced, the relative amounts of Cyt b(559)(+) and Car(+) produced upon illumination at 20 K should depend directly on their relative electron donation rates. The measured similarity in the donation rates thus predicts comparable yields of oxidation for both donors. However, what is observed experimentally is that Cyt b(559) oxidation occurs almost exclusively, and this argues strongly against the parallel pathway. The lack of Car(+) as a detectable intermediate is attributed to rapid electron transfer from Cyt b(559) to Car(+). The trapping of Car(+) at low temperature when Cyt b(559) is preoxidized but its absence when Cyt b(559) is prereduced is taken as an argument against the simple linear pathway. Overall, the data reported here and previously favor the branched pathway over the linear pathway, while the parallel pathway is thought to be unlikely. Structural considerations provide further arguments in favor of the branched model.     

Characterization of photosystem II in salt-stressed cyanobacterial Spirulina platensis cells

PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0-0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20-50 ms. Thermoluminescence measurements revealed that S₂Q_A⁻ and S₂Q_B⁻ recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the Q_B niche at the acceptor side and an increase in the stability of the S₂ state at the donor side, which is associated with a dissociation of the PsbO protein.     

PsbU provides a stable architecture for the oxygen-evolving system in cyanobacterial photosystem II

PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], we investigated the function of PsbU using a psbU deletion mutant (DeltaPsbU) of Synechocystis 6803. In contrast to the wild type, DeltaPsbU did not grow when both Ca2+ and Cl- were eliminated from the growth medium. When only Ca2+ was eliminated, DeltaPsbU grew well, whereas when Cl- was eliminated, the growth rate was highly suppressed. Although DeltaPsbU grew normally in the presence of both ions under moderate light, PS II-related disorders were observed as follows. (1) The mutant cells were highly susceptible to photoinhibition. (2) Both the efficiency of light utilization under low irradiance and the chlorophyll-specific maximum rate of oxygen evolution in DeltaPsbU cells were 60% lower than those of the wild type. (3) The decay of the S2 state in DeltaPsbU cells was decelerated. (4) In isolated PS II complexes from DeltaPsbU cells, the amounts of the other three lumenal extrinsic proteins and the electron donation rate were drastically decreased, indicating that the water oxidation system became significantly labile without PsbU. Furthermore, oxygen-evolving activity in DeltaPsbU thylakoid membranes was highly suppressed in the absence of Cl-, and 60% of the activity was restored by NO3- but not by SO4(2-), indicating that PsbU had functions other than stabilizing Cl-. On the basis of these results, we conclude that PsbU is crucial for the stable architecture of the water-splitting system to optimize the efficiency of the oxygen evolution process.     

Effects of salinity stress on photosystem II function in cyanobacterial Spirulina platensis cells

The changes in PSII photochemistry in Spirulina platensis cells exposed to salinity stress (0–0.8 M NaCl) for 12 h were studied. Salinity stress induced a decrease in oxygen evolution activity, which correlated with the decrease in the quantum yield of PSII electron transport ( Φ _PSII). Phycocyanin content decreased significantly while chlorophyll content remained unchanged in salt-stressed cells. Salinity stress induced an increase in non-photochemical quenching (q_N) and a decrease in photochemical quenching (q_P). Analyses of the polyphasic fluorescence transients (OJIP) showed that with the increase in salt concentration, the fluorescence yield at the phases J, I and P declined sharply and the transient almost levelled off at salt concentration of 0.8 M NaCl. The effects of DCMU on the polyphasic rise of fluorescence transients decreased significantly. Salinity stress resulted in a decrease in the efficiency of electron transfer from Q_A⁻ to Q_B. The slope at the origin of the relative variable fluorescence curves (dV/dt_o) and the relative variable fluorescence at phase J (V_J) increased in the absence of DCMU, but decreased in the presence of DCMU. The shape of the relative variable fluorescence transients in salt-stressed cells was comparable to that of the control cells incubated with DCMU. The results in this study suggest that salt stress inhibited the electron transport at both donor and acceptor sides of PSII, resulted in damage to phycobilisome and shifted the distribution of excitation energy in favour of PSI.     

State transitions,photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F relative fluorescence intensity at emission wavelength nm - F _o fluorescence intensity when all PS II traps are open - light 1 light absorbed preferentially by PS I - light 2 light absorbed preferentially by PS II - PBS phycobilisome - PS photosystem     

A high redox potential form of cytochrome c550 in photosystem II from Thermosynechococcus elongatus

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ～300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.     

The cyanobacterial homologue of HCF136/YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly and repair of photosystem II in Synechocystis sp. PCC 6803

The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair.     

Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II

The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b₅₅₉ (Cyt b₅₅₉) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b₅₅₉, which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b₅₅₉ was oxidized by P680⁺ upon light illumination and re-reduced in the dark, mainly by the electron from the Q_B site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b₅₅₉ and induced its reduction under the illumination. This inhibition of Cyt b₅₅₉ oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b₅₅₉, rather than that of CEF-PSI in photosynthetic organisms.