Primary photosynthetic processes in Heliobacterium chlorum at 15K

Absorbance changes induced by 25-ps laser flashes were measured in membranes of Heliobacterium chlorum at 15 K. Absorbance difference spectra, measured at various times after the flash showed negative bands in the Q_y region at 812, 793 and 665 nm. The first of these bands was attributed to the formation of excited singlet states of a long-wavelength form of antenna bacteriochlorophyll g (BChl g 808). Absorbance changes of shorter wavelength absorbing antenna BChls g were at least an order of magnitude smaller, indicating rapid excitation energy transfer (i.e. within the time resolution of the apparatus) from these BChls to BChl g 808. Excited BChl g 808 showed a bi-exponential decay with time constants of 50 and 200 ps. The bands at 793 and 665 nm may be attributed to the primary charge separation and reflect the photooxidation of the primary electron donor P-798 and photoreduction of a primary electron acceptor absorbing near 670 nm, presumably a BChl c or Chl a-like pigment. The bleaching of this pigment reversed with a time constant of 300 ps at 15 K and of 800 ps at 300 K. This indicates that electron transfer from the primary to the secondary electron acceptor is approximately 2.5 times faster at 15 K than at room temperature.Abbreviations BChl bacteriochlorophyll - FWHM full width at half maximum - P-798 primary electron donor - Tris tris(hydroxymethyl)amino methane     

Composition and optical properties of reaction centre core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum

Photosynthetically active reaction centre core (RCC) complexes were isolated from two species of green sulfur bacteria, Prosthecochloris (Ptc.) aestuarii strain 2K and Chlorobium (Chl.) tepidum, using the same isolation procedure. Both complexes contained the main reaction centre protein PscA and the iron–sulfur protein PscB, but were devoid of Fenna–Matthews–Olson (FMO) protein. The Chl. tepidum RCC preparation contained in addition PscC (cytochrome c). In order to allow accurate determination of the pigment content of the RCC complexes, the extinction coefficients of bacteriochlorophyll (BChl) a in several solvents were redetermined with high precision. They varied between 54.8 mM⁻¹ cm⁻¹ for methanol and 97.0 mM⁻¹ cm⁻¹ for diethylether in the Q_Y maximum. Both preparations appeared to contain 16 BChls a of which two are probably the 13²-epimers, 4 chlorophylls (Chls) a 670 and 2 carotenoids per RCC. The latter were of at least two different types. Quinones were virtually absent. The absorption spectra were similar for the two species, but not identical. Eight bands were present at 6 K in the BChl a Q_Y region, with positions varying from 777 to 837 nm. The linear dichroism spectra showed that the orientation of the BChl a Q_Y transitions is roughly parallel to the membrane plane; most nearly parallel were transitions at 800 and 806 nm. For both species, the circular dichroism spectra were dominated by a strong band at 807–809 nm, indicating strong interactions between at least some of the BChls. The absorption, CD and LD spectra of the four Chls a 670 were virtually identical for both RCC complexes, indicating that their binding sites are highly conserved and that they are an essential part of the RCC complexes, possibly as components of the electron transfer chain. Low temperature absorption spectroscopy indicated that typical FMO–RCC complexes of Ptc. aestuarii and Chl. tepidum contain two FMO trimers per reaction centre. This revised version was published online in June 2006 with corrections to the Cover Date.     

Orientation and excitonic interactions of the Fenna—Matthews—Olson bacteriochlorophyll a protein in membranes of the green sulfur bacterium Chlorobium tepidum

Linear and circular dichroism spectra of isolated bacteriochlorophyll a proteins (FMO proteins) and membrane vesicles containing FMO protein from the green sulfur bacterium Chlorobium tepidum were measured at room temperature and 77 K. The orientation of membranes and isolated FMO protein was obtained by gel squeezing. Linear dichroism (LD) data indicate that isolated FMO protein and membrane vesicles associated with the FMO protein are oriented in a similar way in a squeezed polyacrylamide gel. Both samples show a characteristic negative LD band around 814 nm with flanking positive bands at 802 and 824 nm ascribed to the Q_y excitonic transitions of BChl a of the FMO protein. This confirms that the C3 symmetry axis of the trimer is perpendicular to the membrane plane, which is supported by the model of the disc-like structure of FMO protein trimers of Cb. tepidum [Li Yi-Fen, Zhou W, Blankenship RE, and Allen JP (1997) J Mol Biol 272: 456–471]. The LD data are consistent with either BChl 3 or 6, but not 7 as the principal contributor to the low temperature band at 825 nm. The low temperature linear and circular dichroism spectra of FMO protein trimers from Chlorobium tepidum show significant differences from the low temperature LD and CD spectra of FMO protein trimers from Prosthecochloris aestuarii. The data are interpreted in terms of somewhat different pigment-protein and pigment-pigment interactions in the two complexes.     

Characterization of the FMO protein from the aerobic chlorophototroph, Candidatus Chloracidobacterium thermophilum

Candidatus Chloracidobacterium (Cab.) thermophilum is a recently discovered aerobic chlorophototroph belonging to the phylum Acidobacteria. From analyses of genomic sequence data, this organism was inferred to have type-1 homodimeric reaction centers, chlorosomes, and the bacteriochlorophyll (BChl) a-binding Fenna–Matthews–Olson protein (FMO). Here, we report the purification and characterization of Cab. thermophilum FMO. Absorption, fluorescence emission, and CD spectra of the FMO protein were measured at room temperature and at 77 K. The spectroscopic features of this FMO protein were different from those of the FMO protein of green sulfur bacteria (GSB) and suggested that exciton coupling of the BChls in the FMO protein is weaker than in FMO of GSB especially at room temperature. HPLC analysis of the pigments extracted from the FMO protein only revealed the presence of BChl a esterified with phytol. Despite the distinctive spectroscopic properties, the residues known to bind BChl a molecules in the FMO of GSB are well conserved in the primary structure of the Cab. thermophilum FMO protein. This suggests that the FMO of Cab. thermophilum probably also binds seven or possibly eight BChl a(P) molecules. The results imply that, without changing pigment composition or structure dramatically, the FMO protein has acquired properties that allow it to perform light harvesting efficiently under aerobic conditions.     

A comparative study of the optical characteristics of intact cells of photosynthetic green sulfur bacteria containing bacteriochlorophyll c,d or e

Energy transfer and pigment arrangement in intact cells of the green sulfur bacteria Prosthecochloris aestuarii, Chlorobium vibrioforme and chlorobium phaeovibrioides, containing bacteriochlorophyll (BChl) c, d or e as main light harvesting pigment, respectively, were studied by means of absorption, fluorescence, circular dichroism and linear dichroism spectroscopy at low temperature. The results indicate a very similar composition of the antenna in the three species and a very similar structure of main light harvesting components, the chlorosome and the membrane-bound BChl a protein. In all three species the Q_y transition dipoles of BChl c, d or e are oriented approximately parallel to the long axis of the chlorosome. Absorption and fluorescence excitation spectra demonstrate the presence of at least two BChl c-e pools in the chlorosomes of all three species, long-wavelength absorbing BChls being closest to the membrane. In C. phaeovibrioides, energy from BChl e is transferred with an efficiency of 25% to the chlorosomal BChl a at 6 K, whereas the efficiency of transfer from BChl e to the BChl a protein is 10%. These numbers are compatible with the hypothesis that the chlorosomal BChl a is an intermediary in the energy transfer from the chlorosome to the membrane.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - CD circular dichroism - LD linear dichroism     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Q_y transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Q_y transition of BChl c and the -carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Q_y transition is close to the magic angle (55°).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - LD linear dichroism Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800–B850 gap from 750 cm^\s-1 at 0.1 MPa to 900 cm^-1 at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800–850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800–B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C₉-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (-0.4 cm⁰¹/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm⁰¹/MPa). The shift rate for B850 at 4.2 K is-0.28 cm^–1/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.Abbreviations B800 BChl antenna band absorbing (at room temperature) at 800 nm (B850, B875, B1015 are defined similarly) - CD circular dichroism - FC factor Franck-Condon factor - FMO comple Fenna-Matthews-Olson complex - L-S theory Laird-Skinner theory - LH1 core light-harvesting complex of the BChl antenna complexes - LH2 peripheral light-harvesting complex of the BChl antenna complexes - NPHB non-photochemical hole burning - P960 absorption band of special pair of Rhodopseudomonas viridis absorbing at 960 nm (room temperature). P870 of Rhodobacter sphaeroides is defined similarly - QM/MM results quantum mechanical/molecular mechanical results - RC reaction center - ZPH zero phonon hole     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus. III. Energy transfer in whole cells

The transfer of excitation energy in intact cells of the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus was studied both at low temperature and under more physiological conditions. Analysis of excitation spectra measured at 4K indicates that the minor fraction of bacteriochlorophyll a present in the chlorosome functions as an intermediate in energy transfer between the main light-harvesting pigment BChl c and the membrane-bound B808-866 antenna complex. This supports the hypothesis that BChl a is associated with the base plate which connects the chlorosome with the membrane. The overall efficiency for energy transfer from the chlorosome to the membrane is only 15% at 4K. High efficiencies of close to 100% are observed above 40°C near the temperature where the cultures are grown. Cooling to 20°C resulted in a sudden drop of the transfer efficiency which appeared to originate in the chlorosome. This decrease may be related to a lipid phase transition. Further cooling mainly affected the efficiency of transfer between the chlorosome and the membrane. This effect can only partially be explained by a decreased Förster overlap between the chlorosomal BChl a and BChl a 808 associated with the membrane-bound antenna system. The temperature dependence of the fluorescence yield of BChl a 866 also appeared to be affected by lipid phase transitions, suggesting that this fluorescence can be used as a native probe of the physical state of the membrane.     

The FMO protein is related to PscA in the reaction center of green sulfur bacteria

The Fenna–Matthews–Olson protein is a water-soluble protein found only in green sulfur bacteria. Each subunit contains seven bacteriochlorophyll (BChl) a molecules wrapped in a string bag of protein consisting of mostly β sheet. Most other chlorophyll-binding proteins are water-insoluble proteins containing membrane-spanning α helices. We compared an FMO consensus sequence to well-characterized, membrane-bound chlorophyll-binding proteins: L & M (reaction center proteins of proteobacteria), D1 & D2 (reaction center proteins of PS II), CP43 & CP47 (core proteins of PS II), PsaA & PsaB (reaction center proteins of PS I), PscA (reaction center protein of green sulfur bacteria), and PshA (reaction center protein of heliobacteria). We aligned the FMO sequence with the other sequences using the PAM250 matrix modified for His binding-site identities and found a signature sequence (LxHHxxxGxFxxF) common to FMO and PscA. (The two His residues are BChl a. binding sites in FMO.) This signature sequence is part of a 220-residue C-terminal segment with an identity score of 13%. PRSS (Probability of Random Shuffle) analysis showed that the 220-residue alignment is better than 96% of randomized alignments. This evidence supports the hypothesis that FMO protein is related to PscA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ultrafast multi-pulse transient absorption spectroscopy of fucoxanthin chlorophyll a protein from Phaeodactylum tricornutum

We have applied femtosecond transient absorption spectroscopy in pump-probe and pump-dump-probe regimes to study energy transfer between fucoxanthin and Chl a in fucoxanthin-Chl a complex from the pennate diatom Phaeodactylum tricornutum. Experiments were carried out at room temperature and 77?K to reveal temperature dependence of energy transfer. At both temperatures, the ultrafast (<100?fs) energy transfer channel from the fucoxanthin S₂ state is active and is complemented by the second pathway via the combined S₁/ICT state. The S₁/ICT-Chl a pathway has two channels, the fast one characterized by sub-picosecond energy transfer, and slow having time constants of 4.5?ps at room temperature and 6.6?ps at 77?K. The overall energy transfer via the S₁/ICT is faster at 77?K, because the fast component gains amplitude upon lowering the temperature. The pump-dump-probe regime, with the dump pulse centered in the spectral region of ICT stimulated emission at 950?nm and applied at 2?ps after excitation, proved that the S₁ and ICT states of fucoxanthin in FCP are individual, yet coupled entities. Analysis of the pump-dump-probe data suggested that the main energy donor in the slow S₁/ICT-Chl a route is the S₁ part of the S₁/ICT potential surface.     

Excitation transfer in chlorosomes of green photosynthetic bacteria

The formation of excited states and energy transfer in chlorosomes of the green photosynthetic bacteria Chlorobium limicola and Chloroflexus aurantiacus were studied by measurements of flash-induced absorbance changes and fluorescence. Upon excitation with 35 ps, 532 nm flashes, large absorbance decreases around 750 nm were observed that were due to the disappearance of ground state absorption of the main pigment, bacteriochlorophyll (BChl) c. The absorbance changes decayed after the flash with a time constant of approx. 1 ns, together with faster components. Absorbance changes that could be ascribed to formation of excited BChl a were much smaller than those of BChl c. The yields of BChl c and BChl a fluorescence were measured as a function of the energy density of the exciting flash. At high energy a strong quenching occurred caused by annihilation of singlet excited states. An analysis of the results shows that energy transfer between BChl c molecules is very efficient and that in C. limicola excitations can probably move freely through the entire chlorosome (which contains about 10 000 BChls c). The chlorosome thus serves as a common antenna for several reaction centres. The small amounts of BChl a present in the chlorosomes of both species form clusters of only a few molecules. Upon cooling to 4 K the sizes of the domains of BChl c for energy transfer decreased considerably. The results are discussed in relation to recently suggested models for the pigment organization within chlorosomes.     

The size of the photosynthetic unit in purple bacteria

Pigment analysis was performed by means of normal phase HPLC on a number of bacteriochlorophyll a and b containing species of purple bacteria that contain a core antenna only. At least 99% of the bacteriochlorophyll in Rhodobacter sphaeroides R26, Rhodopseudomonas viridis and Thiocapsa pfennigii was esterified with phytol (BChl a _p and BChl b _p, respectively). Rhodospirillum rubrum contained only BChl a esterified with geranyl-geraniol (BChl a _GG). Rhodospirillum sodomense and Rhodopseudomonas marina contained, in addition to BChl a _p, small amounts of BChl a _GG, and presumably also of BChl a esterified with dihydro and tetrahydro geranyl-geraniol (2,10,14-phytatrienol and probably 2,14-phytadienol). In all species bacteriopheophytin (BPhe) esterified with phytol was present. The BChl/BPhe ratio indicated that in these species a constant number of 25 ± 3 antenna BChls is present per reaction centre. This number supports a model in which the core antenna consists of 12 - heterodimers surrounding the reaction centre. Determination of the in vivo extinction coefficient of BChl in the core-reaction centre complex yielded a value of ca. 140 mM^–1 cm^–1 for BChl a containing species and of 130 mM^–1 cm^–1 for Rhodopseudomonas viridis.Abbreviations BChl bacteriochlorophyll - BPhe bacteriopheophytin - GG geranyl-geraniol - LHI and LHII core and peripheral antenna complexes - P phytol - RC reaction centre Dedicated to the memory of Professor D.I. Arnon.     

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Q_y absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. One-color and two-color pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.     

Redox effects on the bacteriochlorophyll α-containing Fenna-Matthews-Olson protein fromChlorobium tepidum

The BChla-containing Fenna-Matthews-Olson (FMO) protein from the green sulfur bacteriumChlorobium tepidum was purified and characterized. Fluorescence spectra indicate that efficient excited state quenching occurs at neutral or oxidizing redox potentials. The major fluorescence lifetime at room temperature is approximately 60 ps in samples that are in neutral or oxidizing conditions, and approximately 2 ns in samples where the strong reductant sodium dithionite has been added. A similar change is observed in pump-probe picosecond absorbance difference experiments, where the long life time component increases after dithionite addition. A 16 Gauss wide EPR signal with g factor =2.005 is observed in samples without dithionite. This signal largely disappears upon addition of dithionite. Dithionite induces large reversibile changes in the 77 K absorbance spectra of the purified FMO protein and in whole cells. These results indicate that the FMO protein contains redox active groups, which may be involved in the regulation of energy transfer. Room temperature circular dichroism and low temperature absorption spectra show that dithionite also induces conformational or structural changes of the FMO protein complex.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30–35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808–866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Q_y transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Q_y transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808–866 complex of C. aurantiacus and the B800–850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 was derived.Abbreviations BChl bacteriochlorophyll - BPheo bacteriopheophytin - CD circular dichroism - LD linear dichroism - Tris Tris(hydroxymethyl)aminomethane     

Utilization of blue-green light by chlorosomes from the photosynthetic bacterium Chloroflexus aurantiacus: Ultrafast excitation energy conversion and transfer

Chlorosomes of photosynthetic green bacteria are unique molecular assemblies providing efficient light harvesting followed by multi-step transfer of excitation energy to reaction centers. In each chlorosome, 10⁴–10⁵ bacteriochlorophyll (BChl) c/d/e molecules are organized by self-assembly into high-ordered aggregates. We studied the early-time dynamics of the excitation energy flow and energy conversion in chlorosomes isolated from Chloroflexus (Cfx.) aurantiacus bacteria by pump-probe spectroscopy with 30-fs temporal resolution at room temperature. Both the S₂ state of carotenoids (Cars) and the Soret states of BChl c were excited at ~490 nm, and absorption changes were probed at 400–900 nm. A global analysis of spectroscopy data revealed that the excitation energy transfer (EET) from Cars to BChl c aggregates occurred within ~100 fs, and the Soret → Q energy conversion in BChl c occurred faster within ~40 fs. This conclusion was confirmed by a detailed comparison of the early exciton dynamics in chlorosomes with different content of Cars. These processes are accompanied by excitonic and vibrational relaxation within 100–270 fs. The well-known EET from BChl c to the baseplate BChl a proceeded on a ps time-scale. We showed that the S₁ state of Cars does not participate in EET. We discussed the possible presence (or absence) of an intermediate state that might mediates the Soret → Q_y internal conversion in chlorosomal BChl c. We discussed a possible relationship between the observed exciton dynamics and the structural heterogeneity of chlorosomes.     

Determination of the primary charge separation rate in Photosystem II reaction centers at 15 K

We have measured the rate constant for the formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a ^– (Pheo a ^–) following excitation of isolated Photosystem II reaction centers (PS II RC) at 15 K. This PS II RC complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure which stabilizes the protein complex. Transient absorption difference spectra were measured from 450–840 nm as a function of time with 500fs resolution following 610 nm laser excitation. The formation of P680⁺-Pheo a ^– is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 490, 515 and 546 nm due to the formation of Pheo a ^–. The appearance of the 490 nm and 820 nm bands is monoexponenital with =1.4±0.2 ps. Treatment of the PS II RC with sodium dithionite and methyl viologen followed by exposure to laser excitation results in accumulation of Pheo a ^–. Laser excitation of these prereduced RCs at 15 K results in formation of a transient absorption spectrum assigned to ^1*P680. We observe wavelength-dependent kinetics for the recovery of the transient bleach of the Q_y absorption bands of the pigments in both untreated and pre-reduced PS II RCs at 15K. This result is attributed to an energy transfer process within the PS II RC at low temperature that is not connected with charge separation.Abbreviations PS I Photosystem I - PS II Photosystem II - RC reaction center - P680 primary electron donor in Photosystem II - Chl a chlorophyll a - Pheo a pheophytin a     

Linear and nonlinear optical responses in bacteriochlorophyll a

Nonlinear optical responses of bacteriochlorophyll a (BChl a) were investigated by means of the three-pulse four-wave mixing (FWM) technique under the resonant excitation into the Q _y band. The experimental results are explained by a theoretical model calculation including the Brownian oscillation mode of the solvent. We have determined the spectral density, which is the most important function with which to calculate optical signals. The linear absorption spectrum can be reproduced fairly well when the vibronic oscillation modes of the solvent together with those of BChl a are properly taken into consideration. The FWM signal was also calculated using the spectral density. It was found that a simple two-level model could not explain the experimental result. The effect of the higher-order interactions is discussed.     

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.