Reaction Center Complex Of Photosystem Research Articles

Structural and functional analysis of a novel galactose-binding lectin derived from Chlorella sorokiniana MW769776

A freshwater green microalgal strain was isolated and the lectin was identified in it by a strong hemagglutination activity (HA) assay. Characterization of the algal strain was found to be Chlorella sorokiniana (MW769776). A single step affinity chromatographic technique was developed to purify Chlorella sorokiniana lectin (CSL) using guar gum as the affinity matrix. The precipitate showed a single active peak with a titer value of 1024 HU, with a concentration of 1111 U, and a purification fold of 9. The purified protein exhibited a single band in SDS-PAGE with a molecular weight of 16 kDa. Analysis by liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (LC-ESI-Q-TOF-MS) of tryptic-digested purified lectin showed that it was a monomeric protein. A multiple sequence alignment analysis revealed that the peptide sequences of CSL exhibited similarity with the H-type lectin domain of Micractinium conductrix. The structure of CSL was studied by FTIR and homology modeling methods, indicating the presence of α-helix as well as β-sheet in its secondary structure. Whereas the 3D structure exhibited the similarity with the core protein of light-harvesting reaction center complex of photosystem I. The significance of this study suggests that the characteristics of CSL are consistent with its identification as a hemagglutinin, a type of novel lectin, which suggests its candidature for various biological purposes. Communicated by Ramaswamy H. Sarma

Non-conservative circular dichroism of photosystem II reaction centers: Is there an enhancement by a coupling with charge transfer states?

The non-conservative nature of the circular dichroism (CD) spectrum of the Qy spectral region of the D1/D2/Cytb559 reaction center complexes of photosystem II is investigated. Our theory, in addition to the usual excitonic couplings between the Qy transitions of the chlorophyll and pheophytin pigments, takes into account the coupling of the Qy with the higher-energy Bx, By and Nx+xy transitions of these pigments and with S0→S2 transitions of the two β-carotene pigments close to the reaction center. The higher-energy transitions are parameterized by quantum chemical calculations and an analysis of the experimental oscillator strength of the isolated pigments. The excitonic couplings between pigments are obtained with the Poisson-TrEsp method. The coupling between exciton and charge transfer states in the central PD1-PD2 dimer is implicitly taken into account by a scaling factor of the excitonic coupling. The observed non-conservativity of the Qy CD spectrum can be qualitatively understood by the above described mixing of Qy and higher-energy transitions. The analysis suggests that the non-conservativity may be enhanced by coupling to charge transfer states. We expect that the present study will stimulate explicit microscopic studies of the charge transfer states in the reaction center of photosystem II.

Asymmetry in Charge Transfer Pathways Caused by Pigment–Protein Interactions in the Photosystem II Reaction Center Complex

This article discusses the photoinduced charge transfer (CT) kinetics within the reaction center complex of photosystem II (PSII RC). The PSII RC exhibits a structural symmetry in its arrangement of pigments forming two prominent branches, D1 and D2. Despite this symmetry, the CT has been observed to occur exclusively in the D1 branch. The mechanism to realize such functional asymmetry is yet to be understood. To approach this matter, we applied the theoretical tight-binding model of pigment excitations and simulated CT dynamics based upon the framework of an open quantum system. This simulation used a recently developed method of computation based on the quasi-adiabatic propagator path integral. A quantum CT state is found to be dynamically active when its site energy is resonant with the exciton energies of the PSII RC, regardless of the excitonic landscape we utilized. Through our investigation, it was found that the relative displacement between the local molecular energy levels of pigments can play a crucial role in realizing this resonance and therefore greatly affects the CT asymmetry in the PSII RC. Using this mechanism phenomenologically, we demonstrate that a near 100-to-1 ratio of reduction between the pheophytins in the D1 and D2 branches can be realized at both 77 K and 300 K. Our results indicate that the chlorophyll Chl D 1 is the most active precursor of the primary charge separation in the D1 branch and that the reduction of the pheophytins can occur within pico-seconds. Additionally, a broad resonance of the active CT state implies that a large static disorder observed in the CT state originates in the fluctuations of the relative displacements between the local molecular energy levels of the pigments in the PSII RC.

Perturbation of the Structure of P680 and the Charge Distribution on Its Radical Cation in Isolated Reaction Center Complexes of Photosystem II as Revealed by Fourier Transform Infrared Spectroscopy

The structure and the electronic properties of P680 and its radical cation in photosystem II (PSII) were studied by means of Fourier transform infrared spectroscopy (FTIR). Light-induced P680+/P680 FTIR difference spectra in the mid- and near-IR regions were measured using PSII membranes from spinach, core complexes from Thermosynechococcus elongatus, and reaction center (RC) complexes (D1-D2-Cytb559) from spinach. The spectral features of the former two preparations were very similar, indicating that the structures of P680 and its radical cation are virtually identical between membranes and cores and between plants and cyanobacteria. In sharp contrast, the spectrum of the RC complexes exhibited significantly different features. A positive doublet at approximately 1724 and approximately 1710 cm-1 due to the 131-keto C=O stretches of P680+ in the membrane and core preparations were changed to a prominent single peak at 1712 cm-1 in the RC complexes. This observation was interpreted to indicate that a positive charge on P680+ was extensively delocalized over the chlorophyll dimer in RC, whereas it was mostly localized on one chlorophyll molecule (70-80%) in intact P680. The significant change in the electronic structure of P680+ in RC was supported by a dramatic change in the characteristics of a broad intervalence band in the near-IR region and relatively large shifts of chlorin ring bands. It is proposed that the extensive charge delocalization in P680+ mainly causes the decrease in the redox potential of P680+/P680 in isolated RC complexes. This potential decrease explains the well-known phenomenon that YZ is not oxidized by P680+ in RC complexes.

Photosystem Damage and Spatial Architecture of Thylakoids in Chloroplasts of Pea Chlorophyll Mutants

The effect of chlorophyll-protein complexes on the ultrastructure of chloroplasts was studied in the leaves of pea, the parent cultivar Torsdag and mutants chlorotica 2004 and 2014. The mutants were shown to accumulate 80 and 55% of chlorophyll, relative to the control, while the composition of the synthesized photosystem complexes was the same as in the parent cultivar Torsdag. The size of the light-harvesting antenna was similar to the control in the 2014 mutant but considerably increased (by 30%) in the 2004 mutant. These changes were due to a proportional decrease in the number of all complexes (by 40-45%) in the 2014 mutant. At the same time, the number of reaction center complexes of photosystem I (PS I) decreased by 50% while that of photosystem II (PS II) remained virtually constant in the 2004 mutant. A proportional decrease in the number of the PS I and PS II complexes in the chlorotica 2014 mutant was accompanied by a partial reduction of the entire chloroplast membrane system against the background of normal development of both granal and intergranal sites of thylakoids. Conversely, the loss of PS I reaction centers led mainly to the reduction of the intergranal sites of thylakoids in chloroplasts. This effect is attributed to the prevalence of PS I complexes in the intergranal thylakoids.

Chlorophyll triplet states associated with photosystem II of thylakoids.

The analysis of FDMR thylakoid spectra, determined at multiple emission wavelengths, by a global decomposition technique, has revealed the presence of three previously undescribed triplet populations at emission wavelengths characteristic of Photosystem II chlorophyll/protein complexes. Their zero-field splitting parameters have been determined in order to compare them with the well-studied PSII recombination triplet state. None of these triplets have the zero-field splitting parameters characteristic of the recombination triplet and are therefore probably not generated directly in the reaction center. On the basis of their microwave-induced emission spectra, it is suggested that two are probably generated in the core complex(es) while the third may be generated in the external antenna. These triplets are formed under nonreducing redox conditions, when the recombination triplet is undetectable. It is suggested that they may be involved in the photoinhibitory damage of Photosystem II. The triplet-minus-singlet spectrum associated with the recombination triplet state has been determined for thylakoids after reduction of the secondary acceptors. Its main peak is at 685 nm, slightly red shifted with respect to earlier reports, with a weak signal, of opposite sign at approximately 675 nm. The 685 nm peak indicates that at cryogenic temperatures, the triplet is located on the long-wavelength chlorophyll state present in the reaction center complex of Photosystem II (D1.D2.Cytb(559) complex). From the absence of a clear structure in the 680 nm absorption region, this long-wavelength absorbing state does not appear to be strongly coupled to P(680), though it must be associated with one of the "inner core" pigments recently identified in the photosystem II crystallographic structure [Zouni et al. (2001) Nature 408, 739-743].

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.

Comparison of the Excited-State Dynamics of Five- and Six-Chlorophyll Photosystem II Reaction Center Complexes

Spectral hole-burning experiments have been performed at liquid He temperature on the Qy-band of isolated reaction center complexes of photosystem II (PS II RC) containing five (RC-5) and six (RC-6) chlorophyll a (Chl a) molecules. The aim was to investigate the nature of the redmost shoulder in the absorption spectrum of RC-5 and to identify distributions of “trap” pigments. The “effective” homogeneous line width was measured at 682 nm as a function of temperature between 1.2 and 4.2 K. It follows a T1.3±0.1 power law in both complexes and extrapolates to the fluorescence lifetime-limited value, τfl = (4 ± 1) ns, for T → 0. These results indicate that the redmost absorbing pigments act as “traps” for the excitation energy. The spectral distribution of these traps was reconstructed from the hole depth measured as a function of excitation wavelength λexc and compared to that of RC-6 previously obtained by us (Groot, M. L.; et al. J. Phys. Chem. 1996, 100, 11488). The maximum of the RC-5 trap distribution l...

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH − 4). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH − 4 in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13 1-deoxo-13 1-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo − and P680 +. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Q y and Q x transitions of this molecule were determined to be located at 5°C at 679.5–680 nm and 542 nm, respectively.

Structure and Function in the Isolated Reaction Center Complex of Photosystem II. 1. Ultrafast Fluorescence Measurements of PSII

Time-resolved fluorescence measurements of PSII D1−D2−Cytb559 reaction center complexes were made by using an optical signal analyzer based on a streak camera tube synchronized with subpicosecond jitter to a femtosecond 1 kHz titanium:sapphire laser system. The instrumental response function of this analyzer was 4−4.5 ps fwhm under experimental conditions. Synchronization permitted extensive signal averaging and allowed measurements to be made with low incident pulse energy (3−10 nJ) and photon flux ((1.5−5.0) × 1013 photons/cm2). Fluorescence decays were obtained following excitation throughout the Qy band of PSII. The data are inconsistent with an effective or apparent charge separation time scale of ca. 3 ps following direct excitation of P680. Rather, the effective charge separation from a quasi-equilibrium of chromophore excited states is either quite fast (≤1.25 ps) or relatively slow (≥20 ps). The most consistent interpretation of the available data involves rapid energy redistribution processes wi...

Primary Processes and Structure of the Photosystem II Reaction Center. 4. Low-Intensity Femtosecond Transient Absorption Spectra of D1-D2-cyt-b559 Reaction Centers,

Low-intensity transient absorption spectroscopy has been performed on the isolated D1-D2-cyt-b559 reaction center complex of photosystem II from spinach. The excitation intensity was low enough to keep annihilation at a relatively low level (∼11%) and still maintain a very high signal/noise ratio. The kinetics has been measured with ∼200 fs resolution over two time ranges extending up to 200 ps for two different excitation wavelengths (680 nm, preferential primary donor excitation, and 670 nm, preferential external chlorophyll excitation). Detection wavelengths both in the pheophytin Qx region (535−555 nm) and in the chlorin Qy region, including the stimulated emission range (660−760 nm) have been employed. The data for all excitation/detection wavelength pairs and from both time ranges have been analyzed by combined global analysis. A highly complex kinetics, i.e. six lifetimes, has been found necessary for a good description of the data over the entire excitation/detection matrix and time range. These l...

Pressure dependence of primary charge separation in a photosynthetic reaction center

Abstract Spectral hole burning is used to study the pressure dependence of the Qy absorption spectrum and primary charge separation kinetics of the D1–D2-cyt b559 reaction center complex of photosystem II. The 4.2 K lifetime of P680∗, the primary donor state, lengthens from 2.0 ps at 0.1 MPa to 7.0 ps at 267 MPa. Importantly, this effect is irreversible (plastic), in sharp contrast with the elastic effects of pressure on the low-temperature absorption and non-line-narrowed hole spectrum of P680. These observations and data which show that the electron-phonon coupling is weakly dependent on pressure, suggest a model that has the plastic behavior of charge separation kinetics due mainly to the pressure dependence of the energy of the acceptor state and of the variance of the P680∗-acceptor energy gap stemming from structural heterogeneity. Nonadiabatic rate expressions, which take into account the distribution of energy gap values, are used to estimate the linear pressure shift of the acceptor state energy for both the superexchange and two-step mechanisms for primary charge separation. For both mechanisms shifts in the vicinity of 1 cm−1/MPa are required to explain the data, a value which is not unreasonable based on pressure dependent studies of other systems. The results point to the marriage of hole burning and high pressures as having considerable potential for the study of primary transport dynamics in reaction center and antenna complexes.

Optoacoustic and Singlet Oxygen Near-IR Emission Study of the Isolated D1-D2-cyt b-559 Reaction Center Complex of Photosystem II. Protein Movement Associated with Charge Separation

Optoacoustic and Singlet Oxygen Near-IR Emission Study of the Isolated D1-D2-cyt b-559 Reaction Center Complex of Photosystem II. Protein Movement Associated with Charge Separation

Gaussian band analysis of absorption, fluorescence and photobleaching difference spectra of D1/D2/cytb-559 complex

A study of the absorption and fluorescence characteristics of the D1/D2/cytb-559 reaction centre complex of Photosystem II has been carried out by gaussian decomposition of absorption spectra both at room temperature and 72 K and of the room temperature fluorescence spectrum. A five component fit was found in which the absorption and fluorescence sub-bands could be connected by the Stepanov relation. The photobleaching and light-activated degradation in the dark of long wavelength pigments permitted a further characterisation of the absorption bands. The absorption (fluorescence) maxima of the five bands at room temperature are 660 nm (670 nm), 669 nm (675 nm), 675 nm (681 nm), 680 nm (683 nm), 681 nm (689 nm). A novel feature of this analysis is the presence of two approximately isoenergetic absorption bands near 680 nm at room temperature. The narrower one (FWHM=12.5 nm) is attributed to pheophytin while the broader band (FWHM=23 nm) is thought to be P680. The P680 band width is discussed in terms of homogeneous and site inhomogeous band broadening. The P680 fluorescence has a large Stokes shift (≈9 nm) and most fluorescence in the 690-700 nm range is associated with this chromophore.The three accessory pigment bands are broad (FWHM=17-24 nm) and the 660 nm gaussian is largely temperature insensitive thus indicating significant site inhomogeneous broadening.The very slight narrowing of the D1/D2/cytb-559 Qy absorption at crytogenic temperatures is discussed in terms of the coarse spectral inhomogeneity associated with the spectral forms and the apparently large site inhomogeneous broadening of short wavelength accessory pigments.

Site-Selection Spectroscopy of the Reaction Center Complex of Photosystem II. 2. Identification of the Fluorescing Species at 4 K

Fluorescence emission spectra at 4 K of the D1-D2-Cytb559 reaction center complex of photosystem II, presumably containing six chlorophyll a (Chl a) and two pheophytin a (Pheo a) molecules, were obtained by selective excitation with a tunable, continuous laser ([lambda][sub exc] = 680-691 mm). The bands in the vibrational region (700-800 nm) showed a characteristic structure, which could be used as a [open quotes]fingerprint[close quotes] to identify the emitting pigment at low temperature. By comparison of this [open quotes]fingerprint[close quotes] with those of monomeric Chl a and Pheo a in Triton X-100 detergent, it is unambiguously shown that the steady-state emission arises from a Chl a species for [lambda][sub exc] > 683 nm. The nonselectively excited emission spectrum ([lambda][sub exc] < 679 nm) reveals primarily Chl a emission bands, peaking at 683.7 [+-] 0.3 and 744.0 [+-] 0.5 nm. The shape of this spectrum is significantly different from that of earlier reported emission spectra of D1-D2-Cytb559 complexes with presumably a lower Chl a content (four to five Chl/2 Pheo). These show characteristic bands for Pheo a emission. We conclude that D1-D2-Cytb559 complexes with a higher Chl a content (6 Chl/2 Pheo) contain an additional long-wavelength (680-683 nm) Chl a molecule,more » which is not related to P-680. The steady-state fluorescence at low temperatures mainly arises from emission of this Chl and not from charge-recombination fluorescence of P-680. 28 refs., 5 figs., 1 tab.« less

The isolated D1/D2/cyt b-559 reaction centre complex of Photosystem II possesses a serine-type endopeptidase activity

The isolated D1/D2/cyt b-559 reaction centre complex of Photosystem II has been shown to possess an endopeptidase activity which has been investigated using synthetic substrates and proteinase inhibitors. Substrate specificity and inhibition by serine type proteinase inhibitors suggest that the endopeptidase is of a serine type. Divalent cations, especially calcium, enhanced the activity of the intrinsic peptidase which also showed pH and temperature optima of 8.2 and 37°C respectively. Treatment with strong light had little or no effect on the enzyme activity.

Comparison of the Dl/D2/cytochrome b559 reaction centre complex of photosystem two isolated by two different methods

Photosystem 2 reaction centre complexes prepared either by solubilisation with Triton X-100 and subsequent exchange into dodecyl maltoside or by a procedure involving a combination of dodecyl maltoside and LiC10 4, were characterised in terms of chlorophyll a, pheophytin a, β-carotene and cytochrome b559 content. Time-resolved chlorophyll fluorescence decay kinetics were measured using both types of complexes. Our data show that the isolated photosystem two reaction centre complex contain, for two pheophytin a molecules, close to six chlorophyll a, two β-carotene and one cytochrome b559. No major differences were observed in the composition or the kinetic characteristics measured in the samples prepared by the different procedures. Time-resolved fluorescence measurements indicate that more than 94% of the chlorophyll a in both preparations is coupled to the reaction centre complex.

The interaction between the 33 kDa manganese-stabilising protein and the D 1/D 2cytochrome b-559 complex

Using affinity chromatography with the extrinsic 33 kDa protein as the immobilised ligand, it was demonstrated that the reaction centre complex of photosystem II, composed of the D 1, D 2 and cytochrome b-559 polypeptides, can directly interact with the 33 kDa protein. By this approach it was possible to purify the reaction centre from solubilised photosystem II core complexes since neither the 47 kDa nor the 43 kDa protein would bind to the ligand.

Analysis of the relationship between the extrinsic 30-kDa protein, manganese and oxygen evolution in the thylakoid of Chlamydomonas reinhardtii grown under manganese-deficient conditions

The content and stability of an extrinsic protein in the thylakoid of a green alga Chlamydomonas reinhardtii which has a relative molecular weight of 30 000 and corresponds to the ‘33-kDa’ protein involved in oxygen evolution in spinach chloroplast were studied with the cells grown in a manganese-deficient medium. The amount of the 30-kDa protein in the thylakoid did not change even when the content of manganese in the membrane was decreased significantly and the activity of oxygen evolution was reduced concomitantly under manganese-depleted conditions. The stability of the 30-kDa protein in the membrane, which was determined by the susceptibility of the protein to the treatment with an alkaline buffer and sonication, also showed no significant change between the cells grown with and without manganese. The thylakoid membrane of the cells grown under manganese-deficient conditions was devoid of the loosely bound manganese atoms located at the hydrophilic environment of the membrane probably at the interface between the 30-kDa protein and the reaction center complex of Photosystem II, judging from the partitioning behavior of manganese atoms on butanol/water phase partitioning of the thylakoid. These results suggest that the manganese atoms involved in oxygen evolution are not required for the binding of the 30-kDa protein to the Photosystem II membrane.

In vitro transcription and translation of the psbD gene encoding the D-2 protein of photosystem II in barley.

The plastoquinone-binding polypeptide D-2 of the reaction center complex of photosystem II in barley has been expressed in vitro using the expression vector pGem3 containing the psbD gene of chloroplast DNA as template. The full length D-2 polypeptide with an apparent mol. weight of 32 kD is a major product when mRNA is transcribed with SP6 RNA polymerase from an insert containing the psbD gene with a 36 bp 5' leader sequence and a 3' tail of 99 bp downstream of the stop codon. Translation of the mRNA had to be done in a rabbit reticulocyte lysate. Translation also occurred from the internal AUG codons giving rise to truncated D-2 polypeptides with sizes of 30, 25 and 17 kD. They are immunoprecipitable with antisera either raised against amino acid residues 235 to 241 or against 345 residues of the D-2 polypeptide. A truncated translation product of 12 kD probably initiated at codon 247 is not recognized by either antiserum. An additional immunoprecipitable protein with an apparent molecular weight of 46 kD is observed by SDS-PAGE and is interpreted as a homomeric aggregate of D-2 polypeptides. Upon addition of methanol solubilized 2,6 dichloro-p-benzoquinone or other quinones a preferential translation of the full-length and truncated D-2 polypeptides is observed. The use of in vitro synthesized D-2 polypeptides for studies of binding quinones, other electron carriers and chlorophyll chromophores is discussed.