Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

• MeSH
• Chlorophyll metabolism   MeSH
• Pheophytins metabolism   MeSH
• Photosystem I Protein Complex metabolism   MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• Pheophytins MeSH
• Photosystem I Protein Complex MeSH
• Photosystem II Protein Complex * MeSH

Accumulation of reduced pheophytin a (Pheo-D1) in photosystem II reaction center (PSII RC) under illumination at low redox potential is accompanied by changes in absorbance and circular dichroism spectra. The temperature dependences of these spectral changes have the potential to distinguish between changes caused by the excitonic interaction and temperature-dependent processes. We observed a conformational change in the PSII RC protein part and changes in the spatial positions of the PSII RC pigments of the active D1 branch upon reduction of Pheo-D1 only in the case of high temperature (298 K) dynamics. The resulting absorption difference spectra of PSII RC models equilibrated at temperatures of 77 K and 298 K were highly consistent with our previous experiments in which light-induced bleaching of the PSII RC absorbance spectrum was observable only at 298 K. These results support our previous hypothesis that Pheo-D1 does not interact excitonically with the other chlorins of the PSII RC, since the reduced form of Pheo-D1 causes absorption spectra bleaching only due to temperature-dependent processes.

• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Photosystem II Protein Complex chemistry   metabolism   MeSH
• Protein Conformation radiation effects   MeSH
• Models, Molecular MeSH
• Oxidation-Reduction radiation effects   MeSH
• Cyanobacteria metabolism   MeSH
• Spectrophotometry methods   MeSH
• Light * MeSH
• Hot Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Photosystem II Protein Complex MeSH
• photosystem II, psbA subunit MeSH Browser

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.

• MeSH
• beta Carotene metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Ferricyanides metabolism   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Pisum sativum MeSH
• Molybdenum metabolism   MeSH
• Oxidation-Reduction MeSH
• Silicon Compounds metabolism   MeSH
• Light MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta Carotene MeSH
• Chlorophyll MeSH
• Ferricyanides MeSH
• Photosystem II Protein Complex MeSH
• hexacyanoferrate III MeSH Browser
• Molybdenum MeSH
• silicomolybdate MeSH Browser
• Silicon Compounds MeSH

A multichannel kinetic spectrophotometer-fluorimeter with pulsed measuring beam and differential optics has been constructed for measurements of light-induced absorbance and fluorescence yield changes in isolated chlorophyll-proteins, thylakoids and intact cells including algae and photosynthetic bacteria. The measuring beam, provided by a short (2 micros) pulse from a xenon flash lamp, is divided into a sample and reference channel by a broad band beam splitter. The spectrum in each channel is analyzed separately by a photodiode array. The use of flash measuring beam and differential detection yields high signal-to-noise ratio (noise level of 2 x 10(-4) in absorbance units per single flash) with negligible actinic effect. The instrument covers a spectral range between 300 and 1050 nm with a spectral resolution of 2.1, 6.4 or 12.8 nm dependent on the type of grating used. The optical design of the instrument enables measuring of the difference spectra during an actinic irradiation of samples with continuous light and/or saturation flashes. The time resolution of the spectrophotometer is limited by the length of Xe flash lamp pulses to 2 micros.

• MeSH
• Time Factors MeSH
• Fluorometry instrumentation   methods   MeSH
• Photosynthesis physiology   MeSH
• Kinetics MeSH
• Rhodobacter sphaeroides metabolism   MeSH
• Spectrophotometry instrumentation   methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Absorption and circular dichroism spectra of Photosystem II (PS II) reaction centres (RC) were studied and compared with spectra calculated on the basis of point-dipole approximation. Chlorophyll cation was accumulated during a light treatment of PS II RC in the presence of artificial electron acceptor silicomolybdate. Light-induced difference spectra and their calculated counterparts revealed the location of accumulated cation at the accessory chlorophyll of the D1 protein subunit.

• MeSH
• Models, Biological MeSH
• Chlorophyll chemistry   metabolism   MeSH
• Circular Dichroism MeSH
• Photosystem II Protein Complex chemistry   metabolism   MeSH
• Pisum sativum chemistry   metabolism   MeSH
• Cations chemistry   metabolism   MeSH
• Energy Transfer MeSH
• Plant Proteins chemistry   metabolism   MeSH
• Spectrophotometry, Atomic MeSH
• Light MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll MeSH
• Photosystem II Protein Complex MeSH
• Cations MeSH
• Plant Proteins MeSH