Temperature dependence of the kinetics of dark reduction of bacteriochlorophyll P870, oxidized by impulse laser and continuous light in photosynthetic reaction center preparations from Rhodosuedomonas spheroides strain 1760-1]

A comparative study was carried out of temperature dependence of kinetics of dark reduction of bacteriochlorophyll P870 oxidized both by pulsed laser and continuous actinic light in preparations of photosynthetic reaction centres-bacteriochlorophyll-protein complexes from Rhodopseudomonas spheroides, strain 1760-1. Half-time of the recombination of primary products--P870+ and reduced primary electron acceptor A1, which decreases with temperature lowering from 180-240 ms at 120K, is determined. Values of the rate constant of electron transfer from A1 to secondary acceptors at 29,K (2.7.10-1s-1) and the activation energy of this reaction in the range of 298-255K which is 11.8 kcal/mole are calculated.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

The carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the “special pair” of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b(560) was reduced; the dark oxidation of cytochrome b(560) was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b(560), another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b(560). This pigment, tentatively identified as cytochrome b(565), was also detected in spectra at 77 degrees k, after brief illumination at room temperature; the maxima at 77 degrees k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b(565) and an oxidation of cytochrome b(560). Dark oxidation of b(565) was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77 degrees k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77 degrees k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.     

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.     

Primary reactions, plastoquinone and fluorescence yield in subchloroplast fragments prepared with deoxycholate

1. In subchloroplast fragments prepared with the detergent deoxycholate the primary reactions of Photosystem II could be studied at room temperature, because the secondary reactions were largely or completely inhibited.

2. The main quencher of chlorophyll fluorescence in these particles was the photosynthetically active pool of plastoquinone in its oxidized form. Its photoreduction in the presence of artificial electron donors was accompanied by a shift of a chlorophyll a absorption band. Its reoxidation in the dark was very slow, even in the presence of ferricyanide.

3. Of all the artificial electron donors tested MnCl₂ was by far the most efficient.

4. Measurements at room temperature of the C550 absorbance change confirmed its correlation with the primary electron acceptor. Its difference spectrum was broader and its extinction coefficient correspondingly lower than at liquid-N₂ temperature. In chloroplasts the C550 concentration was about 1:360 chlorophylls.

5. In the dark C550 was largely in the reduced state and its oxidation by plastoquinone took place in the presence of an artificial electron donor only, suggesting that the redox potential of C550 was increased by accumulated positive charges at the donor side of the reaction center.

6. The free radical 1,1′-diphenyl-2-picrylhydrazyl oxidized C550 directly in a 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-insensitive reaction. A DCMU-insensitive oxidation of C550 was observed at high ferricyanide concentrations as well, but probably in this case an endogenous electron donor was oxidized, which in turn oxidized C550 via the back reaction of the photochemical reaction.

7. The oxidized form of the primary electron donor, P680⁺, accumulated in the light in the presence of deoxycholate and a low ferricyanide concentration. In chloroplasts the P680 concentration was about 1:360 chlorophylls.

8. The P 680 absorption difference spectrum and electron spin resonance could be explained by the oxidation of a chlorophyll a dimer. Repeated deoxycholate treatments progressively changed the spectra to those of a monomer. The monomer was still photochemically active.

9. A new interpretation of the difference spectrum of P700 is proposed: it may be the same as that of the difference spectrum of P680 if the bleaching at 700 nm is attributed to a band shift.     

Characterization of a redox-active cross-linked complex between cyanobacterial photosystem I and its physiological acceptor flavodoxin.

A covalent complex between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was generated by chemical cross-linking. Laser flash-absorption spectroscopy indicates that the bound flavodoxin of this complex is stabilized in the semiquinone state and is photoreduced to the quinol form upon light excitation. The kinetics of this photoreduction process, which takes place in approximately 50% of the reaction centres, displays three exponential components with half-lives of 9 microsec, 70 microsec and 1 ms. The fully reduced flavodoxin subsequently recombines with P700+ with a t1/2 of 330 ms. A corresponding flavodoxin semiquinone radical signal is readily observed in the dark by room temperature electron paramagnetic resonance, which reversibly disappears upon illumination. In contrast, the light-induced reduction of oxidized flavodoxin can be observed only by first-flash experiments following excessive dark adaptation. In addition, the docking site of flavodoxin on photosystem I was determined by electron microscopy in combination with image analysis. Flavodoxin binds to the cytoplasmic side of photosystem I at a distance of 7 nm from the centre of the trimer and in close contact to a ridge formed by the subunits PsaC, PsaD and PsaE.     

EPR and low temperature spectrophotometric study of the phototransformation products of bacteriorhodopsin]

It is shown that BR and intermediate products of its phototransformation P600, P550 and P415 (the maximum at -196 degrees C at 419 nm) are not paramagnetic. Illumination of samples containing P415 (P419) at -- 196 degrees C with light in the region of 360-480 nm results in the formation of paramagnetic centres with a sunglet spectrum deltaH=18 Oe and g=2.002 (R1). In parallel formation of a new photoproduct P421 in the absorption spectrum is observed. During subsequent heating at -140 degrees C formation of an asymmetric signal with deltaH=45 Oe and g=2.006 and g=2.03 was observed. In the absorption spectra a dark transition. P421-P565 was observed under the same conditions. P565 differs from initial BR P570 as to its photochemical properties. R1 is identified as retinal radical, R2 as a peroxide radical of the BR-complex lipids. Paramagnetic, spectral, and photochemical properties of some products of BR transformation are compared. A scheme of oxidative-phosphorylation processes with participation of Mn ions in BR phototransformation.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review

The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at approximately 730 nm (Frese et al., Biochemistry 42:9205-9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84-91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of Q(A) photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841-851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (B(A) and B(B)) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of B(A) molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P(870)* increased up to approximately 200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the B(A) appears to be lost and B(B) is replaced by bacteriopheophytin Phi(B) with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of approximately 2 ps. These results are discussed in terms of obligatory role of B(A) and Phi(B) molecules located near P for efficient electron transfer from P*.     

Fluorescence excitation spectra and the relative numbers of pigment molecules in reaction centres from Rhodopseudomonas spheroides

1. The excitation spectrum for the bacteriochlorophyll P890 fluorescence in reaction centre preparations was determined at wavelengths ranging from 360 to 890 nm.2. A fluorescence excitation spectrum corresponding to the absorbance spectrum of bacteriopheophytin was also obtained. This spectrum was used in an analysis of the absorbance spectrum of a reaction centre preparation. Based on this spectrum and on literature data, we estimated that the bacteriopheophytin: bacteriochlorophyll ratio in reaction centre particles is at least 1 : 2.3. On the basis of literature data, it is shown that bacteriopheophytin occurs probably as such in reaction centres in vivo.     

Ordered arrays of the photosystem I reaction centre after reconstitution: projections and surface reliefs of the complex at 2 nm resolution.

We present an electron microscopical analysis of the photosystem I reaction centre, the membrane complex involved in the second light-driven step of photosynthetic electron transfer in plants and cyanobacteria. To this end, ordered two-dimensional arrays were reconstituted from detergent solubilized photosystem I reaction centres and phospholipids, and studied by electron microscopy and digital image processing. Small (P1) and large (P3) hexagonal lattices obtained with reaction centres of the thermophilic cyanobacterium Phormidium laminosum had unit cell sizes of a = b = 8.8 nm and 15.8 nm, respectively. Reaction centres of a second thermophilic strain, Synechococcus sp. OD24, gave square lattices (a = b = 14.5 nm; P2(1)). Irrespective of the packing arrangement, projections of negatively stained photosystem I complexes showed elongated asymmetric shapes with a large domain at one end which was tilted with respect to a small domain forming the tip of the other end. Such features were also found in averaged projections of solubilized reaction centre trimers. Surface reliefs reconstructed from freeze-dried metal-shadowed P2(1) lattices revealed that reaction centres had a ridge of 2.5 nm height projecting from one side of the membrane while their other side was rather flat and exhibited a shallow, central indentation.     

Kinetics of pigment-acceptor interaction induced by continuous illumination in Synechocystis spaeroides photosystem I preparations cooled to 160 K in the dark and light

The kinetics of dark reduction of chlorophyll P700 oxidized by continuous light in preparations of photosystem I reaction centers from cyanobacterium Synechosystis spharoides cooled in the dark to 160 K is essentially nonexponential. The characteristic times of the components range from fractions of a second to minutes or more. During the cooling of reaction center preparations under illumination with actinic light, most of the chlorophyll P700 molecules are fixed in the oxidized state at 160 K. The kinetics of dark reduction of P700⁺ in the fraction of reaction centers that retain photochemical activity under these conditions is somewhat faster compared to the samples cooled in the dark. A theoretical analysis of substantial deceleration of P700⁺ dark recovery kinetics was done for preparations of photosystem I reaction centers oxidized by continuous light at 160 K in comparison to the experiments where reaction centers were oxidized by short single light flashes. This slowing down of the kinetics in samples excited by continuous illumination can be explained by microconformational relaxation processes related to proton shifts in the reaction center.     

晴天条件下光、温变化对苹果绿色果皮原初光化学反应的影响

利用快速叶绿素荧光诱导动力学和光谱反射测定技术,研究了晴天条件下,光、温变化对苹果绿色果皮原初光化学反应的影响.结果表明：一天内,随着光、温的增强,金冠苹果果皮在12:00-14:00存在较严重的光抑制.O-J-I-P荧光诱导曲线在300 μs处的相对可变荧光（W_k）几乎没有变化,说明果皮PSⅡ的放氧复合体（OEC）的活性在一天当中没有受到强光和高温的伤害;但是果皮捕获的激子将电子传递到电子传递链中Q_A-下游电子受体的概率（Ψ_o）从8:00-12:00逐渐下降,说明金冠果皮PSⅡ反应中心受体侧的功能受到抑制.强光降低了果皮单位面积上有活性的反应中心（RC/CS）的数量,导致单位反应中心吸收的光能（ABS/RC）增加.果皮的光化学反应（TR_o/RC）不能完全利用所吸收的光能,使单位反应中心的热耗散（DI_o/RC）增加.伴随着光抑制的出现,苹果果皮叶黄素库的脱环化比例增加,表明强光下,果皮启动了叶黄素循环机制,来耗散过剩光能,以减轻过剩光能对光合机构的进一步伤害;一天中,光强和温度的增加均可加重果皮的光抑制程度,但光强对果皮的影响程度显著大于温度对果皮的影响.     

Comparative studies of two membrane fractions isolated from chemotrophically and phototrophically grown cells of Rhodopseudomonas capsulata.

Light and heavy membrane fractions have been isolated by equilibrium sucrose density centrifugation from Rhodopseudomonas capsulata 938 GCM grown aerobically in the dark (chemotrophically) and anaerobically in the light (phototrophically). The densities of the light and heavy fractions from phototrophic cells were 1.1004 to 1.1006 and 1.1478, respectively, and the densities of the light and heavy fractions from chemotrophic cells were 1.0957 to 1.0958 and 1.1315, respectively. Both fractions were active in photochemical and respiratory functions and in electron transport-coupled phosphorylation. The light membrane fraction isolated from chemotrophic cells contained the reaction center and the light-harvesting pigment-protein complex B 870, but not the variable light-harvesting complex B 800-850. A small amount of the complex B 800-850 was present in the light fraction isolated from phototrophically grown cells, but it was not energetically coupled to the photosynthetic apparatus. From inhibitor studies, difference spectroscopy, and measurement of enzyme activities it was tentatively concluded that the light membrane fraction contains only the reduced nicotinamide adenine dinucleotide-oxidizing electron transport chain having a KCN-insensitive, low-potential cytochrome c oxidase, whereas the heavy fraction contains additionally the succinate dehydrogenase and a high-potential cytochrome b terminal oxidase sensitive to KCN. The light membrane fraction was more labile than the heavy fraction in terms of phosphorylating activity.     

Spectral characteristics of PS II reaction centres: as isolated preparations and when integral to PS II core complexes

The discovery that the native PS II enzyme undergoes charge separation via an absorption extending to 730 nm has led us to re-examine the low-temperature absorption spectra of Nanba-Satoh PS II reaction centre preparations with particular focus on the long wavelength region. It is shown that these preparations do not exhibit absorption in the 700-730 nm region at 1.7 K. Absorption in the Nanba-Satoh type preparations analogous to the 'red tail' as observed in functional PS II core complexes is likely shifted to higher energy by >20 nm. Spectral changes associated with the stable reduction of pheo(a) in chemically treated reaction centre preparations are also revisited. Dithionite treatment of PS II preparations in the dark leads to changes of pigment-pigment and/or pigment-protein interactions, as evidenced by changes in absorption and CD spectra. Absorption and CD changes associated with stable Pheo(D1) photo-reduction in PS II core complexes and Nanba-Satoh preparations are compared. For Nanba-Satoh preparations, Q(y) bleaches are approximately 3x broader than in PS II core complexes and are blue-shifted by approximately 4 nm. These data are discussed in terms of current models of PS II, and suggest a need to consider protein-induced changes of some electronic properties of reaction centre pigments.     

Kinetics of pigment-acceptor interaction induced by steady-state illumination in Synechocystis spaeroides photosystem I preparations cooled to 160 K in the dark and on light

The kinetics of dark reduction of chlorophyll P700 oxidized by steady-state illumination in photosystem I reaction center preparations of cyanobacterium Synechocystis sp. coolled in the dark to 160 K is greatly nonexponential. The characteristic times for the components of the reaction are from fractions of a second to minutes and more. During cooling reaction center preparations on actinic light, a great part of chlorophyll P700 is fixed at 160 K in oxidized state. The kinetics of dark reduction of P700+ in the fraction of reaction centers that retain the photochemical activity in these conditions is faster than the kinetics in samples cooled in the dark. A theoretical analysis of the substantial deceleration of the P700+ dark recovery kinetics was done for photosystem I reaction center preparations oxidized by steady-state illumination to 160 K in contrast with situation that arises after the oxidation of reaction centers by single short light pulses. The deceleration of the kinetics in samples activated by steady-state illumination can be explained by processes of microconformational relaxation, connected with proton shifts in the reaction center structure.     

Photosystem 1 preparations from dark-and light-grown cells of the cyanobacterium,Chlorogloea fritschii

Photosystem 1 (PS1) enriched preparations have been extracted from the cyanobacterium Chlorogloea fritschii grown either in darkness or in the light. Absorption spectra show that the main chlorophyll peak has shifted from 678 nm in PS1 from light grown cells to 675 nm in PS1 from dark grown cells. Fluorescence spectra show a similar blue shift in wavelength maximum from 690 nm to 678 nm and the fluorescence intensity is higher in PS1 from dark grown cells. Allophycocyanin is present in PS1 from light grown cells, but absent from preparations from C. fritschii grown in the dark. P700: chlorophyll a ratios of the preparations from light and dark grown cells are 1:35 and 1:80 respectively, all P700 being photoactive. The results are interpreted to suggest that allophycocyanin is not attached to PS1 in dark grown C. fritschii, neither is all chlorophyll arranged in such a way as to ensure efficient energy transfer to P700.     

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.Abbreviations PS 1 (2) photosystem 1 (2) - chl chlorophyll - car carotenoid - Q primary plastoquinone electron acceptor - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - K₃Fe(CN)₆ potassium ferricyanide - DCMU dichlorophenyldimethylurea - DCPIP dichlorophenolindophenol - DPC diphenyl-carbazide     

Effect of infrared and visible light on 2-azidoanthraquinone in the QA binding site of photosynthetic reaction centres. An unusual mode of activation of a photoaffinity label

Quinone-depleted reaction centres of Rhodobacter sphaeroides were reconstituted with 2-azidoanthraquinone and irradiated with short (50 ms) pulses of intense infrared (lambda = 850 +/- 50 nm) or visible light (460 less than lambda less than 630 nm). The irradiations brought about the rapid degradation of the protein-bound photoaffinity label even though it does not absorb light in either spectral region. The decomposition of the label was accompanied by a covalent modification of subunit M and by a loss of photochemical activity of the reaction centre protein (as measured by the light-induced electron transfer onto the primary acceptor, QA). In the case of the photolysis with IR light, these effects were triggered by the reduction of the protein-bound quinone (QA) to the semiquinone (Q-A) in the process of primary charge separation. The resulting reactive species showed properties of both a semiquinone and a triplet nitrene. Efficiency and specificity of the covalent incorporation were markedly improved when visible rather than IR light was used for the photolysis, presumably, because the triplet nitrene resulting from the primary charge separation was further activated in a second light-dependent reaction. The results suggest that, in conventional photoaffinity labeling experiments, the efficiency and specificity of the covalent incorporation of an aryl azide photolabel into a target protein may be improved when the photolysis is carried out with a combination of UV and intense visible light, rather than with UV light alone.     

An alternative pathway of light-induced transmembrane electron transfer in photosynthetic reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the absorption spectrum of Rhodobacter sphaeroides reaction centers, a minor absorption band was found with a maximum at 1053 nm. The amplitude of this band is ~10,000 times less and its half-width is comparable to that of the long-wavelength absorption band of the primary electron donor P₈₇₀. When the primary electron donor is excited by femtosecond light pulses at 870 nm, the absorption band at 1053 nm is increased manifold during the earliest stages of charge separation. The growth of this absorption band in difference absorption spectra precedes the appearance of stimulated emission at 935 nm and the appearance of the absorption band of anion-radical B_A ^– at 1020 nm, reported earlier by several researchers. When reaction centers are illuminated with 1064 nm light, the absorption spectrum undergoes changes indicating reduction of the primary electron acceptor Q_A, with the primary electron donor P₈₇₀ remaining neutral. These photoinduced absorption changes reflect the formation of the long-lived radical state PB_AH_AQ_A ^–.