Chlorophyll Pair Research Articles

Correlation of electronic carotenoid–chlorophyll interactions and fluorescence quenching with the aggregation of native LHC II and chlorophyll deficient mutants

The aggregation dependent correlation between fluorescence quenching and the electronic carotenoid–chlorophyll interactions, ϕ Coupling Car S 1 - Chl , as measured by comparing chlorophyll fluorescence observed after two- and one-photon excitation, has been investigated using native LHC II samples as well as mutants lacking Chl 2 and Chl 13. For native LHC II the same linear correlation between ϕ Coupling Car S 1 - Chl and the fluorescence quenching was observed as previously reported for the pH and Zea-dependent quenching of LHC II [1]. In order to elucidate which carotenoid–chlorophyll pair might dominate this correlation we also investigated the mutants lacking Chl 2 and Chl 13. However, also with these mutants the same linear correlation as for native LHC II was observed. This provides indication that these two chlorophylls play only a minor role for the observed effects. Nevertheless, we also conclude that this does not exclude that their neighboured carotenoids, lutein 1 and neoxanthin, might interact electronically with other chlorophylls close by.

Quantum chemical excited state calculations on pigment–protein complexes require thorough geometry re-optimization of experimental crystal structures

Calculations of vertical excited states of a strongly coupled chlorophyll pair and a carotenoid–chlorophyll complex stemming from the light harvesting complex II of green plants employing the experimentally determined crystal structures and quantum chemically re-optimized model complexes demonstrate the need for preceding re-optimizations of the experimental structures at quantum chemical level. While in the case of the chlorophyll dimers, the re-optimization step is crucial for a correct description of the coupling of the excited states, in carotenoid–chlorophyll complexes the S1 excitation energies of carotenoids depend strongly on the structure, in particular on the correct bond lengths alternation pattern of its conjugated double bond chain, which is not sufficiently accurately reproduced by experimental structures.

Impact of dimeric organization of enzyme on its function: the case of photosynthetic water splitting

It is a question of whether the supramolecular organization of the protein complex has an impact on its function, or not. In the case of the photosystem II (PSII), water splitting might be influenced by cooperation of the PSIIs. Since PSII is the source of the atmospheric oxygen and because better understanding of the water splitting may contribute to the effective use of water as an alternative energy source, possible cooperation should be analyzed and discussed. We suggest that the dimeric organization of the PSII induces cooperation in the water splitting. We show that the model of monomeric PSII is unable to produce the oxygen after the second short flash (associated with the double turnover of the PSII), in contrast to the experimental data and model of dimeric PSII with considered cooperation. On the basis of this fact and partially from the support from other studies, we concluded that the double turnover of the PSII induced by short flashes might be caused by the cooperation in the water splitting. We further discuss a possibility that the known pathway of the electron transport through the PSII might be incomplete and besides D1-Y161, other cofactor which is able to oxidize the special chlorophyll pair (P680) must be considered in the monomeric PSII to explain the oxygen production after the second short flash. Commented SBML codes (.XML files) of the monomeric and dimeric PSII models will be available (at the time of publication) in the BioModels database (www.ebi.ac.uk/biomodels).

Covalently linked zinc chlorophyll dimers as a model of a chlorophyllous pair in photosynthetic reaction centers

A heterodimer, where zinc pyropheophorbide-a was linked with zinc pyropheophorbide-d through ethylene glycol diester, was prepared, as well as the corresponding homodimers. The synthetic dimers were complexed with methanol in benzene to give folded dimers by mutual Zn...O(Me)-H...O=C13(1) bonding. Such complexes had furthest red (Qy) absorption bands at longer wavelengths than the monomeric species. These red-shifts were ascribable to excitonic coupling of the Qy transition states in the chlorin pi-pi stacking conformer. In the heterodimeric system, a minor band was observed at the shorter wavelength side of the main Qy band. This observation can be explained by an additional contribution of Qy vibronic state to the exciton-coupled states. Based on the experimental results, a pair of chlorophyll(Chl)-d with Chl-a as well as a Chl-d homopair were proposed as dimers in reaction centers of Chl-d dominating cyanobacteria.

Semi-continuum electrostatic calculations of redox potentials in photosystem I

The midpoint redox potentials (E(m)) of all cofactors in photosystem I from Synechococcus elongatus as well as of the iron-sulfur (Fe(4)S(4)) clusters in two soluble ferredoxins from Azotobacter vinelandii and Clostridium acidiurici were calculated within the framework of a semi-continuum dielectric approach. The widely used treatment of proteins as uniform media with single dielectric permittivity is oversimplified, particularly, because permanent charges are considered both as a source for intraprotein electric field and as a part of dielectric polarizability. Our approach overcomes this inconsistency by using two dielectric constants: optical epsilon(o)=2.5 for permanent charges pre-existing in crystal structure, and static epsilon(s) for newly formed charges. We also take into account a substantial dielectric heterogeneity of photosystem I revealed by photoelectric measurements and a liquid junction potential correction for E(m) values of relevant redox cofactors measured in aprotic solvents. We show that calculations based on a single permittivity have the discrepancy with experimental data larger than 0.7 V, whereas E(m) values calculated within our approach fall in the range of experimental estimates. The electrostatic analysis combined with quantum chemistry calculations shows that (i) the energy decrease upon chlorophyll dimerization is essential for the downhill mode of primary charge separation between the special pair P(700) and the primary acceptor A(0); (ii) the primary donor is apparently P(700) but not a pair of accessory chlorophylls; (iii) the electron transfer from the A branch quinone Q(A) to the iron-sulfur cluster F(X) is most probably downhill, whereas that from the B branch quinone Q(B) to F(X) is essentially downhill.

Separation of Light-induced Linear, Cyclic and Stroma-sourced Electron Fluxes to P700+ in Cucumber Leaf Discs after Pre-Illumination at a Chilling Temperature

Pre-illumination of cucumber leaf discs at 4 degrees C with low-irradiance white light (i) led to a marked decrease in the extent of photo-oxidation of P700 (the special chlorophyll pair in the PSI reaction center) in actinic light at room temperature and (ii) hastened the post-illumination re-reduction of P700+. Quantifying the linear, cyclic and stroma-sourced electron fluxes to P700+ in two actinic light regimes, we found that there was no increase in cyclic or linear electron fluxes to account for these changes. Rather, we observed a decrease in the maximum extent of P700 photo-oxidation assayed by a strong flash superimposed on continuous, background light of wavelength 723 nm, which we interpret to represent a loss of stable charge separation in PSI due to enhanced charge recombination as a result of the pre-illumination treatment. The funneling of electrons towards fewer non-damaged PSI complexes could explain the hastened post-illumination re-reduction of P700+, aided by a slight increase in a stroma-sourced electron flux after prolonged pre-illumination at 4 degrees C. Quantifying the separate fluxes to P700+ helps to elucidate the effects of chilling of cucumber leaf discs in the light and the reasons for the hastened post-illumination re-reduction of P700+.

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

Identification by time-resolved EPR of the peridinins directly involved in chlorophyll triplet quenching in the peridinin–chlorophyll a–protein from Amphidinium carterae

The mechanism of triplet–triplet energy transfer in the peridinin–chlorophyll–protein (PCP) from Amphidinium carterae was investigated by time-resolved EPR (TR-EPR). The approach exploits the concept of spin conservation during triplet–triplet energy transfer, which leads to spin polarization conservation in the observed TR-EPR spectra. The acceptor (peridinin) inherits the polarization of the donor (chlorophyll) in a way which depends on the relative geometrical arrangement of the donor–acceptor couple. Starting from the initially populated chlorophyll triplet state and taking the relative positions among Chls and peridinins from the X-ray structure of PCP, we calculated the expected triplet state polarization of any peridinin in the complex. Comparison with the experimental data allowed us to propose a path for triplet quenching in the protein. The peridinin–chlorophyll pair directly involved in the triplet–triplet energy transfer coincides with the one having the shortest center to center distance. A water molecule, which is coordinated to the central Mg atom of the Chl, is also placed in close contact with the peridinin. The results support the concept of localization of the triplet state mainly in one specific peridinin in each of the two pigment subclusters related by a pseudo C 2 symmetry.

Detection of single biomolecule fluorescence excited through energy transfer: Application to light-harvesting complexes

The authors show that energy transfer is a feasible mechanism for exciting fluorescence of single light-harvesting complexes with different characters of the fluorescing state. This approach is applied for excitons consisting of 18 strongly coupled bacteriochlorophylls in light-harvesting complex 2 as well as for peridinin-chlorophyll-protein monomers containing either two chlorophyll a molecules or a pair of chlorophyll a and chlorophyll b characterized with the emission energy difference of 400cm−1. Using this method the authors are able to observe fluorescence spectral dynamics on the scale comparable or larger than the emission linewidth of a single chromophore.

Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.

Directing electron transfer within Photosystem I by breaking H-bonds in the cofactor branches

Photosystem I has two branches of cofactors down which light-driven electron transfer (ET) could potentially proceed, each consisting of a pair of chlorophylls (Chls) and a phylloquinone (PhQ). Forward ET from PhQ to the next ET cofactor (FX) is described by two kinetic components with decay times of approximately 20 and approximately 200 ns, which have been proposed to represent ET from PhQB and PhQA, respectively. Immediately preceding each quinone is a Chl (ec3), which receives a H-bond from a nearby tyrosine. To decrease the reduction potential of each of these Chls, and thus modify the relative yield of ET within the targeted branch, this H-bond was removed by conversion of each Tyr to Phe in the green alga Chlamydomonas reinhardtii. Together, transient optical absorption spectroscopy performed in vivo and transient electron paramagnetic resonance data from thylakoid membranes showed that the mutations affect the relative amplitudes, but not the lifetimes, of the two kinetic components representing ET from PhQ to F(X). The mutation near ec3A increases the fraction of the faster component at the expense of the slower component, with the opposite effect seen in the ec3B mutant. We interpret this result as a decrease in the relative use of the targeted branch. This finding suggests that in Photosystem I, unlike type II reaction centers, the relative efficiency of the two branches is extremely sensitive to the energetics of the embedded redox cofactors.

Proton ENDOR study of the primary donor P740 +, a special pair of chlorophyll d in photosystem I reaction center of Acaryochloris marina

Oxidized primary electron donor P740, a special pair of chlorophyll (Chl) d in photosystem (PS) I reaction center of a newly identified cyanobacterium Acaryochloris marina was studied by EPR and proton ENDOR spectroscopy. EPR and ENDOR spectra of P740 + were compared with those of P700 + in PS I reaction center of spinach. The g-factors of the purified Chl a + and Chl d + in CH 2Cl 2/THF, P700 + and P740 + in PS I reaction centers were determined to be 2.0025, 2.0032, 2.0027 and 2.0028, respectively. Hyperfine coupling constants of 1.9, 2.8 and 3.8 MHz that were detected in the ENDOR spectrum of P700 + were absent in the ENDOR spectrum of P740 +. These features of P740 + were mainly ascribed to the difference between the chemical structures of Chl a and Chl d.

Photoinhibition of photosystem I

Photosystem I (PSI) is a large pigment-protein complex consisting of about 18 different subunits in plants. Recently, the structure of PSI isolated from pea was solved by X-ray crystallography at a resolution of 4.4 A (Ben-Shem et al. 2003). This work has highlighted the structural similarities and differences between plant PSI and cyanobacterial PSI, where a 2.5 A structure was published earlier (Jordan et al. 2001). The subunits of PSI are traditionally divided into 14 core subun its—most of which are also found in cyanobacte ria—and four homologous light harvesting complex I (LHCI) subunits, which are specific to plants (Fig. 1). Under certain conditions, other LHCI subunits can be seen—at least in Arabidopsis (Klimmek et al. 2005). The PSI complex binds about 167 chlorophyll molecules (Ben-shem et al. 2003). A special pair of chlorophyll a molecules constitutes the P700 reaction center and

Excited states of pigments in photosystem II reaction centers of photosynthesis: Characterization into a central dimer and remaining monomers

In the reaction center (RC) of Photosystem II in photosynthesis, neighboring pigments are nearly equidistantly separated at 8–10 A center-to-center. This fact has let many people consider that excited states in the pigment assembly therein may be multimers covering more than four pigments. In reality, however, the excited states are dimeric on the central pair (P) of chlorophylls, while monomeric on other pigments. Such characterization was derived from their energy broadening which was obtained by fitting the absorption spectrum of RC, based on recent mutagenesis studies clarifying that P* is not lowest in RC differently from other photosystems.

Insight into the Structural Role of Carotenoids in the Photosystem I: A Quantum Chemical Analysis

The structural stabilization role of carotenoids in the formation of photosynthetic pigment-protein complexes is investigated theoretically. The π– π stacking and CH- π interactions between β-carotenes and their surrounding chlorophylls (and/or aromatic residues) in Photosystem I (PS1) from the cyanobacterium Synechococcus elongatus were studied by means of the supermolecular approach at the level of the second-order Møller-Plesset perturbation method. PS1 features a core integral antenna system consisting of 22 β-carotenes intertwined with 90 chlorophyll molecules. The binding environments of all 22 β-carotenes were systematically analyzed. For 21 out of the 22 cases, one or more chlorophyll molecules exist within van der Waals’ contacts of the β-carotene molecule. The calculated strengths of π– π stacking interactions between the conjugated core of β-carotene and the aromatic tetrapyrrole rings of chlorophyll are substantial, ranging from −3.54 kcal/mol for the perpendicular-positioned BCR4004…CHL1217 pair to −16.01 kcal/mol for the parallel-oriented BCR4007…CHL1122 pair. A strong dependence of the π– π stacking interaction energies on the intermolecular configurations of the two interacting π-planes is observed. The parallel-oriented β-carotene and chlorophyll pair is energetically much more stable than the perpendicular-positioned pair. The larger the extent of π– π overlapping, the stronger the interaction strength. In many cases, the β-ring ends of β-carotene molecules are found to interact with the tetrapyrrole rings of chlorophyll via CH- π interactions. For the latter interactions, the calculated interaction strengths vary from −7.03 to −11.03 kcal/mol, depending on the intermolecular configuration. This work leads to the conclusion that π– π stacking and CH- π interactions between β-carotene and their surrounding chlorophylls and aromatic residues play an essential role in binding β-carotenes in PS1 from S. elongatus. Consequently, the molecular basis of the structural stabilization function of carotenoids in formation of the photosynthetic pigment-protein complexes is established.

Photosystem II: evolutionary perspectives.

Based on the current model of its structure and function, photosystem II (PSII) seems to have evolved from an ancestor that was homodimeric in terms of its protein core and contained a special pair of chlorophylls as the photo-oxidizable cofactor. It is proposed that the key event in the evolution of PSII was a mutation that resulted in the separation of the two pigments that made up the special chlorophyll pair, making them into two chlorophylls that were neither special nor paired. These ordinary chlorophylls, along with the two adjacent monomeric chlorophylls, were very oxidizing: a property proposed to be intrinsic to monomeric chlorophylls in the environment provided by reaction centre (RC) proteins. It seems likely that other (mainly electrostatic) changes in the environments of the pigments probably tuned their redox potentials further but these changes would have been minor compared with the redox jump imposed by splitting of the special pair. This sudden increase in redox potential allowed the development of oxygen evolution. The highly oxidizing homodimeric RC would probably have been not only inefficient in terms of photochemistry and charge storage but also wasteful in terms of protein or pigments undergoing damage due to the oxidative chemistry. These problems would have constituted selective pressures in favour of the lop-sided, heterodimeric system that exists as PSII today, in which the highly oxidized species are limited to only one side of the heterodimer: the sacrificial, rapidly turned-over D1 protein. It is also suggested that one reason for maintaining an oxidizable tyrosine, TyrD, on the D2 side of the RC, is that the proton associated with its tyrosyl radical, has an electrostatic role in confining P(+) to the expendable D1 side.

Modeling of the P700 + Charge Recombination Kinetics with Phylloquinone and Plastoquinone-9 in the A 1 Site of Photosystem I

Light activation of photosystem I (PS I) induces electron transfer from the excited primary electron donor P700 (a special pair of chlorophyll a/ a′ molecules) to three iron–sulfur clusters, F X, F A, and F B via acceptors A 0 (a monomeric chlorophyll a) and A 1 (phylloquinone). PS I complexes isolated from menA and menB mutants contain plastoquinone-9 rather than phylloquinone in the A 1 site and show altered rates of forward electron transfer from A 1 − to [F A/F B] and altered rates of back electron transfer from [F A/F B] − to P700 + (Semenov, A. Y., et al., J. Biol. Chem. 275:23429–23438, 2000). To identify the modified electron transfer steps, we studied the kinetics of flash-induced P700 + reduction in PS I that contains either an intact set or a subset of iron–sulfur clusters F X, F A, and F B and with the A 1 binding site occupied by phylloquinone or plastoquinone-9. A modeling of the forward and backward electron transfer kinetics in P700–F A/F B complexes, P700–F X cores, and P700–A 1 cores shows that the replacement of phylloquinone by plastoquinone-9 induces a decrease in the free energy gap between A 1 and F A/F B from ∼−205 mV in wild-type PS I to ∼−70 mV in menA PS I. The +135 mV increase in the midpoint potential of A 1 explains the acceleration in the rate of P700 + dark reduction in menA PS I, and the resulting uphill electron transfer from A 1 to F X in menA PS I explains the absence of a contribution from F X − to the reduction of P700 +. This fully quantitative description of PS I relates electron transfer rates, equilibrium constants, and redox potentials, and can be used to predict changes in these parameters upon substitution of electron transfer cofactors.

Uphill Energy Trapping by Reaction Center in Bacterial Photosynthesis: Charge Separation Unistep from Antenna Excitation, Virtually Mediated by Special-Pair Excitation

In the primary process of photosynthesis, energy of the electronic excited state, A*, in the core antenna is trapped by the reaction center into a state, B, of charge separation between pigments therein. This energy-trapping process is mediated by the excited state, P*, of the special pair of chlorophylls or bacteriochlorophylls in the reaction center. Energy transfer from A* to P* has been reported to be uphill with an excitation-energy difference, for example, amounting to about 200 and 350−400 cm-1 in Rhodobacter (Rb.) sphaeroides and Rhodopseudomonas (Rps.) viridis, respectively, in purple bacteria. The rate constant of this energy-trapping process was calculated with special attention to these species, reproducing observed data in the whole temperature range. At physiological temperatures, this process proceeds by the ordinary sequential mechanism, in which the second step from P* to B takes place after thermalization of phonons at P*, in both species. The uphill excitation-energy difference in the f...

Observation of the Excited State of the Primary Electron Donor Chlorophyll (P700) and the Ultrafast Charge Separation in the Spinach Photosystem I Reaction Center

Femtosecond transient absorption spectroscopy was applied to the photosystem I reaction center particles containing only 12−13 chlorophylls per primary electron donor chlorophyll (P700), which were prepared from spinach. Upon preferential excitation of P700, an absorption band due to the excited state of P700 was newly observed in the 730−750 nm region. The primary charge separation dynamics at ≈280 K were revealed by the rise of the absorption band of the radical pair of chlorophylls. The rise was biphasic: 0.8 ps (50%) and 9 ps (50%). The 0.8 ps component is the fastest phase of the charge separation ever observed in photosystem I reaction center. On the basis of the observed dynamics and a kinetic model of excitation migration, the rate constant of the charge separation between P700 and the nearest-acceptor chlorophyll is predicted to be (0.5−0.8 ps)-1.

A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls.