B800-850 Complex Research Articles

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of water-soluble and immobilized (on BrCN-activated agarose) manganese-stabilizing protein PsbO with Mn2+ and Mn3+ cations and with preparations of D1/D2/cyt b559 reaction center (RC) of photosystem II was studied. By native electrophoresis, the formation of dimeric and aggregated forms of PsbO protein were found in the presence of Mn2+, Mg2+, or Fe2+ ions. The dimerization of PsbO occurred after ultraviolet irradiation of the protein preparation. The presence of protein-bound Mn3+ cations increased the electrostatic interaction of the immobilized PsbO with the RC. This was evidenced to by higher amounts of CaCl2 that were required for dissociation of the PsbORC complex. It was first demonstrated that the protein exhibited superoxide dismutase (SOD) activity after an electrophoresis in PAAG upon incubation of the gel in an Mn2+-containing solution. Tetrazolium-reductase activity was also ascertained in the protein after its electrophoresis in a mixture with preparations of the oxygen-evolving complex (OEC). It is suggested that the protein interaction with Mn ions and superoxide radicals, as well as short-term UV irradiation, reduces tyrosine and a disulfide bond in the PsbO protein. This yields tyrosil radical and SH-groups participating in redox reactions with ETC components. The interactions of PsbO with Mn cations and UV light, taking place in the chloroplast thylakoids, may regulate the protein binding to RC, modify structural organization of the protein, and promote its participation in alternative pathways of electron transport under the influence of stress factors. The hypothetical scheme of interaction of the immobilized PsbO protein with Mn ions and RC is discussed.

Proteomic analysis of the electron uptake pathway of Rhodopseudomonas palustris CGA009 under different cathodic potentials

Microbial electrosynthesis can convert greenhouse gas CO2 into value-added chemicals at room temperature and mild conditions, and the efficiency of its reaction is closely dependent on the electrode potential and electron transport chain-related functional proteins. In this study, the photosynthetic bacterium Rhodopseudomonas palustris CGA009 was used to explore CO2 bioreduction behavior at different potentials (−0.2 V, −0.5 V, −0.8 V). Label-free quantitative proteomics was used to analyze the mechanism of microbial electron transfer. The results showed that the cathodic potential could positively affect the NAD(P)-binding domain-containing protein of R. palustris CGA009. Ubiquinone and other terpenoid-quinone biosynthesis pathways that are mainly responsible for NAD(P)-binding domain-containing proteins also showed an upregulating trend in the KEGG analysis. The electrons are transferred to the photosynthetic chain through the B800-850 complex to collect light energy, and then the photosynthetic reaction center uses this energy to generate a proton gradient for transmembrane transport, allowing R. palustris CGA009 to use electrons for the fixation process of CO2. Our results enlighten the role of proteins in electron transfer chain of microbial electrosynthesis and provide an insight into how to regulate the microbial process to produce value-added chemicals.

LH2 complexes (B800-850 AND B800-830) are assembled in the cells of sulfur bacterium Thiorhodospira sibirica, strain Kir 3, without any carotenoids

It has been studied the result of assembling the light-harvesting complexes in the cells of purple sulfuric bacterium Thiorhodospira (T.) sibirica, strain Kir‑3, while suppressing biosynthesis of carotenoids by diphenylamine (DPA). LH2 complexes (B800-850 and B800-830) with different carotenoids’ composition were isolated from the cells obtained. Maximal inhibition of carotenoid biosynthesis (~90% of the control) was achieved at the inhibitor concentration of 53,25 mM (9 mg/l). It has been established that changes in qualitative and quantitative composition of carotenoids do not affect the assembling of B800-830 and B800-850 complexes. It is assumed that in the population of DPA-LH2 complexes from T. sibirica, strain Kir‑3, both carotenoidless complexes and the complexes, containing one or two carotenoid molecules, can be assembled. These results support a hypothesis that carotenoids are not required for assembling B800-850 and B800-830 complexes.

The LH2 Complexes (B800-850 and B800-830) Are Assembled in the Cells of the Sulfur Bacterium Thiorhodospira sibirica Strain Kir-3 without Carotenoids.

The results of assembling the light-harvesting complexes in the cells of the purple sulfur bacterium Thiorhodospira (T.) sibirica strain Kir-3 while suppressing the biosynthesis of carotenoids with diphenylamine (DPA) were studied. LH2 complexes (B800-850 and B800-830) with different carotenoid composition were isolated from the cells obtained. Maximum inhibition of carotenoid biosynthesis (~90% of the control) was reached at an inhibitor concentration of 53.25 μM (9 mg/L). It was established that changes in the qualitative and quantitative composition of carotenoids do not affect the assembly of B800-830 and B800-850 complexes. It is assumed that, in the population of DPA-LH2 complexes from T. sibirica strain Kir-3, both the carotenoidless complexes and the complexes containing one or two carotenoid molecules can be assembled. These results support the hypothesis that carotenoids are not required for assembling B800-850 and B800-830 complexes.

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assembly of peripheral light-harvesting complexes in purple sulfur bacteria Allochromatium vinosum ATCC 17899

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain- increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodovibrin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphe- nylamine (DPA) concentrations not exceeding 53 .iM, while at illumination intensity decreased to 500 Ix they could grow at 71 jiM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoine synthetase (resulting in a decrease in the total carotenoid content) and of phyto- ine desturase, which results in formation of neurosporene hydroxy derivatives and ;-carotene. In the cells grown at 500 lx, small amounts of spheroidene and.OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was -15% of the control (without inhibition) for the B800-850 and -20%of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with -carotenoids due to- disintegration of some carotenoid-free complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be Assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.

Bicarbonate stimulates the electron donation from Mn2+ to [formula omitted] in isolated D1/D2/cytochrome b559 complex

Influence of bicarbonate on the efficiency of the electron donation from Mn2+ to P680+ in isolated D1/D2/cytochrome b559 complex was investigated. All the experiments were carried out in a medium depleted of HCO3-/CO2. Kinetics of photoinduced absorbance changes (ΔA) at different wavelengths and decrease of chlorophyll fluorescence yield (−ΔF) related to photoaccumulation of reduced pheophytin, the intermediary electron acceptor of photosystem II (PSII), in the presence of Mn2+ under anaerobic conditions were measured. Addition of bicarbonate (1mM) increased the amplitude of these ΔA and −ΔF at least by a factor of 3. Measurements of the photoinduced ΔA, related to photooxidation of the primary electron donor of PSII, chlorophyll P680, were done in the presence of silicomolybdate as electron acceptor. These results show that the addition of 0.05mM Mn2+ alone or jointly with 1mM bicarbonate induces a 20% and 70%-decrease of the magnitude of the ΔA at 680nm. The effect of Mn2+ (in the presence and absence of bicarbonate) was completely eliminated by the addition of 12mM EDTA. All these bicarbonate effects were not observed if MgCl2 or formate were used instead of MnCl2 and bicarbonate, respectively. In the absence of Mn2+, bicarbonate induced none of the mentioned above effects (increase of photoaccumulation of reduced pheophytin and decrease of photooxidation of P680). The presented data suggest that bicarbonate stimulates the electron donation from Mn2+ to D1/D2/cyt b559 reaction center evidently due to formation of easily oxidizable Mn-bicarbonate complexes.

Bicarbonate stabilizes isolated D1/D2/cytochrome b559 complex of photosystem 2 against thermoinactivation.

It has been shown that thermoinactivation of the isolated D1/D2/cytochrome b(559) complex (RC) of photosystem 2 (PS-2) from pea under anaerobic conditions at 35°C in 20 mM Tris-HCl buffer (pH 7.2) depleted of HCO(3)(-), with 35 mM NaCl and 0.05% n-dodecyl-β-maltoside, results in a decrease in photochemical activity measured by photoreduction of the PS-2 primary electron acceptor, pheophytin (by 50% after 3 min of heating), which is accompanied by aggregation of the D1 and D2 proteins. Bicarbonate, formate, and acetate anions added to the sample under these conditions differently influence the maintenance of photochemical activity: a 50% loss of photochemical activity occurs in 11.5 min of heating in the presence of bicarbonate and in 4 and 4.6 min in the presence of formate and acetate, respectively. The addition of bicarbonate completely prevents aggregation of the D1 and D2 proteins as opposed to formate and acetate (their presence has no effect on the aggregation during thermoinactivation). Since the isolated RCs have neither inorganic Mn/Ca-containing core of the water-oxidizing complex nor nonheme Fe(2+), it is supposed that bicarbonate specifically interacts with the hydrophilic domains of the D1 and D2 proteins, which prevents their structural modification that is a signal for aggregation of these proteins and the loss of photochemical activity.

P 680 (P D1P D2) and Chl D1 as alternative electron donors in photosystem II core complexes and isolated reaction centers

Low temperature (77–90 K) measurements of absorption spectral changes induced by red light illumination in isolated photosystem II (PSII) reaction centers (RCs, D1/D2/Cyt b559 complex) with different external acceptors and in PSII core complexes have shown that two different electron donors can alternatively function in PSII: chlorophyll (Chl) dimer P 680 absorbing at 684 nm and Chl monomer Chl D1 absorbing at 674 nm. Under physiological conditions (278 K) transient absorption difference spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach PSII core complexes excited at 710 nm. It was shown that the initial electron transfer reaction takes place with a time constant of ∼0.9 ps. This kinetics was ascribed to charge separation between P 680 ∗ and Chl D1 absorbing at 670 nm accompanied by the formation of the primary charge-separated state P 680 + Chl DI - , as indicated by 0.9-ps transient bleaching at 670 nm. The subsequent electron transfer from Chl D 1 - occurred within 13–14 ps and was accompanied by relaxation of the 670-nm band, bleaching of the Pheo D1 Q x absorption band at 545 nm, and development of the anion-radical band of Pheo D 1 - at 450–460 nm, the latter two attributable to formation of the secondary radical pair P 680 + Pheo D 1 - . The 14-ps relaxation of the 670-nm band was previously assigned to the Chl D1 absorption in isolated PSII RCs [Shelaev, Gostev, Nadtochenko, Shkuropatov, Zabelin, Mamedov, Semenov, Sarkisov and Shuvalov, Photosynth. Res. 98 (2008) 95–103]. We suggest that the longer wavelength position of P 680 (near 680 nm) as a primary electron donor and the shorter wavelength position of Chl D1 (near 670 nm) as a primary acceptor within the Q y transitions in RC allow an effective competition with an energy transfer and stabilization of separated charges. Although an alternative mechanism of charge separation with Chl D 1 ∗ as the primary electron donor and Pheo D1 as the primary acceptor cannot be ruled out, the 20-fs excitation at the far-red tail of the PSII core complex absorption spectrum at 710 nm appears to induce a transition to a low-energy state P 680 ∗ with charge-transfer character (probably P D 1 δ + P D 2 δ - ) which results in an effective electron transfer from P 680 ∗ (the primary electron donor) to Chl D1 as the intermediary acceptor.

SANS Investigation of the Photosynthetic Machinery of Chloroflexus aurantiacus

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.

Promotion of nano-anatase TiO 2 on the spectral responses and photochemical activities of D1/D2/Cyt b559 complex of spinach

Previous researches approved that photocatalysis activity of nano-TiO 2 could obviously increase photosynthetic effects of spinach, but the mechanism of improving light energy transfer and conversion is still unclear. In the present we investigated effects of nano-anatase TiO 2 on the spectral responses and photochemical activities of D1/D2/Cyt b559 complex of spinach. Several effects of nano-anatase TiO 2 were observed: (1) UV–vis spectrum was blue shifted in both Soret and Q bands, and the absorption intensity was obviously increased; (2) resonance Raman spectrum showed four main peaks, which are ascribed to carotene, and the Raman peak intensity was as 6.98 times as that of the control; (3) the fluorescence emission peak was blue shifted and the intensity was decreased by 23.59%; (4) the DCPIP photoreduction activity showed 129.24% enhancement; (5) the oxygen-evolving rate of PS II was elevated by 51.89%. Taken together, the studies of the experiments showed that nano-anatase TiO 2 had bound to D1/D2/Cyt b559 complex, promoted the spectral responses, leading to the improvement of primary electron separation, electron transfer and light energy conversion of D1/D2/Cyt b559 complex.

Calcium Ions Are Required for the Enhanced Thermal Stability of the Light-harvesting-Reaction Center Core Complex from Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Thermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057-1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca2+ as a cofactor. Removal of the Ca2+ from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca2+ to the Ca2+-depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Qy transition between 915 nm for the native and 880 nm for the Ca2+-depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15 degrees C higher and the enthalpy change is about 28% larger than the Ca2+-depleted LH1-RC. Substitution of the Ca2+ with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca2+ ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources.

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.

Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting B800-850 complex and its dependence on the spectral composition of the light is discussed.

Spectroelectrochemistry of cytochrome b559 in the D1–D2–Cyt b559 complex from spinach

The redox potential of cytochrome b559 (Cyt b559) in the D1–D2–Cyt b559 complex from spinach has been determined to be +90 ± 2 mV vs. SHE at pH 6.0, by thin-layer cell spectroelectrochemistry for the first time. The redox potential, corresponding uniquely to the so-called “low-potential form”, exhibited a sigmoidal pH-dependence from pH 4.0 to 9.0, ranging from +115 to +50 mV. An analysis of the pH-dependence based on model equations suggests that two histidine residues coordinating to the heme iron in the protein subunits may exert electrostatic influence on the redox potential of Cyt b559.

Dimeric and Monomeric Organization of Photosystem II

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.

Purification and Characterization of the B808–866 Light-harvesting Complex from Green Filamentous Bacterium Chloroflexus aurantiacus

The integral membrane light-harvesting complex B808-866 from the thermophilic green filamentous bacterium Chloroflexus aurantiacus has been isolated and characterized. Reversed-phase HPLC analysis demonstrated that the number of bacteriochlorophyll (BChl) in the B808-866 antenna complex is 36 +/- 2 per reaction center. The main carotenoid type is gamma-carotene, and the molar ratio of BChl to carotenoid is 3:2. The steady-state absorption and fluorescence spectroscopy of the B808-866 complex are reminiscent of the well-studied LH2 peripheral antenna of purple bacteria, whereas the protein sequence and the circular dichroism spectrum of B808-866 is more similar to the LH1 inner core antenna. The efficiency of excitation transfer from carotenoid to BChl is about 25%. The above results combined with electron microscopy and dynamic light scattering analysis suggest that the B808-866 antenna is more like the LH1, whereas surrounds the reaction center but probably consists of 24 building blocks with a ring diameter of about 20 nm. The above results suggested that there are probably two reaction centers inside the ring of B808-866. The unique properties of this light-harvesting complex may provide insights on the protein-pigment interactions in bacterial photosynthesis.

Effect of changes in the level of light harvesting complexes of Rhodobacter sphaeroides on the photoheterotrophic production of hydrogen

Increase in levels of photosynthetic spectral complexes by maintaining the plasmids harboring DNA encoding puhA, pufBA, or pucBAC in trans in Rhodobacter sphaeroides resulted in decrease of the photoheterotrophic production of H 2. However, removal of B875 or B800–850 light-harvesting (LH) complexes affected H 2 production differently. Lack of B875 complex following in-frame deletion of pufBA (mutant PUF1) not only slowed photoheterotrophic growth but also decreased H 2 production, indicative of the essential requirement of the complex for LH process. However, the pucBA-deleted mutant, PUC1 lacking of B800–850 complex, increased H 2 production in comparison with its parental cell by approximately twofold, given irradiated with light (10 W/m 2) saturating the growth of wild type. The H 2 production of PUC1 did not increase in proportion to the light intensity, which is also observed with wild type. Thus, we suggest that light is not limited for the H 2 production of the cells under the experimental conditions employed in this work, but the cellular energy to be used for the formation of B800–850 complex may flow into the metabolism leading to the H 2 production in PUC1.

A structural model for the assembly of the reaction centre and the B808-866 complex in the membranes of Chloroflexus aurantiacus based on the calculation of the triplet minus singlet spectrum of the primary donor

A well resolved triplet minus singlet spectrum of the primary donor P870 of the green bacterium Chloroflexus aurantiacus, detected by optically detected magnetic resonance (ODMR), in intact membranes, was reported in our previous work [Photosynth. Res. 71 (2002) 45]. The spectrum is now interpreted and reconstructed by calculating the electronic interactions between the primary donor and the BChl a molecules belonging to the 866 absorption component of the B808-866 light-harvesting complex. The good agreement between the calculation and the experimental data strongly supports a topological model for the B808-866 complex which consists in a circular aggregate structure of a number of polypeptide units binding BChl a molecules assembled as in the LH2 peripheral antenna complex of purple bacteria, giving rise to two spectral forms. The aggregate surrounds the reaction centre (RC) resembling in this respect the LH1 core antenna complex of purple bacteria.

Structural changes upon excitation of D1-D2-Cyt b559 photosystem II reaction centers depend on the beta-carotene content.

Different preparations of D1-D2-Cyt b559 complexes from spinach with different beta-carotene (Car) content [on average from <0.5 to 2 per reaction center (RC)] were studied by means of laser-induced optoacoustic spectroscopy. phiP680(+)Pheo(-) does not depend on the preparation (or on the Car content) inasmuch as the magnitude of the prompt heat (produced within 20 ns) does not vary for the different samples upon excitation at 675 and 620 nm. The energy level of the primary charge-separated state, P680(+)Pheo(-), was determined as EP680(+)Pheo(-) = 1.55 eV. Thus, an enthalpy change accompanying charge separation from excited P680 of deltaH*P680Pheo-->P680(+)Pheo(-) = -0.27 eV is obtained. Calculations using the heat evolved during the time-resolved decay of P680(+)Pheo(-) (< or = 100 ns) affords a triplet (3[P680Pheo]) quantum yield phi3[P680Pheo] = 0.5 +/- 0.14. The structural volume change, deltaV1, corresponding to the formation of P680(+)Pheo(-), strongly depends on the Car content; it is ca. -2.5 A3 molecule(-1) for samples with <0.5 Car on average, decreases (in absolute value) to -0.5 +/- 0.2 A3 for samples with an average of 1 Car, and remains the same for samples with two Cars per RC. This suggests that the Car molecules induce changes in the ground-state RC conformation, an idea which was confirmed by preferential excitation of Car with blue light, which produced different carotene triplet lifetimes in samples with 2 Car compared to those containing less carotene. We conclude that the two beta-carotenes are not structurally equivalent. Upon blue-light excitation (480 nm, preferential carotene absorption) the fraction of energy stored is ca. 60% for the 9Chl-2Car sample, whereas it is 40% for the preparations with one or less Cars on average, indicating different paths of energy distribution after Car excitation in these RCs with remaining chlorophyll antennae.