Photoelectric response generated under non-heme iron reduction on the photosystem II acceptor side.

Proteoliposomes containing oxygen-evolving particles of Photosystem II and associated with a planar phospholipid membrane generate a transmembrane electric potential difference (DeltaPsi) induced by a laser flash. With direct electrometrical technique, it was shown that the direction of the electrical field ("minus" inside the proteoliposome) corresponds to acceptor side of the Photosystem II complex facing inside and donor side facing outside of the liposomes. In addition to the fast phase (tau < 0.1 microsec) of the DeltaPsi generation due to electron transfer between YZ of the water-oxidizing complex and the primary plastoquinone QA, a phase with tau approximately 120 microsec and maximum amplitude approximately 30% of the amplitude of the fast phase was observed under the first flash in proteoliposomes containing potassium ferricyanide, which is known as an oxidant of the non-heme iron (Fenh) on the acceptor side of Photosystem II. This additional phase was absent under the second laser flash but was completely restored after 5 min dark adaptation. The phase of the photoelectric response with tau approximately 120 microsec is probably due to electron transfer from QA to Fenh(III) and likely includes a component related to H+ transfer.

[Mechanism of photosynthetic water oxidation in the dimeric oxygen-evolving complex of chloroplast photosystem II]. / O mekhanizme fotosinteticheskogo okisleniia vody v dimernom kislorodvydeliaiushchem komplekse fotosistemy II khloroplastov.

Based on the analysis of the molecular organization and properties of an isolated oxygen-evolving complex of photosystem II of plant chloroplasts, a mechanism of water oxidation and oxygen release during photosynthesis was proposed. It is suggested that the photolysis of water occurs in a dimeric oxygen-evolving complex consisting of two core complexes. In the region of contact of these complexes, a hydrophobic "boiler" is formed where the conditions for screening and stabilization of Z-linanded manganese cations accumulating positive charges for the oxidation of water molecules are created. A prerequisite to the photolysis of water is the formation of a binuclear [Mn(3+)-OH ... HO-Mn3+] hydroxyl-manganese associate, which appears in the dimeric oxygen-evolving complex after the first two light flashes as a result of photohydrolysis of photochemically oxidized Z-liganded manganese cations. The process is accompanied by the release of the first water protons to the medium. The photosynthetic oxidation of water hydroxyls occurs at the next stage and is considered as synchronous detachment of four electrons from two bound OH-groups of the associate upon photooxidation of Mn3+ cations to Mn4+ cations after two subsequent light flashes. This process is accompanied by the disproportionation of electron density and the formation of a bond between oxygen atoms of hydroxyls followed by the evolution of molecular oxygen and protons, and regeneration of two starting Mn2+ cations and the primary state of the system.

[Efficiency of chromatographic separation of the pigment-protein lipid complexes of the reaction centers of photosystems 1 and 2 from pea chloroplasts on DEAE-cellulose]. / Issledovanie éffektivnosti khromatograficheskogo razdeleniia pigment-belkovolipidnykh kompleksov reaktsionnykh tsentrov fotosistemy 1 i fotosistemy 2 khloroplastov gorokha na DEAE-tselliuloze.

Using a previously developed procedure, further optimization of isolation of the functionally active pigment-protein lipid complex of the reaction center of photosystem 2 (PPLC RC PS-2) by DEAE-cellulose chromatography has been carried out. Preliminary chromatographic separation of PPLC RC PS-1 and PS-2 in the presence of a light-harvesting complex has proved to be the most efficient technique. In the latter case the admixture of P700 within PPLC RC PS-2 decreases (1.8-fold on the average) at chromatographic pH value of 8.0. During chromatography at weakly acid, neutral (6.5-7.0) and weakly alkaline (8.3-9.0) values of pH a further, more than 2-fold decrease of P700 admixture is observed. The increase in the degree of separation of PPLC RC PS-1 and PS-2 is concomitant with the increase in PPLC RC PS-2 of photochemically active pheophytin (Ph), an intermediate electron acceptor in RC PS-2 which functions between P680 and Q. The resulting preparations of PPLC RC PS-2 contain one molecule of Ph and 0.06-0.09 molecule of P700 per 31-48 molecules of chlorophyll. The peculiarities of the complexes separation are discussed in terms of the role of hydrogen ion concentration in regulation of affinity between the reaction center complexes and the light-harvesting complex.

[Study of the effect of specific translation inhibitors on formation of native forms of chlorophyll in pigment-protein-lipid complexes of pea chloroplasts]. / Izuchenie deistviia spetsificheskikh ingibitorov transliatsii na obrazovanie nativnykh form khlorofilla v pigment-belkovo-lipidykh kompleksakh khloroplastov gorokha.

The effect of specific inhibitors of translation in chloroplasts (chloramphenicol) and in cytoplasm (cycloheximide) on the formation of pigment-protein-lipid complexes of photosynthetic membranes, on the chlorophyll state in chloroplasts and isolated membrane complexes had been studied. It is proved that the inhibition of translation blocks chlorophyll incorporation only into the complexes of reaction centres of photosystems without any change of the light-harvesting complex. The action of inhibitors leads to the disappearance of long-wave native forms of pigment in the complexes of reaction centres, which are characteristic for them. But the action of inhibitors does not effect the formation of non-specific short-wave forms which are present in all types of membrane complexes. Chloramphenicol proved to be more active in such processes than cycloheximide. On the basis of the data on localization of biosynthesis of polypeptide components of plastid membranes we suppose that during biogenesis of the photosynthetic apparatus the conditions for self-assembly of non-specific native forms of chlorophyll in light-harvesting complexes are made as the result of polymerazation of the main membrane polypeptides which are synthesized in the cytoplasms. Minor polypeptides of plastid (photosystems 1 and 2) and cytoplasmic (photosystem 1) orgin are necessary for self-assembly of the dense units of pigment in reaction centre complexes.

[Optimization of isolation conditions for three types of pigment protein-lipid complexes from chloroplasts during solubilization with Triton X-100]. / Optimizatsiia uslovii vydeleniia trekh tipov pigment-belkovolipidnykh kompleksov khloroplastov gorokha pri soliubilizatsii s pomoshch'iu tritona X-100.

The maximal total release of pigment protein-lipid complexes (PPLC) during their isolation from pea chloroplasts was achieved by 1-hr solubilization with Triton X-100, the Triton:chlorophyll (T/Chl) ratio being 50 mg/mg/ml. The total yield of the reaction center complexes (sigma PPLC RC) was 22,3%, whereas that of the auxiliary light-accumulating complex (ALA-PPLC) was approximately 32% with respect to Chl. An increase in the solubilization time and of the T/Chl ratio resulted in dissociation of ALA-PPLC. On the contrary, the reaction center complexes steadily maintained their composition and high photochemical activity within a wide range of T/Chl during 24--28 hrs of solubilization. The purest preparations of PPLC RC of phostosystem I (PS-I) were obtained by 24 hr-incubation (T/Chl = 80); their Chl/P700 ratio after a single fractionation on DEAE-cellulose was equal to 36. A considerable increase of T/Chl and of the solubilization time hampered the chromatographical separation of PPLC RC of PS-I and PPLC RC of PS-II. The optimal conditions for isolation of PPLC RC of PS-I and PPLC RC of PS-II were: solubilization at T/Chl 80--120 and prolongation of incubation time from 5 to 7 hrs. The photochemical activity of the complexes obtained was maximal and correlated with the minimal content of admixture P700 (1 molecule of P700 per 450--500 molecules of Chl.).

[Photochemical and spectral properties of photosystem-2 subchloroplast fragments, highly purified from photosystem-1 mixtures]. / Issledovanie fotokhimicheskikh i spektral'nykh svoistv subkhloroplastnykh fragmentov FS-2, vysokoochischennykh ot primesi FS-1

A rather simple method of isolation of photosystem 2 fragments, which are highly purified from Photosystem 1 admixture, has been developed on the basis of combined action of detergents and differential centrifugation. The isolated fragments are characterized by insignificant content of P700 (one molecule per 10500 molecules of chlorophyll) and by high ratio of band values at 685 and 735 nm in the low temperature emission spectrum of fluorescence (F685/F735=5.9). The data on photochemical activity and ability for photoinduced changes in fluorescence prove that the activity of Photosystem 2 is retained both at the level of reaction centre operation and at that of water photooxidation with oxygen evolution.

[Method of isolating photochemically active pigmet-proteolipid complexes from chloroplasts]. / K voprosu o metode vydeleniia fotokhimicheski aktivnykh pigment-belkovolipidnykh kompleksov khloroplastov

The paper deals with the chromatography isolation method of pigment-lipoprotein complexes of photosystem I and II of pea chloroplasts on the DEAE-cellulose. The componenet composition and photochemical properties of isolated complexes are compared with the data available in literature concerning similar objects. The question about the use of pigment-lipoprotein complexes as the model systems for investigating structure organization and functional characteristics of different parts of the chloroplast photosynthetic apparatus is under discussion.