Core Antenna Complexes Research Articles

Changes of absorption spectra during heat-induced denaturation of Photosystem II core antenna complexes CP43 and CP47: revealing the binding states of chlorophyll molecules in these two complexes

The Photosystem II (PSII) core antenna complexes, CP43 and CP47, were prepared from spinach ( Spinacia oleracea L.). The absorption spectra in the red region at room temperature were recorded for the PSII core antenna samples after increased temperature treatment (up to 80°C). Derivative and difference spectra revealed the existence of two groups of chlorophyll a (Chl a) molecules in both CP43 and CP47. The one with the absorption peak in the shorter wavelength region was designated as CP43-669 and CP47-669, while the other with the absorption peak in the longer wavelength region was designated as CP43-682 and CP47-680. The results of the thermal treatment experiment demonstrated that CP43-669 and CP47-669 may exist as monomers of Chl a and that their binding sites on the polypeptides are insensitive to thermal treatment, whereas CP43-682 and CP47-680 may exist as dimers or multimers of Chl a and their binding regions in the polypeptide chains are more sensitive to heat treatment. The excitation energy transfer mechanism between these two different groups of Chl a molecules is also analyzed.

Spectroscopic study of trypsin, heat and Triton X-100-induced denaturation of the chlorophyll-binding protein CP43

Trypsin-, heat- and Triton X-100-induced denaturation of CP43, the core antenna complex of photosystem II purified from spinach, has been investigated using absorption, fluorescence and circular dichroism spectroscopy. Triton X-100 was found to bring about considerable dissolution of pigments from the protein to the monomeric state in solution and destruction of the interactions among the chlorophyll, carotene and protein. Heat induced significant unfolding of the protein secondary structure and loss of excitonic interactions of the pigments, but no apparent dissolution of the pigments from CP43. Trypsin caused structural changes in the extrinsic part of the protein but no change of the native state of the pigments. Trypsin, heat and Triton X-100 treatments increased the light sensitivity of chlorophyll in CP43 to different extents. The results suggest that the protein and β-carotene can protect the chlorophyll from light-induced destruction in CP43.

The excitation energy transfer between β-Car and Chla molecules in PS II core antenna complexes CP43 and CP47

The excitation energy transfer efficiency between β-Car and Chla molecules in purified CP43 and CP47 was calculated by comparing absorption and fluorescence excitation after normalization at 550 nm, CP43 had an energy transfer efficiency of 29.1% while the CP47 had an energy transfer efficiency of 62.8%, proving that excitation energy was transferred between β-Car and Chla molecules in CP43 and CP47 at normal conditions. The excitation energy transfer between β-Car and Chla molecules in CP43 and CP47 may occur through the “Dexter” mechanism and the distance between these two kinds of pigments should be less than 1 nm. In addition, the results were also used to discuss the conformational relationship between β-Car and Chla molecules in CP43 and CP47.

Pigment–protein architecture in the light-harvesting antenna complexes of purple bacteria: does the crystal structure reflect the native pigment–protein arrangement?

Structural analysis of crystallized peripheral (LH2) and core antenna complexes (LH1) of purple bacteria has revealed circular aggregates of high rotational symmetry (C 8, C 9 and C 16, respectively). Quantum-chemical calculations indicate that in particular the waterwheel-like arrangements of pigments should show characteristic structure-sensitive spectroscopic behavior in the near infrared absorption region. Laser-spectroscopic data obtained with non-crystallized, isolated LH2 of Rhodospirillum molischianum are in line with a highly symmetric (C 8) circular aggregate, but deviations have been found for LH2 of Rhodobacter sphaeroides and Rhodopseudomonas acidophila. For both the latter, C-shaped incomplete circular aggregates (as seen only recently in electron micrographs of crystallized LH1–reaction center complexes) may be a suitable preliminary model.

Light- and heat-induced denaturation of photosystem II core-antenna complexes CP43 and CP47

Light- and heat-induced denaturation of the core-antenna complexes of Photosystem II, CP43 and CP47, purified from spinach, has been investigated using absorption, fluorescence and circular dichroism (CD) spectroscopy. Light is found to bring about considerable bleaching of chlorophyll a but no apparent change in the protein secondary structure, while heat induces significant unfolding of the protein secondary structure but no apparent destruction of the chlorophyll a molecule in the two antenna complexes. Both the destruction of chlorophyll a by light and the denaturation of the protein conformation by heat cause the loss of excitonic interaction of chlorophyll a in CP43 and CP47, as measured by visible CD activity. Light induces a larger decrease of the chlorophyll a fluorescence and the CD activity of CP47 than that of CP43, indicating that the native state of chlorophyll a of CP47 is more sensitive to light than that of CP43. The main thermal transitions of protein secondary structure occur at 50°C for CP43 and 63°C for CP47, while the half-loss of chlorophyll a excitonic interaction during heating occurs at 45°C for CP43 and 60°C for CP47, suggesting that CP47 is more thermally stable than CP43.

The Paths of Excitation Energy Deactivation in LH1 Reduced Mutant and Wild-Type Strains of Rhodobacter sphaeroides

The P3 mutant of Rhodobacter sphaeroides had an altered ratio of reaction center to core (LH1) and peripheral (LH2) antenna complexes compared to the wild-type strain. Intracytoplasmic membranes from these two strains were purified and then resuspended in buffer or immobilized in isotropic and stretched polymer film. The absorption, photoacoustic, and delayed luminescence spectra were measured. The ratios of infrared absorption and photoacoustic bands (located at about 880 nm for LH1 and at 850 and about 800 nm for LH2) as well as the half-width of these bands are different for the LH2 and LH1 mutants and wild-type strain. The whole yields of thermal deactivation of the two strains were comparable, but in the absorption region of LH2 it was slightly lower in the case of the mutant than for the wild-type strain. The delayed luminescence main maxima were observed at about 860 and 700 nm. The first one could be due to emission of bacteriochlorophyll a of LH2 complexes. The emission at about 700 nm is probably due to dihydromesochlorophyll, which is usually, to some extent, produced from bacteriochlorophyll a in bacterial complexes. The delayed luminescence emission is competing with excitation energy transfer to the reaction center. The intensity of the delayed luminescence of the mutant strain was higher than that of the wild-type strain when both samples were excited in a region of carotenoid absorption. The mutant contains less carotenoids than the wild-type strain. Carotenoids work as efficient antenna. When they at a lower concentration the excitation can be trapped more easily by some chlorophyll-like pigment isolated from the excitation energy chain. The dependences of delayed luminescence spectra on the light polarization and excitation wavelengths for the wild-type strain and for the mutant were different. The anisotropy of delayed luminescence showed that bacteriochlorophyll a molecules of different orientations were contributing to the mutant and the wild-type strain emission. All the results suggest that the excitation energy transfer from the antenna to the reaction center in the mutant and the wild-type strain is similar.

The effect of pressure on the bacteriochlorophyll a binding sites of the core antenna complex from Rhodospirillum rubrum.

In this paper we examine the effect of pressure on the absorption spectrum and binding site of the core antenna complex from the photosynthetic bacterium Rhodospirillum rubrum. Absorption spectra and Raman spectra in preresonance with the Qy transition of the bacteriochlorophyll a were studied at pressures up to 625 MPa. In agreement with previous work we observe a pressure-induced red shift and broadening of the absorption spectrum. We show that at these pressures the pigments within the protein matrix at room temperature experience little if any distortion, and the hydrogen-bonding network involving the C2 and C9 carbonyl groups of the pigment molecules are undisturbed. Having shown the lack of sensitivity to pressure of the binding site interactions, which are known to modulate the absorption spectrum, we feel that it is relatively safe to attribute the pressure-induced red shift broadly to solvatochromic effects and, in particular, to the modulation of the pigment-pigment interactions by the pressure. This paper represents the first vibrational study of photosynthetic complexes at high pressure and the first application of FT Raman spectroscopy to biological molecules at high pressure.

Pigment Binding-Site and Electronic Properties in Light-Harvesting Proteins of Purple Bacteria

Comparison of the absorption maxima with the hydrogen bonding of the C2 acetyl groups for various mutants and wild-type bacterial antenna complexes has demonstrated a role for this molecular interaction in the tuning of the absorption properties of these complexes. There is a consistent linear relationship, between the downshift in the bacteriochlorophyll a C2 acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H-bonding to the red shifts of these complexes and the sensitivity of the absorption to changes in the C2 acetyl group environment. Investigation of changes in the Raman spectra caused by altering the excitation conditions reveal either no or only minor changes in the positions of the methine bridge, the C2 acetyl, and the C9 keto stretching modes. We interpret the lack of sensitivity of these bands to the resonance condition as indicating that neither differences in ring distortion nor in the H-bonding environment of these groups are important in determining the heterogeneous absorption bandwidth of the absorption spectrum of these complexes. We suggest that the differences in coupling between the pigments might be responsible for the static disorder and this may also be partially reflected in the small differences in the C9 keto group stretching band we observe in the Raman spectra.

Functions of conserved tryptophan residues of the core light-harvesting complex of Rhodobacter sphaeroides.

We have examined mutants in the core light-harvesting complex of Rhodobacter sphaeroides in which the tryptophan residues located at positions alpha+11, beta+6, and beta+9 have been mutated to each of the three other aromatic amino acids, namely tyrosine, phenylalanine, and histidine. We confirm that the alpha+11 residue and show that the beta+9 residue each form a hydrogen bond to a C2-acetyl group of a BChl molecule. Mutation of either of these residues to a phenylalanine results in a breakage of the normal hydrogen bond, whereas a histidine in either of these positions is able to form a hydrogen bond to the BChl. Comparison of the absorption spectra with the hydrogen bonding of the C2-acetyl groups for the various mutants demonstrates a role for this molecular interaction in the tuning of the absorption properties of the complex. We further demonstrate that there is a consistent linear relationship between the downshift in the C2-acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H bonding to the red shifts of these complexes. Though the residue beta+6 is found not to be directly involved in interactions with the pigment molecules, mutation of this residue is shown in some cases to result in both a destabilization of the complex and a decrease in the binding site homogeneity. Finally, a consideration of the amount of antenna complex present in the various mutants shows an important role for the reaction center and/or the pufX gene product in the assembly or stabilization of this membrane protein.

The theory of Forster-type migration between clusters of strongly interacting molecules: application to light-harvesting complexes of purple bacteria

Excitation dynamics in the light-harvesting core antenna of purple bacteria was studied theoretically using a model of excitation delocalization over the circular aggregate of strongly interacting bacteriochlorophyll molecules (‘DCA model’) (V.I. Novoderzhkin and A.P. Razjivin, FEBS Lett. 330 (1993) 5). Energy transfer from the core antenna complex to the reaction center (RC) and between core antenna complexes was treated as a Forster-type process. The assumption of the DCA model for the core antenna offers an explanation of the experimental data on the excitation trapping by RCs (60 ps antenna excitation decay; quasi-irreversible trapping). The rate of excitation migration between neighboring antenna core complexes is well suited to the realization of the ‘lake model’. The advantages of the proposed model as compared with other models are discussed.

Ultrafast dynamics within the B820 subunit from the core (LH-1) antenna complex of Rs. rubrum

Femtosecond transient dynamics within the dimeric subunit of a bacterial light-harvesting complex have been investigated using magic angle and polarized pump-probe spectroscopy. Oscillations of 170 cm −1 and ≈ 20 cm −1, 50 cm −1 and 115 cm −1 are observed in the isotropic signal, and of ≈ 500 cm −1 in the anisotropy; the Stokes shift and dephasing of the dimer exciton levels occurs on a sub-50 fs timescale. The re-assembled aggregate exhibits ultrafast spectral evolution and biphasic anisotropy decay. Collectively, the results support a view of energy migration within LH-1 complexes as proceeding by strongly scattered excitation transfer between states of the dimeric subunits.

Matrix and temperature effects on absorption spectra of β-carotene and pheophytin a in solution and in green plant photosystem II

Absorption spectra of β-carotene (β-Car) and pheophytin (Pheo) a have been recorded in a number of solvents and polymers and the properties are compared with those observed in the isolated reaction centre complex of green plant photosystem II and in its core antenna complex CP47. The peak maxima of the investigated absorption bands shift to the red with increasing polarizability of the medium. The extent of the shift is in the order β-Car > Pheo (Soret) > Pheo (Q x ) > Pheo (Q y ), and it exhibits a linear relationship with the Lorentz-Lorenz function ( n 2 − 1)/( n 2 + 2) of the refractive index of the solvent. The maxima also depend on the polarity of the solvent, and for liquids with similar refractive indices the shift in the maxima of the absorption bands was found to be proportional to the dielectric permittivity function ( ϵ − 1)/( ϵ + 2). Most absorption bands shift strongly (β-Car) or weakly (Pheo Soret and Q x ) to the red when the polarity is increased, but the Pheo Q y band shifts to the blue. All investigated absorption bands also broaden with increasing polarity of the medium. The temperature dependence of the absorption properties was recorded in poly (vinyl butyral) and polystyrene matrices between 80 and 295 K. The purely thermal shift of the absorption maxima was calculated from the difference between the observed shift and the estimated dispersive shift. The thermal and dispersive effects cause in most cases blue and red shifts respectively on cooling, but for β-Car both effects lead to red shifts on cooling from room temperature to 110–160 K. The absorption bands of β-Car in CP47 are remarkably narrow, suggesting a non-polar and highly uniform environment. The temperature-induced band shift is much larger in CP47 than in the polymer matrices, which probably is related to a phase transition of the protein matrix at about 200 K. At room temperature, the effective refractive index of the CP47 host was estimated to be 1.51 ± 0.04. The absorption bands of β-Car in the isolated photosystem II reaction centre complex are distorted because of excitonic interactions but exhibit roughly the same temperature dependence as those in CP47. The Pheo a Q x absorption band is considerably red shifted compared with that in solvents and polymers, which is ascribed to specific interactions with the reaction centre proteins.

A Null Mutant of Synechococcus sp. PCC7942 Deficient in the Sulfolipid Sulfoquinovosyl Diacylglycerol

The sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol is associated with the thylakoid membranes of many photosynthetic organisms. Previously, genes involved in sulfolipid biosynthesis have been characterized only in the purple bacterium Rhodobacter sphaeroides. Unlike plants and cyanobacteria, photosynthesis in this bacterium is anoxygenic due to the lack of a water splitting photosystem II. To test the function of sulfolipid in an organism with oxygenic photosynthesis, we isolated and inactivated a sulfolipid gene of the cyanobacterium Synechococcus sp. PCC7942. Extensive analysis of the sulfolipid-deficient null mutant revealed subtle changes in photosynthesis related biochemistry of O2. In addition, a slight increase in the variable room temperature chlorophyll fluorescence yield was observed. Regardless of these changes, it seems unlikely that sulfolipid is an essential constituent of a functional competent water oxidase or the core antenna complex of photosystem II. However, reduced growth of the mutant under phosphate-limiting conditions supports the hypothesis that sulfolipid acts as a surrogate for anionic phospholipids under phosphate-limiting growth conditions.

Platinization: a novel technique to anchor photosystem I reaction centres onto a metal surface at biological temperature and pH

Photosystem I (PSI) reaction centre core antenna complexes containing about 40 chlorophylls per P700 (PSI-40) are 6 nm biological photovoltaic device. PSI particles carry positively-charged domain(s) and can therefore interact electrostatically with a negatively-charged surface. PSI particles were platinized by reduction of [PtCl 6] −2 at neutral pH and 20°C. The presence of metallic platinum on PSI particles was observed directly by scanning tunnelling microscopy (STM). The platinized PSI particles were larger than control PSIs (without platinization), due to the presence of metallic platinum. The electric properties of PSI and metallic platinum were characterized by STM tunnelling conductance (I–V) measurements. Diodic response was obtained in platinized PSI. Metallic platinum formed during the reduction of [PtCl 6] 2− caused bonding between PSI particles and a gold metal surface. Using the technique of chemical platinization, PSI particles can be “welded” onto gold plates under biologically compatible conditions, resulting in a 2-dimensional spatial array of PSI particles anchored on the metal surface. Metallic platinum is not only a good conductor but also an active catalyst. This platinization “welding” technique is potentially important for both bioelectronics and biometallocatalysis.

Temporally and spectrally resolved subpicosecond energy transfer within the peripheral antenna complex (LH2) and from LH2 to the core antenna complex in photosynthetic purple bacteria.

We report studies of energy transfer from the 800-nm absorbing pigment (B800) to the 850-nm absorbing pigment (B850) of the LH2 peripheral antenna complex and from LH2 to the core antenna complex (LH1) in Rhodobacter (Rb.) sphaeroides. The B800 to B850 process was studied in membranes from a LH2-reaction center (no LH1) mutant of Rb. sphaeroides and the LH2 to LH1 transfer was studied in both the wild-type species and in LH2 mutants with blue-shifted B850. The measurements were performed by using approximately 100-fs pulses to probe the formation of acceptor excitations in a two-color pump-probe measurement. Our experiments reveal a B800 to B850 transfer time of approximately 0.7 ps at 296 K and energy transfer from LH2 to LH1 is characterized by a time constant of approximately 3 ps at 296 K and approximately 5 ps at 77 K. In the blue-shifted B850 mutants, the transfer time from B850 to LH1 becomes gradually longer with increasing blue-shift of the B850 band as a result of the decreasing spectral overlap between the antennae. The results have been used to produce a model for the association between the ring-like structures that are characteristic of both the LH2 and LH1 antennae.

Triplet and fluorescing states of the CP47 antenna complex of photosystem II studied as a function of temperature

Fluorescence emission and triplet-minus-singlet (T-S) absorption difference spectra of the CP47 core antenna complex of photosystem II were measured as a function of temperature and compared to those of chlorophyll a in Triton X-100. Two spectral species were found in the chlorophyll T-S spectra of CP47, which may arise from a difference in ligation of the pigments or from an additional hydrogen bond, similar to what has been found for Chl molecules in a variety of solvents. The T-S spectra show that the lowest lying state in CP47 is at approximately 685 nm and gives rise to fluorescence at 690 nm at 4 K. The fluorescence quantum yield is 0.11 +/- 0.03 at 4 K, the chlorophyll triplet yield is 0.16 +/- 0.03. Carotenoid triplets are formed efficiently at 4 K through triplet transfer from chlorophyll with a yield of 0.15 +/- 0.02. The major decay channel of the lowest excited state in CP47 is internal conversion, with a quantum yield of about 0.58. Increase of the temperature results in a broadening and blue shift of the spectra due to the equilibration of the excitation over the antenna pigments. Upon increasing the temperature, a decrease of the fluorescence and triplet yields is observed to, at 270 K, a value of about 55% of the low temperature value. This decrease is significantly larger than of chlorophyll a in Triton X-100. Although the coupling to low-frequency phonon or vibration modes of the pigments is probably intermediate in CP47, the temperature dependence of the triplet and fluorescence quantum yield can be modeled using the energy gap law in the strong coupling limit of Englman and Jortner (1970. J. Mol. Phys. 18:145-164) for non-radiative decays. This yields for CP47 an average frequency of the promoting/accepting modes of 350 cm-1 with an activation energy of 650 cm-1 for internal conversion and activationless intersystem crossing to the triplet state through a promoting mode with a frequency of 180 cm-1. For chlorophyll a in Triton X-100 the average frequency of the promoting modes for non-radiative decay is very similar, but the activation energy (300 cm-1) is significantly smaller.

Core Antenna Complexes, CP43 and CP47, of Higher Plant Photosystem II. Spectral Properties, Pigment Stoichiometry, and Amino Acid Composition

The core antenna complexes of photosystem II, CP43 and CP47, were purified from two higher plants by anion-exchange chromatography, using a combination of the chaotropic agent LiClO4 and the nonionic detergent beta-dodecyl maltoside. The Qy transition was resolved at 48 K into two main bands near 682.3 and 671.5 nm for CP43, while the CP47 spectrum showed a more complex structure with main bands at 688, 681.2, 676, 667, and 661 nm. Emission bands (77 K) were detected at 683 and 695 nm for CP43 and CP47, respectively. Fluorescence excitation spectra showed high efficiency of energy transfer between the different transitions of the chlorophylls and a somewhat lower efficiency from beta-carotene. The circular dichroism spectrum of CP47 indicated the presence of excitonic interactions between some chlorophylls. In contrast, CP43 showed a single negative circular dichroism band at 670 nm. The pigment content of the complexes was determined by both spectroscopic measurements and HPLC. Contents of 18 chlorophylls a and 5 beta-carotenes per CP43 polypeptide and 19 chlorophylls a and 3 beta-carotenes per CP47 polypeptide were found, using the methods of Lowry or Bradford for protein quantitation. When the protein concentration was determined from the amino acid analysis, 20 chlorophylls a and 5 beta-carotenes per CP43 and 21-22 chlorophylls a and 4 beta-carotenes per CP47 were obtained. Thus, a content of 46-48 chlorophylls a was obtained for the core complex, assuming 4-6 chlorophylls per reaction center, in agreement with the composition obtained experimentally using a highly purified oxygen-evolving core complex.(ABSTRACT TRUNCATED AT 250 WORDS)

POLARIZED SITE‐SELECTION SPECTROSCOPY OF CHLOROPHYLL a IN DETERGENT

AbstractMonomeric chlorophyll a (Chl a) was obtained from the isolated core antenna complex CP47 of photo‐system II after incubation with the detergent triton X‐100 and was studied by low‐temperature polarized light spectroscopy with the aim to obtain model spectra for Chi a in intact photosynthetic complexes. Evidence is presented by circular dichroism and anisotropy measurements that the isolated chlorophyll is monomeric. The absorption bandwidths are relatively large compared to those found in photosynthetic complexes due to inhomogeneous broadening introduced by the detergent. By selective laser excitation at low temperature, considerable narrowing can be achieved. A number of vibrational bands are resolved in the site‐selected, polarized absorption and fluorescence emission spectra. The emission spectrum of Chi a in detergent‐damaged CP47 is compared with that of Chi a in the intact light‐harvesting complex of photosystem II (LHC‐II) from green plants. The spectra are remarkably similar indicating that the low‐temperature thermal emitter in LHC‐II has spectral properties that are very similar to those of monomeric Chl a.

Spectral hole burning in pigment-protein complexes of photosynthetic bacteria

The excited-state dynamics of the peripheral light-harvesting complex LH2 (B800-850) of Rb. sphaeroides is compared to that of the dimer subunit (B8200(OG) of the core antenna complex LH1 of Rs. rubrum G9 at liquid helium temperatures by means of spectral hole burning. Both the B800 band of the LH2 complex and the absorption band of the B820(GO) subunit are inhomogeneously broadened. The homogeneous line width, Γ hom, of B800 is determined by energy transfer within the LH2 complex (√ transfer ~~0.8 to 2.5 ps); it is temperature independent between 1.2 and 30K and a factor of about 60 larger than that of B820(OG). By contrast, for B820(OG)Γ hom is only ∼ 1 GHz at 1.2 K; it depends on temperature and is predominatly given by dephasing.

Resonance Raman spectroscopy of the B820 subunit of the core antenna from Rhodospirillum rubrum G9

The resonance Raman spectrum in the carbonyl-stretch region (1600–1700 cm −1) of the B820(OG) subunit form is completely different from the original core antenna complex of Rhodospirillum rubrum G9. The observed changes appear to be fully reversible upon reassociation of the B820(OG) subunits into the native-like B873(reassoc). This allows direct conclusions regarding the BChl a binding-site in the antenna complex. In the native complex all 2-acetyl carbonyl groups are hydrogen bonded since only a single resonance peak is observed at 1640 cm −1. When the core antenna is dissociated into its subunit B820(OG) form, half of this vibrational band is shifted to 1625 cm −1, indicating that approx. 50% of the 2-acetyl groups of the BChl a porphyrin rings have changed their hydrogen bond interaction. Besides these changes, there are also effects in the resonance frequency of the 9-keto carbonyl group that indicate that, upon dissociation, a fraction of these groups becomes free from interactions. Similar changes are observed for the B820(ODPS) subunit that is prepared using a different detergent. The spectral shifts upon dissociation cannot be attributed solely to the change in hydrogen bonding, suggesting that an additional perturbation of the BChl a binding-site takes place. Possible models for the hydrogen bonding of these groups are discussed.