The antenna reaction center complex of heliobacteria: composition, energy conversion and electron transfer.

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.

A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm.

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.

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum.

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.

Electron transfer in reaction center core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum.

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 acceptors 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 (P840(+)) 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 acceptors, 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 micros 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.

Pathways of energy transformation in antenna reaction center complexes of Heliobacillus mobilis.

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.

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 13(2)-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.

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium.

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the 'two-color' measurements at 820 and 835 nm upon excitation at 800 nm, whereas the 'one-color' kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps.

Dynamics of energy conversion in reaction center core complexes of the green sulfur bacterium Prosthecochloris aestuarii at low temperature.

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.

Excited states and trapping in reaction center complexes of the green sulfur bacterium Prosthecochloris aestuarii.

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.

A permanent hole burning study of the FMO antenna complex of the green sulfur bacterium Prosthecochloris aestuarii.

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.

Isolation and properties of photochemically active reaction center complexes from the green sulfur bacterium Prosthecochloris aestuarii.

A new and rapid procedure was developed for the isolation of the reaction center core (RCC)-complex from the green sulfur bacterium Prosthecochloris aestuarii. Reaction center preparations containing the Fenna Matthews Olson (FMO) protein were also obtained. The procedure involved incubation of broken cells with the detergents Triton X-100 and SB12, sucrose gradient centrifugation and hydroxyapatite chromatography. Three different pigment protein complexes were obtained: one containing (about) three FMO trimers per RCC, one with one FMO per RCC and one consisting of RCC only. The last one contained polypeptides with apparent molecular masses of 64 kDa (pscA) and 35 kDa (pscB, the FA/FB, FeS subunit), but no cytochrome. Bacteriochlorophyll a and the chlorophyll a isomer functioning as primary electron acceptor were present at a ratio of 4.8:1. The complexes were also characterized spectroscopically and in terms of photochemical activity, at room temperature as well as at cryogenic temperatures. Illumination caused oxidation of the primary donor P840, with the highest activity in the RCC complex (DeltaA840/A810 = 0.06). At room temperature in the RCC complex essentially all of the P840+ produced in a flash was re-reduced slowly in the dark (several seconds). At low temperatures (150-10 K) a triplet was formed in a fraction of the reaction centers, presumably by a reversal of the charge separation, whereas in others P840+ formed in the light was re-reduced in 40-50 ms.