Energy equilibration and primary charge separation in chlorophyll d-based photosystem I reaction center isolated from Acaryochloris marina

Primary photochemistry in photosystem I (PS I) reaction center complex from Acaryochloris marina that uses chlorophyll d instead of chlorophyll a has been studied with a femtosecond spectroscopy. Upon excitation at 630 nm, almost full excitation equilibration among antenna chlorophylls and 40% of the excitation quenching by the reaction center are completed with time constants of 0.6(±0.1) and 4.9(±0.6) ps, respectively. The rise and decay of the primary charge-separated state proceed with apparent time constants of 7.2(±0.9) and 50(±10) ps, suggesting the reduction of the primary electron acceptor chlorophyll (A₀) and its reoxidation by phylloquinone (A₁), respectively.     

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

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.     

An electron paramagnetic resonance investigation of the electron transfer reactions in the chlorophyll d containing photosystem I of Acaryochloris marina

Electron paramagnetic resonance (EPR) spectroscopy reveals functional and structural similarities between the reaction centres of the chlorophyll d-binding photosystem I (PS I) and chlorophyll a-binding PS I. Continuous wave EPR spectrometry at 12K identifies iron-sulphur centres as terminal electron acceptors of chlorophyll d-binding PS I. A transient light-induced electron spin echo (ESE) signal indicates the presence of a quinone as the secondary electron acceptor (Q) between P(740)(+) and the iron-sulphur centres. The distance between P(740)(+) and Q(-) was estimated within point-dipole approximation as 25.23+/-0.05A, by the analysis of the electron spin echo envelope modulation.     

Photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina

Fourier transform infrared spectroscopy (FTIR) difference spectroscopy in combination with deuterium exchange experiments has been used to study the photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina. Comparison of (P740(+)-P740) and (P700(+)-P700) FTIR difference spectra show that P700 and P740 share many structural similarities. However, there are several distinct differences also: 1), The (P740(+)-P740) FTIR difference spectrum is significantly altered upon proton exchange, considerably more so than the (P700(+)-P700) FTIR difference spectrum. The P740 binding pocket is therefore more accessible than the P700 binding pocket. 2), Broad, "dimer" absorption bands are observed for both P700(+) and P740(+). These bands differ significantly in substructure, however, suggesting differences in the electronic organization of P700(+) and P740(+). 3), Bands are observed at 2727(-) and 2715(-) cm(-1) in the (P740(+)-P740) FTIR difference spectrum, but are absent in the (P700(+)-P700) FTIR difference spectrum. These bands are due to formyl CH modes of chlorophyll d. Therefore, P740 consists of two chlorophyll d molecules. Deuterium-induced modification of the (P740(+)-P740) FTIR difference spectrum indicates that only the highest frequency 13(3) ester carbonyl mode of P740 downshifts, indicating that this ester mode is weakly H-bonded. In contrast, the highest frequency ester carbonyl mode of P700 is free from H-bonding. Deuterium-induced changes in (P740(+)-P740) FTIR difference spectrum could also indicate that one of the chlorophyll d 3(1) carbonyls of P740 is hydrogen bonded.     

Structure,assembly and energy transfer of plant photosystem II supercomplex

Around photosystem II (PSII), the peripheral antenna system absorbs sunlight energy and transfers it to the core complex where the water-splitting and oxygen-evolving reaction takes place. The peripheral antennae in plants are composed of various light-harvesting complexes II (LHCII). Recently, the three-dimensional structure of the C₂S₂M₂-type PSII-LHCII supercomplex from Pisum sativum (PsPSII) has been solved at 2.7-Å resolution using the single-particle cryo-electron microscopy method. The large homodimeric supercomplex has a total molecular weight of >1400?kDa. Each monomer has a core complex surrounded by strongly and moderately bound LHCII trimers, as well as CP29, CP26, and CP24 monomers. Here, we review and present a detailed analysis of the structural features of this supramolecular machinery. Specifically, we discuss the structural differences around the oxygen-evolving center of PSII from different species. Furthermore, we summarize the existing knowledge of the structures and locations of peripheral antenna complexes, and dissect the excitation energy transfer pathways from the peripheral antennae to the core complex. This detailed high-resolution structural information provides a solid basis for understanding the functional behavior of plant PSII-LHCII supercomplex.     

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.     

Excitation energy transfer in thylakoid membranes from Chlamydomonas reinhardtii lacking chlorophyll b and with mutant Photosystem I

Energy trapping in Photosystem I (PS I) was studied by time-resolved fluorescence spectroscopy of PS II-deleted Chl b-minus thylakoid membranes isolated from site-directed mutants of Chlamydomonas reinhardtii with specific amino acid substitutions of a histidine ligand to P₇₀₀. In vivo the fluorescence of the PS I core antenna in mutant thylakoids with His-656 of PsaB replaced by asparagine, serine or phenylalanine is characterized by an increase in the lifetime of the fast decay component ascribed to the energy trapping in PS I (25 ps in wild type PS I with intact histidine-656, 50 ps in the mutant PS I with asparagine-656 and 70 ps in the mutant PS I with phenylalanine-656). Assuming that the excitation dynamics in the PS I antenna are trap-limited, the increase in the trapping time suggests a decrease in the primary charge separation rate. Western blot analysis showed that the mutants accumulate significantly less PS I than wild type. Spectroscopically, the mutations lead to a decrease in relative quantum yield of the trapping in the PS I core and increase in relative quantum yield of the fluorescence decay phase ascribed to uncoupled chlorophyll–protein complexes which suggests that improper assembly of PS I and LHC in the mutant thylakoids may result in energy uncoupling in PS I.     

Oligomerization and pigmentation dependent excitation energy transfer in fucoxanthin-chlorophyll proteins

The ultrafast caroteonid to chlorophyll a energy transfer dynamics of the isolated fucoxanthin-chlorophyll proteins FCPa and FCPb from the diatom Cyclotella meneghiniana was investigated in a comprehensive study using transient absorption in the visible and near infrared spectral region as well as static fluorescence spectroscopy. The altered oligomerization state of both antenna systems results in a more efficient energy transfer for FCPa, which is also reflected in the different chlorophyll a fluorescence quantum yields. We therefore assume an increased quenching in the higher oligomers of FCPb. The influence of the carotenoid composition was investigated using FCPa and FCPb samples grown under different light conditions and excitation wavelengths at the blue (500 nm) and red (550 nm) wings of the carotenoid absorption. The different light conditions yield in altered amounts of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Since no significant dynamic changes are observed for high light and low light samples, the contribution of the xanthophyll cycle pigments to the energy transfer is most likely negligible. On the contrary, the observed dynamics change drastically for the different excitation wavelengths. The analyses of the decay associated spectra of FCPb suggest an altered energy transfer pathway. For FCPa even an additional time constant was found after excitation at 500 nm. It is assigned to the intrinsic lifetime of either the xanthophyll cycle carotenoids or more probable the blue absorbing fucoxanthins. Based on our studies we propose a detailed model explaining the different excitation energy transfer pathways in FCPa.     

Structure and function in the isolated reaction center complex of Photosystem II: energy and charge transfer dynamics and mechanism

The dynamics of energy and charge transfer in the Photosystem II reaction center complex is an area of great interest today. These processes occur on a time scale ranging from femtoseconds to tens of picoseconds or longer. Steady-state and ultrafast spectroscopy techniques have provided a great deal of quantitative and qualitative data that have led to varied interpretations and phenomenological models. More recently, microscopic models that identify specific charge separated states have been introduced, and offer more insight into the charge transfer mechanism. The structure and energetics of PS II reaction centers are reviewed, emphasizing the effects on the dynamics of the initial charge transfer. This revised version was published online in June 2006 with corrections to the Cover Date.     

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002

Phycobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.     

Photosystem II reaction center with altered pigment-composition: reconstitution of a complex containing five chlorophyll a per two pheophytin a with modified chlorophylls

Pigment-depleted Photosystem II reaction centers (PS II-RCs) from a higher plant (pea) containing five chlorophyll a (Chl) per two pheophytin a (Phe), were treated with Chl and several derivatives under exchange conditions [FEBS Lett. 434 (1998) 88]. The resulting reconstituted complexes were compared to those obtained by pigment exchange of “conventional” PS II-RCs containing six Chl per two Phe. (1) The extraction of one Chl is fully reversible. (2) The site of extraction is the same as the one into which previously extraneous pigments have been exchanged, most likely the peripheral D1-H118. (3) Introducing an efficient quencher (Ni-Chl) into this site results in only 25% reduction of fluorescence, indicating incomplete energy equilibration among the “core” and peripheral chlorophylls.     

Analysis of the electron transfer from Pheo to QA in PS II membrane fragments from spinach by time resolved 325 nm absorption changes in the picosecond domain

Absorption changes at 325 nm (delta A325) induced by 15 ps laser flashes (lambda = 650 nm) in PS II membrane fragments were measured with picosecond time-resolution. In samples with the reaction centers (RCs) kept in the open state (P I QA) the signals are characterized by a very fast rise (not resolvable by our equipment) followed by only small changes within our time window of 1.6 ns. In the closed state (PI QA-) of the reaction center the signal decays with an average half-life time of about 250 ps. It is shown that under our excitation conditions (E = 2 x 10(14) photons/cm2 per pulse) subtraction of the absorption changes in closed RCs (delta A closed 325) from those in open RCs (delta A open 325) leads to a difference signal which is dominated by the reduction kinetics of QA. From the rise kinetics of this signal and by comparison with data in the literature it is inferred that QA becomes reduced by direct electron transfer from Pheo- with a time constant of about 350 +/- 100 ps.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Study on energy transfer between carotenoid and chlorophyll a in cytochrome b 6 f complex from Bryopsis corticulans

The excitation energy transfer between carotenoid and chlorophyll (Chl) in the cytochrome b ₆ f complex from Bryopsis corticulans (B. corticulans), in which the carotenoid is 9-cis-α-carotene, was investigated by means of fluorescence excitation and sub-microsecond time-resolved absorption spectroscopies. The presence of efficient singlet excitation transfer from α-carotene to Chl a was found with an overall efficiency as high as ∼ ∼24%, meanwhile the Chl a-to-α-carotene triplet excitation transfer was also evidenced. Circular dichroism spectroscopy showed that α-carotene molecule existed in an asymmetric environment and Chl a molecule had a certain orientation in this complex.Bin-Xing Li and Ping Zuo contributed equally to this work.     

On the efficiency of energy transfer and the different pathways of electron transfer in mutant reaction centers of Rhodobacter sphaeroides

The efficiency of energy transfer from the monomeric pigments to the primary donor was determined from 77 K steady-state fluorescence excitation spectra of three mutant reaction centers, YM210L, YM210F and LM160H / FM197H. For all three reaction centers this efficiency was not 100% and ranged between 55 and 70%. For the YM210L mutant it was shown using pump-probe spectroscopy with B band excitation at 798 nm that the excitations which are not transferred to P give rise to efficient charge separation. The results can be interpreted with a model in which excitation of the B absorbance band leads to direct formation of the radical pair state B_A ⁺H _A ^– in addition to energy transfer to P. It is also possible that some P⁺B_A ^– is formed from B^*. In previous publications we have demonstrated the operation of such alternative pathways for transmembrane electron transfer in a YM210W mutant reaction center [van Brederode et al. (1996) The Reaction center of Photosynthetic Bacteria, pp 225–238; (1997a,b) Chem Phys Lett 268: 143–149; Biochemistry 36: 6855–6861]. The results presented here demonstrate that these alternative mechanisms are not peculiar to the YM210W reaction center.     

Spectral dependence of energy transfer in wild-type peripheral light-harvesting complexes of photosynthetic bacteria

The precise position of the upper exciton component and relevant vibronic transitions of the B850 ring in peripheral light-harvesting complexes from purple photosynthetic bacteria are important values for determining the exciton bandwidth and electronic structure of the B850 ring. To determine the presence of these components in wild-type LH2 complexes the pump-probe femtosecond transient spectra obtained with excitation into the 730-840 nm spectral range are analyzed. We show that at excitation wavelengths less than 780 nm B850 absorption bands are present and that, in accordance with exciton theory, these bands peak further in the blue when the lowest optically allowed transition is more red-shifted.