[Kinetics and spectra of photo-induced changes in the absorption of pigment-protein complexes of photosystem 1 in a picosecond range]. / Kinetiki i spektry fotoindutsirovannykh izmenenii pogloshcheniia u pigment-belkovykh kompleksov fotosistemy 1 v pikosekundnom diapazone.

The energy transfer from the light-harvesting antenna chlorophylls to the reaction center molecules and subsequent charge separation were investigated using a difference picosecond spectrophotometer with selective excitation. The objects were the pigment-protein complexes of photosystem 1 (Chl/P700 = 60) isolated from bean leaves. The difference absorption spectra of the excited states of light-harvesting antenna chlorophylls and the P700 photooxidation were measured. It was shown that the excited states of antenna chlorophylls were generated within 10 ps and deactivated with three-component kinetics: tau 1 = 20--45 ps, tau 2 = 100--300 ps, tau 3 greater than 500 ps. The process of the P700 photooxidation induced by the 650 nm exciting pulse was approximately monoexponential with tau equal to 15--30 ps. It is established that the P700 photooxidation is due to the efficient transfer of excitation energy from antenna chlorophylls to reaction centers.

[Photooxidation of P700 in photosystem 1 preparations with various amounts of antenna chlorophyll a]. / Fotookislenie P700 v preparatakh fotosistemy 1 s raznym soderzhaniem antennogo khlorofilla a.

A number of membrane fragments and pigment-protein complexes of photosystem 1 was obtained from pea chloroplasts, using ionic and non-ionic detergents (SDS, digitonin, Triton X-100, lauryldimethylamine-N-oxide). The ratio of chlorophyll (Chl) a to P700 varied from 220 to 30. For non-dialyzed preparations the quantum yield of P700 photooxidation (phi e) measured by the initial rate of photobleaching at 696-698 nm with excitation at the Soret band of Chla was equal to 40-60%. When the P700 photooxidation was measured at 432 nm, the phi e value showed a further decrease to 20-40% during red light excitation over the range of 660-680 nm but rose to 70-90% at the exciting light wavelengths of greater than or equal to 695 nm. On the basis of the observed dependences the red absorption band was approximated by a sum of two spectra: the spectrum of Chla photoactive in P700 photooxidation and that of photoinactive Chla. Both spectra had maxima near the absorption peak of the object. The photoinactive fraction was additionally enriched by the long-wavelength absorption forms of Chla with an absorption maximum over the range of 684-690 nm. The amount of the bulk Chla in the photoinactive fraction was no less than 40%. The phi e value for freshly dialyzed preparation at a Chla/P700 ratio of 30 was equal to 50-60% independent of the exciting light wavelength. An addition of 0.05% Triton X-100 to this preparation caused: i) a blue shift of the absorption and fluorescence maxima; ii) a decrease of the long-wavelength absorption forms content of Chla and, iii) a considerable increase in fluorescence lifetime and quantum yield due to deaggregation of Chla and its solubilization by detergent micelles. The same phenomenon seems to be responsible for the formation of photoinactive fraction of a pigment, since after addition of a detergent the above-mentioned spectral dependence of phi e appeared, i.e. phi e showed a 3-fold decrease (down to 18%) within the region of 660-680 nm and a 1,6-fold increase (up to 90%) at 705-730 nm. These results suggest that the detergents destroy the intact construction of a light-harvesting antenna rather than that of the photosystem 1 reaction center.

The detergent and salt effect on the light-harvesting chlorophyll a/b complex from green plants.

The light harvesting accessory pigment-protein complex (LHC) with a chlorophyll (Chl) a/b ratio of 1.2 was isolated by treating pea chloroplasts with Triton X-100. The LHC was used to investigate the action of ionic (sodium dodecyl sulfate) and non-ionic (Triton X-100) detergents. By optical methods (absorption and fluorescence spectra, measurements of fluorescence yield, phi, and lifetime, tau) two successive stages of the process were demonstrated, namely (1) interaction between detergent monomers and proteins and (2) solubilization of pigments into detergent micelles, which is facilitated by the presence of salts. The concentration ranges, characteristic of these stages, differ by 1.5-2 orders of magnitude for SDS, but slightly overlap for Triton X-100. At the second stage, certain changes occur in LHC absorption and fluorescence spectra. Several stable states of the LHC were established: (1) an aggregated state formed the presence of 10 mM MgSO4 with tau approximately 0.6 ns; (2) the dialyzed LHC with tau approximately 0.9 ns; (3) the states of the LHC in detergent solution with tau approximately 2.3, 2.9, 3.4 ns; (4) a 30 kilodalton monomer obtained by SDS-polyacrylamide gel electrophoresis with tau approximately 4.1 ns. The fluorescence parameters of the LHC states were compared with those of Chl a in detergent micelles (for the micelles tau = 5.6-6.0 ns). The tau/phi ratio (the criterion for emission heterogeneity) for the LHC in the absence of a detergent was shown to be higher at least by a factor of 3.5 than that for Chl a in the presence of a detergent. Successive additions of the detergent to the LHC cause gradual decrease in the tau/phi ratio, and for the LHC monomer it reaches practically the same value as for Chl a in detergent micelles. The results are discussed on the basis of the data obtained previously, It is suggested that in vivo LHCs do not form such aggregates as in water solution without a detergent.

The fractionation of plant photoactive pigment-protein complexes I and II.

The pigment-protein complexes enriched with Photosystem I (PPC-I) and Photosystem II (PPC-II) were obtained using sievorptive chromatography on DEAE-Sephadex column. Both types of complexes contain Chlorophyll a, beta-carotene and minor quantities of Chl b. Red absorbance maxima are located at 676 nm and 673 nm for PPC-I and PPC-II, respectively. The degrees of reaction centre enrichment were measured by the method of differential spectrophotometry: PPC-I has one P-700 per 35 bulk Chl a molecules, PPC-II contains one P-680 per 18 bulk Chl a molecules. The yield of PPC-II is 7--10 times lower than that of PPC-I. After one chromatographic procedure the amount of P-680 in PPC-I preparation does not exceed 7% of that of P-700, the amount of P-700 in PPC-II preparation 2% of that of P-680. The product of PPC-II degradation was studied.

[Separation of plant photosystems by sievorptive chromatography]. / Razdelenie fotosistem rastenii metodom sitosorbtsionnoi khromatografii.

The pigment-protein complexes enriched with photosystem I (PPC-1) and photosystem II (PPC-2) were obtained using sievorptive chromatography on DEAE-Sephadex. Both types of complexes contain chlorophyll a, beta-carotene and minor quantities of chlorophyll b. Red absorption maxima for PPC-1 and PPC-2 are located at 676 and 673 nm, respectively. Using differential spectrophotometry, the degree of the reaction centre enrichment was established: PPC-1 has one P700 per 35 bulk chlorophyll a molecules, PPC-2 contains one P680 per 18 bulk chlorophyll a molecules. The yield of PPC-2 is 7-10 times lower than that of PPC-1 and equals to 0,3% of the chlorophyll content of the initial chloroplasts. The amount of P680 in PPC-1 preparations does not exceed 7% after a single chromatographic procedure; the amount of P700 in PPC-2 makes up to 2%. The method proposed is more advantageous with respect to higher reaction centre enrichment of PPC-1 and PPC-2 as compared to ion-exchange chromatography on DEAE-cellulose.

[Effect of cations on the quantum yield and the lifetime of the chloroplast fluorescence]. / Izmeneniia vykhoda i vremeni zhizni fluorestsentsii khloroplastov pod deistviem kationov.

The interrelationship between the cation-induced fluorescence changes and the state of the photosystem 2 (PS-2) reaction centers for pea chloroplasts and their osmotic fragments was studied. The effects of K+ and Mg2+ on the fluorescence quantum yield (phi f1) under varying light intensities as well as on the fluorescence lifetime (tau f1) in the saturating light were demonstrated. K+ induces the decrease in tau f1; Mg2+ exerts an opposite effect. The effects were more pronounced when the reaction centers of PS-2 were converted into an inactive state by illuminating the sample with a saturating light or by adding DCMU. Under these conditions the cations' effect on tau f1 was accompanied by proportional changes in tau f1. It was concluded that in Mg-deficient chloroplasts an efficient channel of the excitation quenching appears in antenna chlorophyll of PS-2 with the rate constant of 7 . 10(8) s-1. The simultaneous measurements of tau f1 by phase and modulation type techniques allowed to reveal the emission heterogeneity within the nanosecond time interval and the DCMU-sensitive delayed fluorescence with the lifetime exceeding 10(-7) s and the overall quantum yield approximately equal to 2 . 10(-3).

Reconstitution of biological molecular generators of electric current. Bacteriochlorophyll and plant chlorophyll complexes.

1. Electric generation by bacteriochlorophyll reaction center complexes from Rhodospirillum rubrum and by photosystem I complexes from pea chloroplasts has been studied. 2. The methods for the proteoliposome reconstitution from azolectin and bacteriochlorophyll- or plant chlorophyll-containing protein complexes have been elaborated. Light-dependent electric responses of the proteoliposomes were detected using (a) phenyldicarbaundecarborane anion (PCB-) probe and (b) direct measurement by a voltmeter in the proteoliposome-planar phospholipid membrane system. 3. Both PCB- and direct measurements demonstrated that bacteriochlorophyll proteoliposomes are competent in light-dependent electric generation (plus outside proteoliposomes). The photoelectric effect was shown to increase on addition of tetramethyl-p-phenylenediamine (TMPD), CoQ6, and vitamin K3, and to decrease on addition of ferricyanide, o-phenanthroline and a protonophorous uncoupler. Estimation of the photoelectromotive force of the bacteriochlorophyll proteoliposome-planar membrane system gave a value of about 0.2 V. The action spectrum of the photoelectric effect was similar to the absorption spectrum of the bacteriochlorophyll complex. 4. Reconstitution of proteoliposomes containing bacteriochlorophyll centers and bacteriorhodopsin resulted in the system generating an electric field whose direction can be changed by varying the spectral composition of the light: the red light, exciting bacteriochlorophyll, induces negative, and the green light, exciting bacteriorhodopsin, induces positive charging of the proteoliposome interior. 5. Association of isolated R. rubrum chromatophores with planar phospholipid membrane was found to give a system demonstrating light-induced electric generation as high as 215 mV in the presence of napthoquinone, TMPD (or phenazine methosulfate, PMS), and ascorbate. Under the same conditions, addition of inorganic pyrophosphate or ATP results in formation of an electric field of the same direction as that induced by light. 6. Proteoliposomes with plant chlorophyll complexes of photosystem I demonstrated light-induced PCB- responses indicating formation of the electric field with plus inside vesicles. The effect required PMS addition. A protonophorous uncoupler and o-phenanthroline were inhibitory. Electric responses in the chlorophyll proteoliposome-planar membrane system were very small (not higher than 10 mV).

[Kinetics and mechanism of inactivation of isolated chloroplasts]. / Kinetika i mekhanizm inaktivatsii izolirovannykh khloroplastov

A detailed kinetic analysis has been performed of a multistep inactivation of chloroplasts. The kinetic model suggested involves the formation of chloroplast forms differing in stability and activity. A comparison of the kinetic model with the experimental data shows that the mechanism of inactivation of isolated pea chloroplasts consists of at least two forms displaying different activity in the Hill reaction and different stability in solution. The effect was studied of the nature of the buffer and destruction products on the kinetics of chloroplast inactivation in the process of "ageing". In phosphate buffer, where the concentration of phosphate exceeds 40 mM, the effect of the destruction products of chloroplasts on their inactivation is insignificant. The pH dependence on the inactivation kinetics suggests that the pH region from 6 to 9 affects only the rapid kinetic process resulting in the increase in the chloroplast activity; irreversible inactivation of chloroplasts is pH-independent. The temperature dependence of the irreversible inactivation kinetics has been studied and the activation parametres of this stage have been determined. Possible molecular mechanism of the limiting stages of the inactivation of isolated chloroplasts are discussed, which can explain the kinetic data obtained.

[Technique for the determination of the quantum yield of the primary process in the course of photosynthesis energy transformation. III. Experimental data obtained for different photosynthetic organisms]. / Opredelenie kvantovogo vykhoda pervichnogo preobrazovaniia energii pri fotosintez III. Eksperimental'nye dannye dlia razlichnykh fotosinteziruiushchikh organizmov

A relative method in two modifications has been developed for determination of the quantum yield of primary photosynthesis energy conversion. The purple bacteria E. chaposhnikovii and Chr. minutissimum, R. rubrum chromatophores, cell extracts of E. shaposhnikovii, Rps. spheroides strains GA and R-26, pigment-protein complexes from green bacterium Chl. limicola and light subchloroplast particles, enriched in the photosystem I, were investigated. The quantum yields for all these objects were shown to be 90-98%. The accuracy of the relative method used is very high (within 2--4+). The photosynthetic units were proved to be of a multicentral type for all these systems. The quantum yield values permit estimation of the coefficient of efficiency of the primary conversion process as being approximately equal to 35-38% for bacteria and approximately equal to 50% plants.