The effect of temperature on the aggregation of 3(l)R-8,12-diethyl farnesyl bacteriochlorophyll c in a mixture of n-pentane and methylcyclohexane (1/1, v/v) was studied by means of absorption, circular dichroism and fluorescence spectroscopy. At room temperature essentially only two aggregate species, absorbing at 702 nm (A-702) and 719 nm (A-719), were present. Upon cooling to 219 K, A-702 was quantitatively converted to A-719. Further lowering of the temperature led to the stepwise formation of larger aggregates by the conversion of A-719 to aggregate species absorbing at 743 nm (A-743) and 755 nm (A-755). All absorption changes were reversible. A-719 was highly fluorescent (maximum at 192 K: 744 nm), while A-743 and especially A-755 were weakly fluorescent. Below 130 K the mixture solidified, and no major changes in the absorption spectrum were observed upon further cooling. At 45 K, however, a relatively strong emission at 775 nm was observed. Below 200 K, the absorption, fluorescence and circular dichroism spectra resembled that of the chlorosome. These results open up the possibility to study higher aggregates of BChl c as models for the chlorosome by various methods at low temperature, thus avoiding interference by thermal processes.

A recently isolated species of the photosynthetic purple sulfur bacteria, provisionally called strain 970, was investigated with respect to its antenna function by means of various spectroscopic techniques, including fluorescence and pump-probe absorption difference spectroscopy. The bacterium contains bacteriochlorophyll a and an as yet unidentified carotenoid, perhaps 3,4,3',4'-tetrahydrospirilloxanthin. It has a single antenna complex of the LH1 type, with a Q(y) absorption band situated at the unusually long wavelength of 963 nm at room temperature and 990 nm at 6 K. In contrast to many other species, the reaction center showed two well-separated absorption bands of bacteriopheophytin at 6 K, located at 747 and 762 nm. The primary electron donor showed a bleaching band centered at 925 nm upon photooxidation. Thus, the energy gap between LH1 and the primary electron donor is quite large in this strain: 425 cm(-1). Nevertheless, trapping occurred with a time constant of 65 +/- 5 ps, similar to the rates observed in other purple bacteria. As in other species, no back-transfer from the reaction center to the antenna was observed. Our results show that strain 970 is a unique subject for the study of antenna and reaction center function and organization.

A survey is given of various aspects of the photosynthetic processes in heliobacteria. The review mainly refers to results obtained since 1995, which had not been covered earlier. It first discusses the antenna organization and pigmentation. The pigments of heliobacteria include some unusual species: bacteriochlorophyll (BChl) g, the main pigment, 8(1) hydroxy chlorophyll a, which acts as primary electron acceptor, and 4,4'-diaponeurosporene, a carotenoid with 30 carbon atoms. Energy conversion within the antenna is very fast: at room temperature thermal equilibrium among the approx. 35 BChls g of the antenna is largely completed within a few ps. This is then followed by primary charge separation, involving a dimer of BChl g (P798) as donor, but recent evidence indicates that excitation of the acceptor pigment 8(1) hydroxy chlorophyll a gives rise to an alternative primary reaction not involving excited P798. The final section of the review concerns secondary electron transfer, an area that is relatively poorly known in heliobacteria.

The conversion of excitation energy in the antenna reaction center complex of Heliobacillus mobilis was investigated at 10 K as well as at 275 K by means of time-resolved absorbance difference spectroscopy of isolated membranes in the (sub)picosecond time range. Selective excitation of the primary electron acceptor, chlorophyll (Chl) a 670, and of the different spectral pools of bacteriochlorophyll (BChl) g (BChl g 778, BChl g 793, and BChl g 808) was applied. At 10 K, excitation at 770 or 793 nm resulted on the one hand in rapid energy transfer to BChl g 808 and on the other hand in fast charge separation from excited BChl g 793 ( approximately 1 ps). Once the excitations were on BChl g 808, the bleaching band shifted gradually to the red, from 806 to 813 nm, and charge separation from excited BChl g 808 occurred by a very slow process ( approximately 500 ps). The main purpose of our experiments was to answer the question whether an "alternative" pathway for charge separation exists upon excitation of Chl a 670. Our measurements showed that the amount of oxidized primary donor (P798(+)) relative to that of excited BChl g produced by excitation of Chl a 670 was considerably larger than upon direct excitation of BChl g. This indicates the existence of an alternative pathway for charge separation that does not involve excited antenna BChl g. This effect occurred at 10 K as well as at 275 K. The mechanism for this process is discussed in relation to different trapping models; it is concluded that charge separation occurs directly from excited Chl a 670.

We have studied energy transfer in chlorosomes of Chlorobium limicola UdG6040 containing a mixture of about 50% bacteriochlorophyll (BChl) c and BChl d each. BChl d-depleted chlorosomes were obtained by acid treatment. The energy transfer between the different pigment pools was studied using both steady-state and time-resolved fluorescence spectroscopy at room temperature and low temperature. The steady-state emission of the intact chlorosome originated mainly from BChl c, as judged by comparison of fluorescence emission spectra of intact and BChl d-depleted chlorosomes. This indicated that efficient energy transfer from BChl d to BChl c takes place. At room temperature BChl c/d to BChl a excitation energy transfer (EET) was characterized by two components of 27 and 74 ps. At low temperature we could also observe EET from BChl d to BChl c with a time constant of approximately 4 ps. Kinetic modeling of the low temperature data indicated heterogeneous fluorescence kinetics and suggested the presence of an additional BChl c pool, E790, which is more or less decoupled from the baseplate BChl a. This E790 pool is either a low-lying exciton state of BChl c which acts as a trap at low temperature or alternatively represents the red edge of a broad inhomogeneous absorption band of BChl c. We present a refined model for the organization of the spatially separated pigment pools in chlorosomes of Cb. limicola UdG6040 in which BChl d is situated distal and BChl c proximal with respect to the baseplate.

Electron transfer in reaction center core (RCC) complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum was studied by measuring flash-induced absorbance changes. The first preparation contained approximately three iron-sulfur centers, indicating that the three putative electron accepters F-X, F-A, and F-B were present; the Chl. tepidum complex contained on the average only one. In the RCC complex of Ptc. aestuarii at 277 K essentially all of the oxidized primary donor (Ps40(+)) created by a flash was rereduced in several seconds by N-methylphenazonium methosulfate. In RCC complexes of Chl. tepidum two decay components, one of 0.7 ms and a smaller one of about 2 s, with identical absorbance difference spectra were observed. The fast component might be due to a back reaction of P840(+) with a reduced electron acceptor, in agreement with the notion that the terminal electron accepters, F-A and F-B, were lost in most of the Chl. tepidum complexes. In both complexes the terminal electron acceptor (F-A or F-B) could be reduced by dithionite, yielding a back reaction of 170 ms with P840(+). At 10 K in the RCC complexes of both species P840(+) was rereduced in 40 ms, presumably by a back reaction with F-X(-). In addition, a 350 mu s component occurred that can be ascribed to decay of the triplet of P840, formed in part of the complexes. For P840(+) rereduction a pronounced temperature dependence was observed, indicating that electron transfer is blocked after F-X at temperatures below 200 K.

Energy relaxation on the bacteriochlorophyll B850 aggregate of the isolated light-harvesting antenna complex LH2 of Rhodopseudomonas acidophila at 7 K was examined by means of two-color femtosecond absorption spectroscopy. Upon excitation at 860 nm, at the blue side of the B850 absorption maximum, two kinetic components were observed in the subpicosecond time region: a major component of 100 fs and a minor one of 700 fs. The spectrum of the first component was typical for energy relaxation in an excitonically coupled system. The same two components were observed in the position of the isosbestic wavelength of the absorbance difference spectrum, which shifted to the red by 4 nm with time. Anisotropy decay to a final value of 0.07 mainly occurred within our time resolution of 70 fs. This corresponds to a homogeneous line width of B850 of at least 80 cm(-1). In addition, a weak slow phase in the anisotropy decay of 700 fs with an amplitude of 5-10% of the total decay was observed. In the absorbance kinetics, time constants of 100 and 700 fs were also observed upon excitation at 880 nm, which is at the red side of the absorption band, but now the spectrum only shifted by about 0.5 nm with time. In this case the decay of the anisotropy occurred within the time resolution to a final value of 0.12-0.14. The final position of the difference spectrum upon excitation at 880 nm was red-shifted by 1 nm with respect to that excited at 860 nm.

Excited-state and electron-transfer dynamics at cryogenic temperature in reaction center core (RCC) complexes of the photosynthetic green sulfur bacterium Prosthecochloris aestuarii were studied by means of time-resolved absorption spectroscopy, using selective excitaton of bacteriochlorophyll (BChl) a and of chlorophyll (Chl) a 670. The results indicate that the BChls a of the RCC complex form an excitonically coupled system. Relaxation of the excitation energy within the ensemble of BChl a molecules occurred within 2 ps. A time constant of about 25 ps was ascribed to charge separation. Absorption changes in the 670 nm region, where Chl a 670 absorbs, were fairly complicated. They showed various time constants and were dependent on the wavelength of excitation and they did not lead to a simple picture of the electron acceptor reaction. Energy transfer from Chl a 670 to BChl a occurred with a time constant of 1.5 ps. However, upon excitation of Chl a 670 the amount of oxidized primary electron donor, P840(+), formed relative to that of excited BChl a was considerably larger than upon direct excitation of BChl a. This indicates the existence of an alternative pathway for charge separation which does not involve excited BChl a.

Photoaccumulation at 205 K in the presence of dithionite produces EPR signals in anaerobically prepared membranes from Chlorobium limicola and Heliobacterium chlorum that resemble the EPR spectrum of phyllosemiquinone (A1*-) photoaccumulated in photosystem I. We have used ENDOR and special TRIPLE resonance spectroscopy to demonstrate conclusively that these signals arise from menasemiquinone electron acceptors reduced by photoaccumulation. Hyperfine couplings to two protons H-bonded to the semiquinone oxygens have been identified by exchange of H. chlorum into D2O, and hyperfine couplings to the methyl group, and the methylene group of the phytyl side chain, of the semiquinone have also been assigned. The electronic structure of these menasemiquinones in these reaction centers is very similar to that of phyllosemiquinone in PSI, and shows a distorted electron spin density distribution relative to that of phyllosemiquinone in vitro. Special TRIPLE resonance spectrometry has been used to investigate the effect of detergents and oxygen on membranes of C. limicola. Triton X-100 and oxygen affect the menaquinone binding site, but n-dodecyl beta-D-maltoside preparations exhibit a relatively unaltered special TRIPLE spectrum for the photoaccumulated menasemiquinone.

Green sulfur bacteria contain reaction centre core (RCC) complexes of the iron-sulfur type. The RCC contains a homodimer of the 82 kD PscA protein, that binds the primary donor P840 (a BChl a dimer), the primary acceptor Chi a 670 and the Fe-S centre Fx. Two more Fe-S centres (FA, and FB) are bound by the 32 kD PscB protein (1–3). Earlier we described the isolation of photochemically active RCC complexes from Prosthecochloris aestuarii (4). Here we describe some of the biochemical and spectroscopic properties of this preparation and of a new preparation from Chlorobium tepidum.

An improved method for fusion of liposomes to intracytoplasmic membrane vesicles of Rhodobacter sphaeroides was developed that involves repeated cycles of freeze-thaw-sonication and provides a controlled procedure for phospholipid enrichment of up to 15-fold. In freeze-fracture replicas, the fusion products appeared as closed vesicles of increased size and reduced intramembrane particle densities. Fluorescence yield measurements at 300 and 4 K showed that the gradual bilayer dilution was accompanied by reductions in energy transfer between the peripheral LH2 and core LH1 antennae, as well as from LH1 to reaction centers. Singlet-singlet annihilation at 4 K revealed a two-fold decrease in the cluster size of core antenna BChls, which was also reflected by changes in fluorescence polarization spectra. Energy transfer dynamics and structural considerations suggested that the annihilation curves were affected by non-uniformities. When taken into account, this led to the conclusion that in native membranes, on average two LH1-reaction center complexes are associated, that most peripheral antenna complexes are adjacent to at least one core assembly, and that fusion induces a separation of single LH1 and LH2 rings. At 4 K, a relatively large Stokes shift severely limits transfer between LH2 complexes in the native bilayer, while restricted transfer among two or three LH1 complexes arises mainly from spectral inhomogeneity. This explanation also implies that the anisotropic long-wavelength component of the LH1 absorption spectrum, which acts as an energy trap at 4 K, exists as an excitonic state involving 6–8 BChls.

The light harvesting 2 (LH2) complex of purple bacteria shows a circular symmetry with 2 rings of closely spaced bacteriochlorophyll (BChl) molecules (1). One ring contains 9 BChl a molecules absorbing at 800 nm (B800), the other ring contains 18 BChl a molecules absorbing at 870 nm (B850). This particular structure makes it an attractive model system for studies of energy transfer and of the influence of static and dynamic disorder on the electronic structure of strongly coupled pigment systems.

A permanent hole burning study on the Fenna-Matthews-Olson, or FMO, antenna complex of the green sulfur bacterium Prosthecochloris aestuarii was carried out at 6 K. Excitation resulted not only in relatively sharp features resonant with the burn wavelength but also in broad absorbance changes in the wavelength region of 800-820 nm. The shape of the latter changes was almost independent of the wavelength of excitation. Evidence is given that they are induced by a different mechanism than that which causes the resonant holes and that they may be due to a conformational change of the protein. The original spectrum was restored upon warming to 60 K. The effective dephasing times T2, as obtained from the homogeneous line widths, increased from about 0.5 ps at 803 nm to >/=20 ps at 830 nm and are in good agreement with recent measurements of accumulated photon-echo and time-resolved absorbance changes.

The Fenna-Matthew-Olson (FMO) complex of green sulfur bacteria consists of an arrangement of 3 identical subunits, with a 3-fold rotational symmetry axis (1-3). Each subunit contains 7 bacteriochlorophylls (BChls) a, which gives a total of 21 pigments for the entire complex. The nearest-neighbor distances (center-to-center) within one subunii vary from 11 to 14 Å, while the distance between nearest neighbors in different subunits of the trimer is about 24 Å. Only minor differences were found in the positions and orientations of the various BChls in the FMO-complexes of Prosthecochloris (P.) aestuari (1,2) and Chlorobium (C.) tepidum (3). However, the amino acid sequences show only 78

We have demonstrated [1] that photoaccumulation under reducing conditions at 205K produces an electron paramagnetic resonance (EPR) signal arising from a stable semiquinone form of the phylloquinone electron acceptor (A1•−) in Photosystem I (PSI) preparations. Electron Nuclear Double Resonance (ENDOR) studies of the phyllosemiquinone [2] have detected hyperfine couplings arising from protons, and thus given information on the electronic structure of the phyllosemiquinone in PSI.

Absorption difference spectra for the singlet excited states of the Fenna-Matthews-Olson (FMO) complex from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum were simulated by exciton theory. The same assumptions and parameters were used as applied earlier (Louwe, R. J. W.; Vrieze, J.; Hoff, A. J.; Aartsma, T. J. J. Phys. Chem. B 1997, 101, 11280. Vulto, S. I. E.; de Baat, M. A.; Louwe, R. T. W.; Permentier, H. P.; Neef, T.; Miller, M.; van Amerongen, H.; Aartsma, T. J. J. Phys. Chem. B 1998, 102, 9577). The difference spectra show a bleaching near the wavelength of excitation, due to ground-state bleaching and stimulated emission. Additional negative and positive bands reflect changes in interaction with other bacteriochlorophylls than the one that is mainly excited at the transition frequency. Simulated spectra were compared with experimental difference spectra obtained by pump-probe experiments in the femto- and picosecond time region with excitation pulses in the spectral range 800-828 nm. In general, good agreement was obtained. Various difference spectra developed during the first 1 to 2 ps, but in all cases the system relaxed to the lowest energy state, which was largely completed in 10 ps.

Rapid conversion of bacteriochlorophyll g (BChl g) to chlorophyll a (Chl a) was observed in acetone on addition of acid in the dark. The product, Chl a esterified with farnesol (Chl aF), was identified by liquid chromatography and fast atom bombardment mass spectrometry. Acid-catalyzed formation of 81-OH-Chl aF, a primary electron acceptor in the heliobacterial reaction center, was also observed in diethyl ether in the dark. These results suggest that acid-catalyzed isomerization is a candidate for the chemical evolution of BChl g to the more stable Chl a and that 81-OH-Chl aF can easily be synthesized from BChl g under weakly acidic conditions in the dark.

The antenna system of green sulfur bacteria consists of three components: the chlorosome, the Fenna Matthews Olson (FMO) protein, and the reaction center core (RCC) complex. The RCC complex resembles the reaction centers of photosystem I and of heliobacteria. The primary donor, P840, is a bacteriochlorophyll (BChl) a dimer and the primary acceptor, A0, is a Chi a-like pigment absorbing near 670 nm. The role of a menaquinone as secondary electron acceptor, A1, is still a matter of debate. As in photosystem I, the further electron transfer involves three iron-sulfur (Fe-S) centers, Fx, FA and FB (1).

The excited states of bacteriochlorophyll (BChl) a were studied by pump-probe transient absorption spectroscopy in reaction center core (RCC), Fenna-Matthews-Olson (FMO) and FMO-RCC complexes of the green sulfur bacterium Prosthecochloris aestuarii. Excitation at 790 or 835 nm resulted in rapid equilibration of the energy between the BChl a molecules of the RCC complex: within 1 ps, most of the excitations had relaxed to the lowest energy level (835 nm), as a result of strong interactions between the BChls. Excitation of chlorophyll a 670 resulted in energy transfer to BChl a with a time constant of 1.2 ps, followed by thermal equilibration. Independent of the wavelength of excitation, the decay at 835 nm could be fitted with a time constant of about 25 ps, comparable to the 30 ps measured earlier with membrane fragments, which is ascribed to trapping in the reaction centers. Similar results were obtained with the FMO-RCC complex upon excitation at 835 or 670 nm, but the results upon 790 nm excitation were quite different. Again an equilibrium was rapidly reached, but now most of the excitations remained within the FMO complex, with a maximum bleaching at 813 nm, the same as observed in the isolated FMO. Even after 100 ps there was no bleaching at 835 nm and no evidence for charge separation. We conclude that there is no equilibration of the energy between the FMO and the RCC complex and that the efficiency of energy transfer from FMO to the reaction center core is low.