A New 4.8-kDa Polypeptide Intrinsic to the PS II Reaction Center, as Revealed by Modified SDS-PAGE with Improved Resolution of Low-Molecular-Weight Proteins

Low-molecular-weight polypeptides in various PS II preparationsfrom spinach and wheat were analyzed by modified SDS-PAGE, whichgave good resolution of low-molecular-weight proteins with minimizedinterference by lipids. PS II membrane fragments contained atleast nine low-molecular-weight polypeptides of between 3.9kDa and 11 kDa, and all of them were identified in thylakoidmembranes. Of these nine polypeptides, the 10-kDa phosphoprotein,the 5-kDa, 4.8-kDa, and 4.1-kDa polypeptides, and the two subunitsof cytochrome b₅₅₉ were commonly found in O₂-evolving core complexesof wheat and spinach. In contrast, PS II reaction center complexesthat consisted of D1, D2 and two cytochrome b₅₅₉ polypeptidesretained only the 4.8- kDa polypeptide. Analysis by Westernblotting revealed that the 4.8-kDa polypeptide is an intrinsiccomponent of the PS II reaction center. (Received May 30, 1988; Accepted August 19, 1988)     

Control of electron flow in intact chloroplasts by the intrathylakoid pH,not by the phosphorylation potential

In the presence of nitrite or oxaloacetate, intact chloroplasts evolved oxygen at a significant rate for the initial 1 to 2 min of illumination. Subsequently, oxygen evolution was suppressed progressively. The suppressed oxygen evolution was stimulated strikingly by NH₄Cl. The results indicate that coupled electron flow in intact chloroplasts is controlled in the light, and the control is released by NH₄Cl. However, at low concentrations, NH₄Cl was not an effective uncoupler of photophosphorylation in intact chloroplasts. Intrachloroplast ATP levels and ATP/ADP ratios were not significantly influenced by NH₄Cl. In contrast, the quenching of 9-aminoacridine fluorescence, which can be used to indicate the intrathylakoid pH in intact chloroplasts, was reduced drastically even by low concentrations of NH₄Cl. This suggests that the chloroplast phosphorylation potential is not in equilibrium with the proton gradient. In coupled chloroplasts, the intrathylakoid pH was lower in the light with nitrite than with oxaloacetate as electron acceptor. Electron flow was also more effectively controlled in chloroplasts illuminated with nitrite than with oxaloacetate. It is concluded that the intrathylakoid pH, not the phosphorylation potential, is a factor in the control of the rate of electron flow in intact chloroplasts.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - OAA oxalo-acetate - MES 2-(N-morpholino)-ethanesulfonic acid - HEPES N-2-hyroxyethylpiperazine-N-2-ethanesulfonic acid Postal address     

Energy charge,phosphorylation potential and proton motive force in chloroplasts

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M^?1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M^?1 in the dark and only 300 M^?1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. ΔG′_ATP was ?42 kJ/mol in darkened intact chloroplasts, ?46 kJ/mol in illuminated intact chloroplasts and ?60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH₄Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO₂ reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO₂ reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.     

Directed inactivation of the psbl gene does not affect Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25–30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80–90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

A His-tagged PSII core complex was purified from recombinantChlamydomonas reinhardtii D2-H thylakoids by single-step Ni²⁺-affinitycolumn chromatography and its properties were partially characterizedin terms of their PSII functions and chemical compositions.The PSII core complex that has a His-tag extension at the C-terminusof the D2 protein evolved oxygen at a high rate of 2,400 µmol(mg Chl)^–1h^–1 at the optimum pH of 6.5 with ferricyanideand 2,6-dichlorobenzoquinone as electron acceptors in the presenceof Ca²⁺ as an essential cofactor, and approximately 90% of theactivity was blocked by 10 µM DCMU. The core complex exhibitedthe thermoluminescence Q-band but not the B-band regardlessof the presence or absence of DCMU, although both bands wereobserved in the His-tagged thylakoids. The core complex wasfree from PSI and contained one Y_D, Tyr 160 of the D2 protein,four Mn atoms, two cytochrome b-559, about 46 Chl a molecules,and probably one Q_A, the primary acceptor quinone of PSII. Itwas inferred from these results that His-tagging at the C-terminusof the D2 protein does not affect the functional and structuralintegrity of the PSII core complex, and that the ‘His-tagstrategy’ is highly useful for biochemical, physicochemical,and structural studies of Chlamydomonas PSII. (Received October 22, 1998; Accepted December 25, 1998)     

pH dependent stabilization of S2QA − and S2QB − charge pairs studied by thermoluminescence

The pH dependence of emission peak temperature and decay time of thermoluminescence arising from S₂Q_B ^– and S₂Q_A ^– recombinations demonstrates that a stabilization of S₂Q_B ^– occurs at low pH whereas stabilization of S₂Q_A ^– occurs at high pH. Based on comparative analysis of thermoluminescence parameters of the two types of recombination, we suggest that in the pH range between 5.3 and 7.5, E_m(S₂/S₁) and E_m(Q_A/Q_A ^–) are constant, but E_m(Q_B/Q_B ^–) gradually increases with decreasing pH, while in the pH range between 7.5 and 8.5, an unusual change occurs on S₂Q_A ^– charge pair, which is interpreted as either a decrease in E_m(S₂/S₁) or an increase in E_m(Q_A/Q_A ^–).     

Photosystem I-initiated postillumination CO2 burst in a cyanobacterium,Anabaena variabilis

In Anabaena variabilis, a postillumination CO₂ burst originating from a pool of HCO₃^? is described here. This burst is insensitive to the electron-transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but is abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and N,N′-dicyclohexylcarbodiimide (inhibitors of photophosphorylation). The action spectrum for the burst shows that only Photosystem I is involved.     

Inhibition of Respiration of Yeast by Photosynthesis Inhibiting Herbicides

In order to elucidate the mode of action of some herbicides, effect of several anilide type herbicides on the respiration of yeast cells was studied. The results obtained were as follows: 1) DCPA (3,4-dichloropropionanilide) and DCMU (3-(3,4-dichlorophenyl)-1,1- dimethylurea), the powerful inhibitors of the Hill reaction in photosynthesis, inhibited the oxygen uptake of yeast cells at low concentrations. 2) DCPA and DCMU inhibited the enzymic reduction of cytochrome-c by the yeast cell-free preparation, but not the reduction of dye. 3) The oxidation of cytochrome-b was inhibited in the yeast cells treated with DCPA or DCMU.     

Light-dependent fluorescence variations in intact leaves

Transient variations in the fluorescence from intact Phytolaccaamericana leaves after the onset of illumination were measuredunder various light and dark conditions. Dark-adapted leaveswhen illuminated with strong light underwent an intensity variationwith a peak; the fluorescence intensity reaching its peak severalseconds after the onset of illumination then decreasing to asteady level. The peak height relative to the steady level increasedwith the increasing intensity of actinic light. Pre-illuminationof the dark-adapted leaves with strong light caused a markedlowering of the peak. About 20 min of dark incubation was requiredfor the light-adapted leaves to return to the dark-adapted state.All of the action spectra, for the peak, the steady level andthe effect of light in post-illumination to inhibit recoveryto the dark state, showed high bands due to chlorophyll b andcarotenoid absorption and low bands due to chlorophyll a absorption.We concluded that the light absorbed by photosystem 2 is responsiblefor these phenomena. (Received April 21, 1975; )