Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Photoinduced formation of pheophytin/chlorophyll-containing complexes during the greening of plant leaves

Illumination of etiolated maize leaves with low-intensity light produces a chlorophyll/pheophytin-containing complex. The complex contains two native chlorophyll forms Chl 671/668 and Chl 675/668 as well as pheophytin Pheo 679/675 (with chlorophyll/pheophytin ratio of 2/1). The complex is formed in the course of two successive reactions: reaction of protochlorophyllide Pchlde 655/650 photoreduction resulted in chlorophyllide Chlde 684/676 formation, and the subsequent dark reaction of Chlde 684/676 involving Mg substitution by H₂ in pigment chromophore and pigment esterification by phytol. Out data show that the reaction leading to chlorophyll/pheophytin-containing complex formation is not destructive. The reaction is in fact biosynthetic, and is competitive with the known reactions of biosynthesis of the bulk of chlorophyll molecules. The relationship between chlorophyll and pheophytin biosynthesis reactions is controlled by temperature, light intensity and exposure duration.The native complex containing pheophytin a and chlorophyll a is supposed to be a direct precursor of the PS II reaction centre in plant leaves.Abbreviations Chl chlorophyll - Chlde chlorophyllide - Pchl protochlorophyll - Pchlde protochloropyllide - Pheo pheophytin - PS II RC Photosystem II reaction centres. Abbreviations for native pigment forms: the first number after pigment symbol corresponds to the maximum position of low-temperature fluorescence band (nm); the second number corresponds to the maximum position of long wave absorption band     

Interactions of Chlorophyll and Polypeptide Mixture with Bacterial Reaction Centres

In aqueous solutions of chlorophyll (Chl) a with synthesized polypeptides, at high ratios of Chl to polypeptides (about 75–150 µM to 500 µM) clusters of polypeptides and pigment molecules were formed. The main absorption maxima of more than one formed cluster were located at about 500 nm (Soret band) and in the region of 720–806 nm (red band). The formation of these clusters was fairly slow (some hours) at room temperature and even slower at 4 °C. The rate of cluster formation increased with the increase in Chl concentration. The addition of the even low amount of reaction centres (RCs), separated from the purple bacteria Rhodobacter sphaeroides, to the sample of Chl with polypeptides caused a very strong decrease in the efficiency of cluster formation, and a change in concentration ratios of various pigment-polypeptide aggregates. It was probably a competition between the interaction of Chl with polypeptides and with the RCs. The yield of thermal deactivation of the clusters was high, much higher than that for the RCs alone and it was different for various types of cluster. The clusters absorbing at 725–750 nm were fluorescent with maximum of emission at about 770 nm, whereas clusters absorbing at about 800 nm were nonfluorescent.     

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu²⁺ ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO₄ and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (A of 430–440 nm) and by the flash induced formation of P680⁺ (A at 820 nm). The relatively slow relaxation kinetics of the latter signal (t_1/2 1 ms) may suggest that in a substantial fraction of the RCs Q_A remains bound to the complex.Abbreviations Car -carotene - Chl a chlorophyll a - CP43, CP47 chlorophyll-proteins, with R_m 43 and 47 kDa - DBMIB dibromothymoquinone,2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone - DM -dodecyl-d-maltoside - HPLC high-performance liquid chromatography - OG n-octyl--d-glucopyranoside - IMAC immobilied metal affinity chromatography - Pheo a pheophytin a - PQ-9 plastoquinone-9 - P680 primary electron donor in PS II - PS II RC Photosystem II reaction centre - Q_A primary electron acceptor in PS II - SB-12 N-dodecyl-N,N-dimethyl-3-amino-1-propanesulphonate, (sulphobetain 12)     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.     

Study of precise pigment composition of photosystem 1-type reaction centers by means of normal-phase HPLC

Based on precise pigment analyses of a series of photosystem (PS) 1-type plants, a novel hypothesis is proposed that chiorophyll (Chl)a′ and bacteriochlorophyll (BChl)g′, the 13²-epimers of Chla and BChlg, constitute the primary electron donors of PS1 and heliobacteria, respectively. Interestingly PS 1-type reaction centers do not have (B) Pheo but have Chl a-like pigments as primary electron acceptors. Recipient of the Botanical Society Award of Young Scientists, 1994.     

Functional characteristics of chlorophyll d-predominating photosynthetic apparatus in intact cells of Acaryochloris marina

Functional organization of the photosynthetic apparatus in the unique chlorophyll d-predominating prokaryote, Acaryochloris marina, was studied using polarographic measurements of single-turnover flash yields, action spectra and optical cross sections for PS-specific reactions. O₂ evolution was indicative of PS II activity, while reversible photoinhibition of respiratory O₂ uptake under aerobic conditions in the presence of DCMU and H₂ photoevolution by anaerobically adapted cells were the indicatives of PS I activity. O₂ evolution in the cells upon single-turnover flashes followed the normal S-state cycle with a period-4 oscillation. Analysis of action spectra for the partial reactions of photosynthesis revealed that: (1) distinct spectral forms of Chl d are nonuniformly distributed between PS I and PS II, e.g. Chl d-695 and Chl d-735 are preferentially located in PS II and PS I, respectively; (2) a minor fraction of Chl a in the cells belongs mostly to PS II; (3) biliproteins transfer excitation energy both to PS II and, with a lower efficiency, PS I; (4) the efficiency of energy transfer from biliproteins to PS II depends on the light quality growth conditions and is larger in white light (WL)-grown cells compared to the red light (RL)-grown cells. Content of functional O₂ evolving PS II centers decreases 2 times in the RL-grown cells relative to the WL-grown cells, whereas content of competent PS I centers involved in photoinhibition of respiration remains almost the same in both the cultures. The effective antenna size of PS I was estimated to be 80–90 Chl d including 3–10 molecules absorbing at 735 nm. The effective optical cross-section of PS II corresponded to 90–100 Chl d and, presumably, 4 Chl a + 2 Pheo a [Mimuro et al. (1999) Biochim Biophys Acta 1412: 37–46]. Optical cross-section measurements indicated that the functional PS II units of A. marina attach one rod of four hexameric units of biliproteins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermoluminescence properties of the isolated photosystem two reaction centre

Formation of thermoluminescence signals is characteristics of energy- and charge storage in Photosystem II. In isolated D1/D2/cytochrome b-559 Photosystem II reaction centre preparation four thermoluminescence components were found. These appear at -180 (Z band), between -80 and -50 (Z_v band), at -30 and at +35°C. The Z band arises from pigment molecules but not correlated with photosynthetic activity. The Z_v and -30°C bands arise from the recombination of charge pairs stabilized in the Photosystem II reaction centre complex. The +35°C band probably corresponds to the artefact glow peak resulting from a pigment-protein-detergent interaction in subchloroplast preparations (Rózsa Zs, Droppa M and Horváth G (1989) Biochim Biophys Acta 973, 350–353).Abbreviations Chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - D1 psbA gene product - D2 psbD gene product - P680 primary electron donor of PS II - Pheo pheophytin - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - RC reaction centre of PS II - TL thermoluminescence     

An overview on chlorophylls and quinones in the photosystem I-type reaction centers

Minor but key chlorophylls (Chls) and quinones in photosystem (PS) I-type reaction centers (RCs) are overviewed in regard to their molecular structures. In the PS I-type RCs, the prime-type chlorophylls, namely, bacteriochlorophyll (BChl) a′ in green sulfur bacteria, BChl g′ in heliobacteria, Chl a′ in Chl a-type PS I, and Chl d′ in Chl d-type PS I, function as the special pairs, either as homodimers, (BChl a′)₂ and (BChl g′)₂ in anoxygenic organisms, or heterodimers, Chl a/a′ and Chl d/d′ in oxygenic photosynthesis. Conversions of BChl g to Chl a and Chl a to Chl d take place spontaneously under mild condition in vitro. The primary electron acceptors, A ₀, are Chl a-derivatives even in anoxygenic PS I-type RCs. The secondary electron acceptors are naphthoquinones, whereas the side chains may have been modified after the birth of cyanobacteria, leading to succession from menaquinone to phylloquinone in oxygenic PS I.     

Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 131-deoxo-131-hydroxy-pheophytin a and comparison of their 6 K absorption spectra

Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 13¹-deoxo-13¹-hydroxy-pheophytin a (13¹-OH-Pheo). The absorption bands of 13¹-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC_1×) or twice (RC_2×). HPLC analysis and Pheo Q_X absorption at 543 nm show that in RC_1× about 50% of Pheo is replaced and in RC_2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo⁻ is unmodified in RC_1× and decreased only in RC_2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the Q_Y region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC_1× or RC_2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of chlorophyll–protein complexes isolated from a Siphonous green alga, Bryopsis corticulans

Six chlorophyll–protein complexes are isolated from thylakoid membranes of Bryopsis corticulans by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. Unlike that of higher plants, the 77 K fluorescence emission spectrum of the CP1 band, the PSI core complexes of B. corticulans, presents two peaks, one at 675 nm and the other at 715–717 nm. The emission peak at 715–717 nm is slightly higher than that at 675 nm in the CP1 band when excited at 438 or 540 nm. However, the peak at 715 nm is obviously lower than that at 675 nm when excited at 480 nm. The excitation spectra of CP1 demonstrate that the peak at 675 nm is mainly attributed to energy from Chl b while it is the energy from Chl a that plays an important role in exciting the peak at 715–717 nm. Siphonaxanthin is found to contribute to both the 675 nm and 715–717 nm peaks. We propose from the above results that chlorophyll a and siphonaxanthin are mainly responsible for the transfer of energy to the far-red region of PSI while it is Chl b that contributes most of the transfer of energy to the red region of PSI. The analysis of chlorophyll composition and spectral characteristics of LHCP¹ and LHCP³ also indicate that higher content of Chl b and siphonaxanthin, mainly presented in LHCP¹, the trimeric form of LHCII, are evolved by B. corticulans to absorb an appropriate amount of light energy so as to adapt to their natural habitats.     

Characterization of the Porphyridium cruentum Chl a-binding LHC by in vitro reconstitution: LHCaR1 binds 8 Chl a molecules and proportionately more carotenoids than CAB proteins

The Porphyridium cruentum light harvesting complex (LHC) binds Chl a, zeaxanthin and -carotene and comprises at least 6 polypeptides of a multigene family. We describe the first in vitro reconstitution of a red algal light-harvesting protein (LHCaR1) with Chl a/carotenoid extracts from P. cruentum. The reconstituted pigment complex (rLHCaR1) is spectrally similar to the native LHC I, with an absorption maximum at 670 nm, a 77 K fluorescence emission peak at 677 nm (ex. 440 nm), and similar circular dichroism spectra. Molar ratios of 4.0 zeaxanthin, 0.3 -carotene and 8.2 Chl a per polypeptide for rLHCaR1 are similar to those of the native LHC I complex (3.1 zeaxanthin, 0.5 -carotene, 8.5 Chl a). The binding of 8 Chl a molecules per apoprotein is consistent with 8 putative Chl-binding sites in the predicted transmembrane helices of LHCaR1. Two of the putative Chl a binding sites (helix 2) in LHCaR1 were assigned to Chl b in Chl a/b-binding (CAB) LHC II [Kühlbrandt et al. (1994) Nature 367: 614–21]. This suggests either that discrimination for binding of Chl a or Chl b is not very specific at these sites or that specificity of binding sites evolved separately in CAB proteins. LHCaR1 can be reconstituted with varying ratios of carotenoids, consistent with our previous observation that the carotenoid to Chl ratio is substantially higher in P. cruentum grown under high irradiance. Also notable is that zeaxanthin does not act as an accessory light-harvesting pigment, even though it is highly likely that it occupies the position assigned to lutein in the CAB LHCs.This revised version was published online in October 2005 with corrections to the Cover Date.     

Chromatic photoacclimation extends utilisable photosynthetically active radiation in the chlorophyll <Emphasis Type="Italic">d</Emphasis>-containing cyanobacterium, <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Chromatic photoacclimation and photosynthesis were examined in two strains of Acaryochloris marina (MBIC11017 and CCMEE5410) and in Synechococcus PCC7942. Acaryochloris contains Chl d, which has an absorption peak at ca 710 nm in vivo. Cultures were grown in one of the three wavelengths (525 nm, 625 nm and 720 nm) of light from narrow-band photodiodes to determine the effects on pigment composition, growth rate and photosynthesis: no growth occurred in 525 nm light. Synechococcus did not grow in 720 nm light because Chl a does not absorb effectively at this long wavelength. Acaryochloris did grow in 720 nm light, although strain MBIC11017 showed a decrease in phycobilins over time. Both Synechococcus and Acaryochloris MBIC11017 showed a dramatic increase in phycobilin content when grown in 625 nm light. Acaryochloris CCMEE5410, which lacks phycobilins, would not grow satisfactorily under 625 nm light. The cells adjusted their pigment composition in response to the light spectral conditions under which they were grown. Photoacclimation and the Q _y peak of Chl d could be understood in terms of the ecological niche of Acaryochloris, i.e. habitats enriched in near infrared radiation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chemical modification of photosystem II core complex pigments with sodium borohydride

The reaction of the irreversible chemical reduction of the 13¹-keto C=O group of pheophytin a (Pheo a) with sodium borohydride in reaction centers (RCs) of functionally active spinach photosystem II (PS II) core complexes was studied. Stable, chromatographically purified PS II core complex preparations with altered chromophore composition are obtained in which ～25% of Pheo a molecules are modified to 13¹-deoxo-13¹-hydroxy-Pheo a. Some of the chlorophyll a molecules in the complexes were also irreversibly reduced with borohydride to 13¹-deoxo-13¹-hydroxy-chlorophyll a. Based on the results of comparative study of spectral, biochemical, and photochemical properties of NaBH₄-treated and control preparations, it was concluded that: (i) the borohydride treatment did not result in significant dissociation of the PS II core complex protein ensemble; (ii) the modified complexes retained the ability to photoaccumulate the radical anion of the pheophytin electron acceptor in the presence of exogenous electron donor; (iii) only the photochemically inactive pheo-phytin Pheo_D2 is subjected to the borohydride treatment; (iv) the Q_x optical transition of the Pheo_D2 molecule in the RC of PS II core complexes is located at 543 nm; (v) in the Q_y spectral region, Pheo_D2 probably absorbs at ～680 nm.     

Light-induced Damage of Photosystem Ⅱ Primary Electron Donor P680: a High Performance Liquid Chromatographic Analysis of Pigment Content in D1/D2/Cytochrome b559 Complex Under Photoinhibitory Conditions

By HPLC analytical method, the change of PS Ⅱ RC' s pigment content in the process of photodamage under strong illumination from spinach ( Spinacia oleracea Mill. ) was comparatively studied. The experimental results show that: (1) In authors' analytical conditions, (of which, [Chl] = 150 µg/mL, and the illumination strength was put at 2.3 ×10 6 mJ·m－2·s－1 ), 45 rain of illumination could cause almost the whole loss of A680 in the fourth derivative absorption spectra, while A670 decreased to about one half of its original intensity; the absorption maximum in red, concurrently, was shifted from 676 nm to 671 nm, representing the loss of more than 90% of the photochemical activities of the PS Il RC. (2) During the period of continuous illumination, the Chl concentration decreased in a 3-period style, which meant that the first [Chl] decreased to the 2/3 of its original amount from 20 min to 40 rain after illumination had started, then became stabilized up to about 60 min of illumination, there after a second decrease of [ Chl ] in another about 20 min until it reached about 30 % of the original level and remained unchanged from about 80 min on. The original pigment components of D1/D2/Cyt b559 was approximately as 6 Chl a:2 Pheo:2β-Car which are in support of authors' previous proposal about the minimum Chl/Pheo ratio of 4: 2 in PS Ⅱ RC’s pigment contents. (3) After about 40 min of illumination, a newly appeared elution peak was found between the Pheo andβ-Car peaks in HPLC profile, at the retention time of 7.2 min, a little later than that (6.9 rain) of Pheo molecules, the newly appeared elution peak was supposed to be a kind of accumulated and stable product of the PS II RC's photodamage process and very much possible the Pheo-like molecules.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. I. Structural studies

Reaction centers (RCs) fromRhodopseudomonas sphaeroides were reconstituted into asolectin vesicles by cosonication. Equilibrium centrifugation on sucrose gradients showed that the vesicles were homogeneous in density (i.e., lipid-to-protein ratio) when reconstituted at a molar lipid-to-protein ratio between 500 to 1000. At lower ratios, a considerable fraction of RCs was not incorporated into closed vesicles, while at higher ratios, an increasing population of liposomes was protein-free. The average vesicle size decreased with increasing lipid-to-protein ratio, exhibiting considerable size heterogeneity within a sample. The average diameter of the largest and smallest population of vesicles, reconstituted at a molar lipid-to-protein ratio of 560, was 1200 and 400 nm, respectively. The orientation of reconstituted RCs with respect to the plane of the membrane was determined from the flash-induced rereduction kinetics of the special-pair bacteriochlorophyll dimer in the presence of reduced cytochromec. The predominant orientation of RCs was such that the cytochromec binding sites faced the external medium. The net orientation of RCs in reconstituted vesicles decreased with vesicle size and was strongly influenced by the ionic strength during reconstitution.Abbreviations RC reaction center - LDAO lauryldimethylamine-N-oxide - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - CCCP carbonyl-cyanide-trichloromethoxy phenylhydrazone - D/D⁺ reduced and oxidized form of the primary electron donor of the reaction centers. During the course of this study K. J. H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Excitation energy transfer and charge separation in the isolated Photosystem II reaction center

The nature of excitation energy transfer and charge separation in isolated Photosystem II reaction centers is an area of considerable interest and controversy. Excitation energy transfer from accessory chlorophyll a to the primary electron donor P680 takes place in tens of picoseconds, although there is some evidence that thermal equilibration of the excitation between P680 and a subset of the accessory chlorophyll a occurs on a 100-fs timescale. The intrinsic rate for charge separation at low temperature is accepted to be ca. (2 ps)^–1, and is based on several measurements using different experimental techniques. This rate is in good agreement with estimates based on larger sized particles, and is similar to the rate observed with bacterial reaction centers. However, near room temperature there is considerable disagreement as to the observed rate for charge separation, with several experiments pointing to a ca. (3 ps)^–1 rate, and others to a ca. (20 ps)^-1 rate. These processes and the experiments used to measure them will be reviewed.Abbreviations Chl chlorophyll - FWHM full-width at half-maximum - Pheo pheophytin - PS II Photosystem II - P680 primary electron donor of the Photosystem II reaction center - RC reaction center The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Pigment organization and their interactions in reaction centers of photosystem II: optical spectroscopy at 6 K of reaction centers with modified pheophytin composition

Photosystem II reaction centers (RC) with selectively exchanged pheophytin (Pheo) molecules as described in [Germano, M., Shkuropatov, A. Ya., Permentier, H., Khatypov, R. A., Shuvalov, V. A., Hoff, A. J., and van Gorkom, H. J. (2000) Photosynth. Res. 64, 189-198] were studied by low-temperature absorption, linear and circular dichroism, and triplet-minus-singlet absorption-difference spectroscopy. The ratio of extinction coefficients epsilon(Pheo)/epsilon(Chl) for Q(Y) absorption in the RC is approximately 0.40 at 6 K and approximately 0.45 at room temperature. The presence of 2 beta-carotenes, one parallel and one perpendicular to the membrane plane, is confirmed. Absorption at 670 nm is due to the perpendicular Q(Y) transitions of the two peripheral chlorophylls (Chl) and not to either Pheo. The "core" pigments, two Pheo and four Chl absorb in the 676-685 nm range. Delocalized excited states as predicted by the "multimer model" are seen in the active branch. The inactive Pheo and the nearby Chl, however, mainly contribute localized transitions at 676 and 680 nm, respectively, although large CD changes indicate that exciton interactions are present on both branches. Replacement of the active Pheo prevents triplet formation, causes an LD increase at 676 and 681 nm, a blue-shift of 680 nm absorbance, and a bleach of the 685 nm exciton band. The triplet state is mainly localized on the Chl corresponding to B(A) in purple bacteria. Both Pheo Q(Y) transitions are oriented out of the membrane plane. Their Q(X) transitions are parallel to that plane, so that the Pheos in PSII are structurally similar to their homologues in purple bacteria.     

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm²) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure—polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC?+?QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC?+?QD) complexes.

     

Photochemical and Thermal Phases of Chlorophyll a Fluorescence

The measurement of variable chlorophyll (Chl) a fluorescence is widely used as a convenient and versatile tool in photosynthesis research. In many applications empirical correlations and simplified models of Chl a fluorescence are used with success. Nevertheless, variable Chl a fluorescence provides only indirect and complex image of processes occurring within photosynthetic membranes and such simplifications have only limited validity. In this review we elucidate some controversial and still unresolved questions about the origin and interpretation of the variable Chl a fluorescence induction and the proper use of variable Chl a fluorescence for studies of photochemical events in photosystem 2 (PS2). Although the major part of variable Chl a fluorescence reflects the photochemical closure of the PS2 reaction centers (RCs) and can be considered as a function of the redox state of the primary acceptor Q_A, up to 50 % of the change in the Chl a fluorescence yield can be of secondary, nonphotochemical origin. We review the possible sources of the inherent heterogeneity in the origin of variable Chl a fluorescence. We also comment on the practical implications this bears for the use of variable Chl a fluorescence. This revised version was published online in August 2006 with corrections to the Cover Date.