Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.     

The oligomeric states of the photosystems and the light-harvesting complexes in the Chl b-less mutant

The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.     

Harvesting far-red light: Functional integration of chlorophyll f into Photosystem I complexes of Synechococcus sp. PCC 7002

The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.     

Fluorescence F 0 of photosystems II and I in developing C3 and C4 leaves,and implications on regulation of excitation balance

This work addresses the question of occurrence and function of photosystem II (PSII) in bundle sheath (BS) cells of leaves possessing NADP-malic enzyme-type C₄ photosynthesis (Zea mays). Although no requirement for PSII activity in the BS has been established, several component proteins of PSII have been detected in BS cells of developing maize leaves exhibiting O₂-insensitive photosynthesis. We used the basal fluorescence emissions of PSI (F _0I) and PSII (F _0II) as quantitative indicators of the respective relative photosystem densities. Chl fluorescence induction was measured simultaneously at 680 and 750 nm. In mature leaves, the F _m(680)/F ₀(680) ratio was 10.5 but less in immature leaves. We propose that the lower ratio was caused by the presence of a distinct non-variable component, F _c, emitting at 680 and 750 nm. After F _c was subtracted, the fluorescence of PSI (F _0I) was detected as a non-variable component at 750 nm and was undetectably low at 680 nm. Contents of Chls a and b were measured in addition to Chl fluorescence. The Chl b/(a + b) was relatively stable in developing sunflower leaves (0.25–0.26), but in maize it increased from 0.09 to 0.21 with leaf tissue age. In sunflower, the F _0I/(F _0I + F _0II) was 0.39 ± 0.01 independent of leaf age, but in maize, this parameter was 0.65 in young tissue of very low Chl content (20–50 mg m^?2) falling to a stable level of 0.53 ± 0.01 at Chl contents >100 mg m^?2. The values of F _0I/(F _0I + F _0II) showed that in sunflower, excitation was partitioned between PSII and PSI in a ratio of 2:1, but the same ratio was 1:1 in the C₄ plant. The latter is consistent with a PSII:PSI ratio of 2:1 in maize mesophyll cells and PSI only in BS cells (2:1:1 distribution). We suggest, moreover, that redox mediation of Chl synthesis, rather than protein accumulation, regulates photosystem assembly to ensure optimum excitation balance between functional PSII and PSI. Indeed, the apparent necessity for two Chls (a and b) may reside in their targeted functions in influencing accumulation of PSI and PSII, respectively, as opposed to their spectral differences.     

Enhanced production of hairy root metabolites using microbubble generator

Previously, increased partitioning of the natural product nicotine from tobacco hairy roots into the culture media was achieved by altering the expression of the nicotine uptake permease gene. The present study demonstrated that further increases in nicotine yield in the media were attained by using surfactant-stabilized microbubbles. Compared to other non-ionic surfactants (Tween 20 and Tween 80) and the ionic surfactant SDS, Triton X-100 (TX100) both increased total nicotine production and exudation into the hairy root culture media. In comparison to surfactant-free medium, TX100 at 10, 25, and 50 mg l^?1 did not show strong inhibition of hairy root growth. At 4,000 rpm shear speed, microbubbles stabilized by 10, 25, and 50 mg l^?1 TX100 had k _L a of 22.3, 36.2, and 44.1 h^?1 in Gamborg’s B5 medium, respectively, in comparison to 16.4 h^?1 with conventional air sparging. In a 1-l bioreactor, microbubbles stabilized by TX100 were applied to hairy roots after the inoculated root tips were self-immobilized by branching. With microbubble dispersion, dissolved oxygen rapidly increased from 60 to 85 %, and hairy root growth rate increased. Nicotine accumulation in culture medium with microbubbles reached 146 mg l^?1 after 30 days cultivation. These results show that combining genetic modification with surfactant-stabilized microbubble dispersion can substantially increase levels of nicotine in the media of hairy root cultures.     

Solvent-induced changes in photochemical activity and conformation of photosystem I particles by glycerol

It has been shown that a large number of water molecules coordinate with the pigments and subunits of photosystem I (PSI); however, the function of these water molecules remains to be clarified. In this study, the photosynthetic properties of PSI from spinach were investigated using different spectroscopic and activity measurements under conditions of decreasing water content caused by increasing concentrations of glycerol. The results show that glycerol addition caused pronounced changes in the photochemical activity of PSI particles. At low concentrations (<60%, v/v), glycerol stimulated the rate of oxygen uptake in PSI particles, while higher concentrations of glycerol cause inhibition of PSI activity. The capacity of P700 photooxidation also increased with glycerol concentrations lower than 60%. In contrast, this capacity decreased at higher glycerol concentrations. On the other hand, glycerol addition considerably affected the distribution of the bulk and red antenna chlorophyll (Chl) forms or states, with the population of red-shifted Chl forms augmented with increasing glycerol. In addition, glycerol-treated PSI particles showed a blue shift of the tryptophan fluorescence emission maximum and an increase in their capacity to bind the hydrophobic probe 1-anilino-8-naphthalene sulfonate, indicating a more non-polar environment for tryptophan residues and increased exposure of hydrophobic surfaces.     

Comparison of excitation energy transfer in cyanobacterial photosystem I in solution and immobilized on conducting glass

Excitation energy transfer in monomeric and trimeric forms of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 in solution or immobilized on FTO conducting glass was compared using time-resolved fluorescence. Deposition of PSI on glass preserves bi-exponential excitation decay of ~4–7 and ~21–25 ps lifetimes characteristic of PSI in solution. The faster phase was assigned in part to photochemical quenching (charge separation) of excited bulk chlorophylls and in part to energy transfer from bulk to low-energy (red) chlorophylls. The slower phase was assigned to photochemical quenching of the excitation equilibrated over bulk and red chlorophylls. The main differences between dissolved and immobilized PSI (iPSI) are: (1) the average excitation decay in iPSI is about 11 ps, which is faster by a few ps than for PSI in solution due to significantly faster excitation quenching of bulk chlorophylls by charge separation (~10 ps instead of ~15 ps) accompanied by slightly weaker coupling of bulk and red chlorophylls; (2) the number of red chlorophylls in monomeric PSI increases twice—from 3 in solution to 6 after immobilization—as a result of interaction with neighboring monomers and conducting glass; despite the increased number of red chlorophylls, the excitation decay accelerates in iPSI; (3) the number of red chlorophylls in trimeric PSI is 4 (per monomer) and remains unchanged after immobilization; (4) in all the samples under study, the free energy gap between mean red (emission at ~710 nm) and mean bulk (emission at ~686 nm) emitting states of chlorophylls was estimated at a similar level of 17–27 meV. All these observations indicate that despite slight modifications, dried PSI complexes adsorbed on the FTO surface remain fully functional in terms of excitation energy transfer and primary charge separation that is particularly important in the view of photovoltaic applications of this photosystem.     

The cause of PSI photoinhibition at low temperatures in leaves of Cucumis sativus, a chilling‐sensitive plant

In a chilling-sensitive plant, cucumber, chilling of leaves in the light results in irreversible damage to PSI. Recent in vitro studies suggested that hydroxyl radicals, which are formed in the presence of H₂O₂ and reduced Fe-S centers, are involved in the PSI inhibition. We therefore examined this possibility in vivo. Chilling of leaves at 5°C in the light caused a temporary increase in H₂O₂ concentration, which was probably due to the net H₂O₂ production in vivo. The activity, measured at 5°C, of the thylakoid ascorbate peroxidase (APX), a key enzyme of the H₂O₂-scavenging system, was about 20% of that measured at 25°C. The isolated thylakoids retaining high thylakoid APX activity did not show light-dependent net H₂O₂ production at 25°C. However, at 5°C, net production of H₂O₂ was observed. Since the rate of electron flow to molecular oxygen in the isolated thylakoids was ca 5 mmol e^? mol^?1 Chl s^?1 at 5°C, the H₂O₂-scavenging capacity was below this level. When intact leaves were illuminated at 5°C at an irradiance of 100 µmol m^?2 s^?1, the rate of electron transport through PSII was ca 20 mmol e^? mol^?1 Chl s^?1 and more than 80% of Q_A was in the reduced state. Since thylakoids are uncoupled in cucumber leaves at 5°C in the light. ATP is not formed and energy dissipation in the form of heat is suppressed. Therefore, the electron flow to molecular oxygen would be greater than 5 mmol e^? mol^?1 Chl s^?1. Moreover, under such conditions, components in the electron transport chain, including Fe-S centers in PSI, were probably reduced. These features indicate that, when cucumber leaves are chilled in the light, hydroxyl radicals can be produced by the Fenton reaction and cause damage to PSI.     

Association of chlorophyll a/b-binding Pcb proteins with photosystems I and II in Prochlorothrix hollandica

Action spectra for photosystem II (PSII)-driven oxygen evolution and of photosystem I (PSI)-mediated H₂ photoproduction and photoinhibition of respiration were used to determine the participation of chlorophyll (Chl) a/b-binding Pcb proteins in the functions of pigment apparatus of Prochlorothrix hollandica. Comparison of the in situ action spectra with absorption spectra of PSII and PSI complexes isolated from the cyanobacterium Synechocystis 6803 revealed a shoulder at 650 nm that indicated presence of Chl b in the both photosystems of P. hollandica. Fitting of two action spectra to absorption spectrum of the cells showed a chlorophyll ratio of 4:1 in favor of PSI. Effective antenna sizes estimated from photochemical cross-sections of the relevant photoreactions were found to be 192 ± 28 and 139 ± 15 chlorophyll molecules for the competent PSI and PSII reaction centers, respectively. The value for PSI is in a quite good agreement with previous electron microscopy data for isolated Pcb-PSI supercomplexes from P. hollandica that show a trimeric PSI core surrounded by a ring of 18 Pcb subunits. The antenna size of PSII implies that the PSII core dimers are associated with ∼ 14 Pcb light-harvesting proteins, and form the largest known Pcb-PSII supercomplexes.     

Analysis of the Molecular Organization of Photosystem I During Light-Dependent Chloroplast Differentiation in Mutant C-6D of Scenedesmus obliquus*

Cells of pigment mutant C-6D of the green alga Scenedesmus obliquus synthesize only Chl a and precursors of carotenoids during heterotrophic growth in the dark. These cells exhibit high PSI-activity per Chl and a low Chl/P700-ratio. After transfer to light, Chl a, Chl b and carotenoids are formed with different kinetics. Analysis of chlorophyll fluorescence emission and excitation spectra revealed a sevenfold increase in the amount of the long wavelength antenna of PSI (720 nm) resulting in an increase in the absorption cross section of PSI during illumination. The underlying changes in molecular organization of PSI were investigated by sucrose density centrifugation of solubilized thylakoids after digitonin treatment and subsequent identification of the components by gel electrophoresis, HPLC and fluorescence. In dark grown cells one blue-green band (0-II) could be resolved. This band contained only Chl a and the reaction center complex of PSI, CPI. After 24 hours of illumination three pigmented zones and a small amount of free pigment were observed. One of the zones (24-I) was identified as a light-harvesting fraction containing the pigment-protein complexes LHCP₁ and LHCP₃. In the second fraction (24-II) the reaction center complexes of PSI and PSII were found. The highest molecular weight fraction (24-III) was enriched in PSI-complexes of higher molecular weight and contained a high amount of long wavelength fluorescence antenna (720 nm) attributed to PSI. In contrast to band 24-II which contained a high percentage of β-carotene and a high Chl a/b-ratio, the Chl a/b-ratio of fraction 24-III was lower and the xanthophyll content increased. Our data demonstrate an increase in the PSI-unit size during chloroplast development in mutant C-6D of Scenedesmus obliquus. Dark-grown cultures have small functional PSI-units composed of the chlorophylls involved in charge separation and the core antenna. This unit contains only Chl a and no carotenoids. After transfer to light Chl b and carotenoids are formed. Simultaneously with the appearance of carotenoids and Chl b, PSI-complexes of higher molecular weight are synthesized indicating the addition of a LHC to the reaction center complex of PSI.     

pH dependence of photosynthetic behavior of plant photosystem I particles

The effect of pH on the photosynthetic properties of photosystem I (PSI) particles isolated from spinach chloroplasts were studied using various spectroscopic and activity measurements. The results indicated that the PSI light energy absorption was not affected by changing pH of suspending media. The low-temperature fluorescence yield of the dominating long-wavelength emission band at 734 nm was decreased with increasing pH, whereas it did not exhibit changes in the major peak position at pHs studied except for pH 12, where the major peak in low-temperature chlorophyll (Chl) fluorescence emission spectra was shifted toward the blue light by 5 nm. Pronounced changes were found in PSI photochemical activities. Mild alkalinity (pH 8–10) in suspending media stimulated the rate of oxygen uptake with a maximum activity of oxygen consumption at about pH 9, while the other pHs exhibited an inhibition as compared to the control at pH 7.8. The rate of P700 photooxidation increased with the increasing pH, and the optimum for the reaction activity was in the region of pH 9–11. Circular dichroism spectra revealed that a progressive increase occurred in the conformation of the α-helices as pH value decreased from pH 7.8 to 3.0 or increased from pH 7.8 to 12.0. The results demonstrated that the Chl states in PSI particles were highly stable, while the photochemical activities and protein secondary structures were very sensitive to the pH stimuli of external medium.     

Highly reactive intermediates in photochemistry of chlorophyll

It is shown that chlorophyll (Chl) has photochemical activity in the reduction of O₂ to H₂O₂. In the case of the monomeric forms of Chl, H₂O₂ formation occurs with the participation of ¹O₂, ^⊙OH and O ₂ ^⊙? . Activity of Chl depends on its microenvironment and pH of the system. Associated forms of Chl do not photosensitize ¹O₂ formation, but are active in transfer of electrons to oxygen that leads to H₂O₂ generation. Also it is found that Chl associates are able to act as electron donors to reduce H₂P ₄ ^? and subsequently form hydrogen atom.     

Optical effects of sodium dodecyl sulfate treatment of the isolated light harvesting complex of higher plants

The light-harvesting complex (LHC) of higher plants isolated using Triton X-100 has been studied during its transformation into a monomeric form known as CPII. The change was accomplished by gradually increasing the concentration of the detergent, sodium dodecyl sulfate (SDS). Changes in the red spectral region of the absorption, circular dichroism (CD), and linear dichroism spectra occurring during this treatment have been observed at room temperature. According to a current hypothesis the main features of the visible region absorption and CD spectra of CPII can be explained reasonably successfully in terms of an exciton coupling among its chlorophyll (Chl) b molecules. We suggest that the spectral differences between the isolated LHC and the CPII may be understood basically in terms of an exciton coupling between the Chl b core of a given CPII unit and at least one of the Chla's of either the same or the adjacent CPII. We propose that this Chl a-Chl b coupling existing in LHC disappears upon segregation into CPII, probably as a result of a detergent-related overall rotation of the strongly coupled Chl b core which changes the relative orientations of the two types of pigments and thus the nature of their coupling.Abbreviations Chl Chlorophyll - CD Circular dichroism - LD Linear dichroism - LHC Light-harvesting complex - SDS Sodium dodecyl sulfate - CPII A solubilized form of LHC obtained with SDS polyacrylamide gel electrophoresis Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Structure and function of wild-type and subunit-depleted photosystem I in Synechocystis

The ability of photosynthetic organisms to use the sun's light as a sole source of energy sustains life on our planet. Photosystems I (PSI) and II (PSII) are large, multi-subunit, pigment–protein complexes that enable photosynthesis, but this intriguing process remains to be explained fully. Currently, crystal structures of these complexes are available for thermophilic prokaryotic cyanobacteria. The mega-Dalton trimeric PSI complex from thermophilic cyanobacterium, Thermosynechococcus elongatus, was solved at 2.5?Å resolution with X-ray crystallography. That structure revealed the positions of 12 protein subunits (PsaA-F, PsaI-M, and PsaX) and 127 cofactors.Although mesophilic organisms perform most of the world's photosynthesis, no well-resolved trimeric structure of a mesophilic organism exists. Our research model for a mesophilic cyanobacterium was Synechocystis sp. PCC6803. This study aimed to obtain well-resolved crystal structures of [1] a monomeric PSI with all subunits, [2] a trimeric PSI with a reduced number of subunits, and [3] the full, trimeric wild-type PSI complex. We only partially succeeded with the first two structures, but we successfully produced the trimeric PSI structure at 2.5?Å resolution. This structure was comparable to that of the thermophilic species, but we provided more detail. The PSI trimeric supercomplex consisted of 33 protein subunits, 72 carotenoids, 285 chlorophyll a molecules, 51 lipids, 9 iron-sulfur clusters, 6 plastoquinones, 6 putative calcium ions, and over 870 water molecules.This study showed that the structure of the PSI in Synechocystis sp. PCC6803 differed from previously described PSI structures. These findings have broadened our understanding of PSI structure.     

Changes in the photosynthetic characteristics and photosystem stoichiometries in wild-type and Chl <Emphasis Type="Italic">b</Emphasis>-deficient mutant rice seedlings under various irradiances

By using a wild-type rice (Oryza sativa L. cv. Norin No. 8) and the chlorophyll (Chl) b-deficient mutant derived from Norin No. 8 (chlorina 11), the present study monitored the oxygen evolution, contents of Chl a and b, β-carotene, and lutein in leaf and the contents of cytochrome f, and the reaction centres of photosystem I (PSI) and photosystem II (PSII) in thylakoids. The oxygen evolution, maximal quantum yield of PSII (F_v/F_m) and Chl concentration remained constant in both Norin No. 8 and chlorina 11 under 5 and 2% of full sunlight for six days. On the other hand, on the thylakoid level, the PSII reaction centre of chlorina 11 was more stable even under high irradiance, while approximately 40% decrease in levels of the PSII reaction centre occurred under 2% of full sunlight for six days. However, under such conditions, by regulating the stoichiometry of active PSII and PSI centres, the light absorption balance in both rice types was adjusted between the two photosystems. The present study attempted to examine whether the light absorption balance between PSII and PSI is altered to effectively conduct photosynthesis in the wild-type and Chl b-deficient mutant rice seedlings.     

Excitation wavelength dependence of the fluorescence kinetics in Photosystem I particles from Synechocystis PCC 6803 and Synechococcus elongatus

The excitation-wavelength dependence of the excited-state dynamics of monomeric and trimeric Photosystem I (PSI) particles from Synechocystis PCC 6803 as well as trimeric PSI particles from Synechococcus elongatus has been studied at room temperature using time-resolved fluorescence spectroscopy. For aselective (400 nm), carotenoid (505 nm), and bulk chlorophyll (approximately 650 nm) excitation in all species, a downhill energy-transfer component is observed, corresponding to a lifetime of 3.4-5.5 ps. For selective red excitation (702-719 nm) in all species, a significantly faster, an approximately 1-ps, uphill transfer component was recorded. In Synechococcus PSI, an additional approximately 10-ps downhill energy-transfer component is found for all wavelengths of excitation, except 719 nm. Each of the species exhibits its own characteristic trap spectrum, the shape of which is independent of the wavelength of excitation. This trap spectrum decays in approximately 23 ps in both monomeric and trimeric Synechocystis PSI and in approximately 35 ps in trimeric Synechococcus PSI. The data were simulated based on the 2.5 A structural model of PSI of Synechococcus elongatus using the F?rster equation for energy transfer, and using the 0.6-1-ps charge-separation time and the value of 1.2-1.3 for the index of refraction that were obtained from the dynamics of a hypothetical PSI particle without red chls. The experimentally obtained lifetimes and spectra were reproduced well by assigning three of the chlorophyll-a (chla) dimers observed in the structure to the C708/C702RT pool of red chls present in PSI from both species. Essential for the simulation of the dynamics of Synechococcus PSI is the assignment of the single chla trimer in the structure to the C719/C708RT pool present in this species.     

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI) [A. Wilde, K. Lünser, F. Ossenbühl, J. Nickelsen, T. Börner, Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content, Biochem. J. 357 (2001) 211-216]. With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Δycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Δycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.     

Photosystem I: a search for green plant trimers.

Recent blue-native gel electrophoresis studies gave evidence for the existence of dimeric and trimeric PSI complexes in green plants. We used single particle electron microscopy to investigate all the larger particles from the thylakoid membrane of pea (Pisum sativum var. Charmette). Peak fractions with monomeric, dimeric and trimeric Photosystem I were obtained after solubilization with digitonin and size-exclusion chromatography. The analysis showed that only a few percent of dimers and trimers were present. In the best resolved trimers some of the monomers were oriented upside down. Many classes were fuzzy, indicating a non-specific or flexible orientation. From these results we conclude that the green plant PSI is monomeric within the green plant membrane.     

PSI-SMALP,a Detergent-free Cyanobacterial Photosystem I,Reveals Faster Femtosecond Photochemistry

Cyanobacterial photosystem I (PSI) functions as a light-driven cyt c₆-ferredoxin/oxidoreductase located in the thylakoid membrane. In this work, the energy and charge transfer processes in PSI complexes isolated from Thermosynechococcus elongatus via conventional n-dodecyl-β-D-maltoside solubilization (DM-PSI) and a, to our knowledge, new detergent-free method using styrene-maleic acid copolymers (SMA-PSI) have been investigated by pump-to-probe femtosecond laser spectroscopy. In DM-PSI preparations excited at 740 nm, the excitation remained localized on the long-wavelength chlorophyll forms within 0.1–20 ps and revealed little or no charge separation and oxidation of the special pair, P700. The formation of ion-radical pair P700⁺A1⁻ occurred with a characteristic time of 36 ps, being kinetically controlled by energy transfer from the long-wavelength chlorophyll to P₇₀₀. Quite surprisingly, the detergent-free SMA-PSI complexes upon excitation by these long-wave pulses undergo an ultrafast (<100 fs) charge separation in ∼45% of particles. In the remaining complexes (∼55%), the energy transfer to P₇₀₀ occurred at ∼36 ps, similar to the DM-PSI. Both isolation methods result in a trimeric form of PSI, yet the SMA-PSI complexes display a heterogenous kinetic behavior. The much faster rate of charge separation suggests the existence of an ultrafast pathway for charge separation in the SMA-PSI that may be disrupted during detergent isolation.