Effect of low pH values on chromatophores,core complexes,and peripheral light harvesting complexes isolated from the sulfur bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis>

Spectral methods have been used to trace pheophytinization of bacteriochlorophyll (BChl) in the membranes of chromatophores isolated from normal and carotenoidless cells of the purple bacterium Allochromatium minutissimum as well as in the core complexes and peripheral light harvesting complexes in the media with different detergents at low pH values. The well-marked staging of damage of native BChl forms with the absorption band of 885 nm has been revealed: (1) the formation and increase of the absorption band of monomeric BChl (785 nm); (2) pheophytinization of resultant monomeric BChl, and (3) aggregation of bacteriopheophytin (BPheo). Compared to the initial carotenoid complexes, carotenoidless pigment protein complexes were less resistant to the effect of low pH values, especially at the stages of BChl monomerization and pheophytinization. However, BPheo aggregation in them was slower. The electrophoresis in PAAG has shown that BChl pheophytinization in peripheral light harvesting complexes is accompanied by disruption of the ring-shaped structures of the complexes, with appearance of typical fragments consisting of α- and β- peptides and carrying monomeric BPheo, and by formation of α-peptide aggregates carrying BPheo aggregates.     

The photosystem of Rhodobacter sphaeroides assembles with zinc bacteriochlorophyll in a bchD (magnesium chelatase) mutant

A Rhodobacter sphaeroides bchD (magnesium chelatase) mutant was studied to determine the properties of its photosystem in the absence of bacteriochlorophyll (BChl). Western blots of reaction center H, M, and L (RC H/M/L) proteins from mutant membranes showed levels of 12% RC H, 32% RC L, and 46% RC M relative to those of the wild type. Tricine-SDS-PAGE revealed 52% light-harvesting complex alpha chain and 14% beta chain proteins compared to those of the wild type. Pigment analysis of bchD cells showed the absence of BChl and bacteriopheophytin (BPhe), but zinc bacteriochlorophyll (Zn-BChl) was discovered. Zn-BChl binds to light-harvesting 1 (LH1) and 2 (LH2) complexes in place of BChl in bchD membranes, with a LH2:LH1 ratio resembling that of wild-type cells under BChl-limiting conditions. Furthermore, the RC from the bchD mutant contained Zn-BChl in the special pair and accessory BChl binding sites, as well as carotenoid and quinone, but BPhe was absent. Comparison of the bchD mutant RC absorption spectrum to that of Acidiphilium rubrum, which contains Zn-BChl in the RC, suggests the RC protein environment at L168 contributes to A. rubrum special pair absorption characteristics rather than solely Zn-BChl. We speculate that Zn-BChl is synthesized via the normal BChl biosynthetic pathway, but with ferrochelatase supplying zinc protoporphyrin IX for enzymatic steps following the nonfunctional magnesium chelatase. The absence of BPhe in bchD cells is likely related to Zn2+ stability in the chlorin macrocycle and consequently high resistance of Zn-BChl to pheophytinization (dechelation). Possible agents prevented from dechelating Zn-BChl include the RC itself, a hypothetical dechelatase enzyme, and spontaneous processes.     

Examination of stability of mutant photosynthetic reaction center of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> I(L177)H and determination of location of bacteriochlorophyll covalently bound to the protein

We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48°C were examined. It is shown that the I(L177)H mutation decreases the RC stability. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl B_B can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl P_A is the molecule of BChl tightly bound with the L-sub- unit in mutant RC as it was supposed earlier.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q(y) transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (alpha and beta) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q(y) transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 A.     

Characterization of the core complex of Rubrivivax gelatinosus in a mutant devoid of the LH2 antenna

The core complex of purple bacteria is a supramolecular assembly consisting of an array of light-harvesting LH1 antenna organized around the reaction center. It has been isolated and characterized in this work using a Rubrivivax gelatinosus mutant lacking the peripheral LH2 antenna. The purification did not modify the organization of the complex as shown by comparison with the intact membranes of the mutant. The protein components consisted exclusively of the reaction center, the associated tetraheme cyt c and the LH1 αβ subunits; no other protein which could play the role of pufX could be detected. The complex migrated as a single band in a sucrose gradient, and as a monomer in a native Blue gel electrophoresis. Comparison of its absorbance spectrum with those of the isolated RC and of the LH1 antenna as well as measurements of the bacteriochlorophyll/tetraheme cyt c ratio indicated that the mean number of LH1 subunits per RC-cyt c is near 16. The polypeptides of the LH1 antenna were shown to present several modifications. The α one was formylated at its N-terminal residue and the N-terminal methionine of β was cleaved, as already observed for other Rubrivivax gelatinosus strains. Both modifications occurred possibly by post-translational processing. Furthermore the α polypeptides were heterogeneous, some of them having lost the 15 last residues of their C-terminus. This truncation of the hydrophobic C-terminal extension is similar to that observed previously for the α polypeptide of the Rubrivivax gelatinosus LH2 antenna and is probably due to proteolysis or to instability of this extension.     

Purification, characterization and crystallization of the core complex from thermophilic purple sulfur bacterium Thermochromatium tepidum

A light-harvesting-reaction center (LH1-RC) core complex has been highly purified from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. The bacteriochlorophyll (BChl) a molecules in the LH1 exhibit a Q_y transition at 914 nm, more than 25 nm red-shift from those of its mesophilic counterparts. The LH1-RC complex was isolated in a monomeric form as confirmed by sucrose density gradient centrifugation, blue native PAGE and size-exclusion chromatography. Four subunits (L, M, H and a tetraheme cytochrome) in RC and two polypeptides (α and β) in LH1 were identified. Spirilloxanthin was determined to be the predominant carotenoid in the core complex. The purified core complex was highly stable, no significant change in the LH1 Q_y transition was observed over 10 days of incubation at room temperature in dark. Circular dichroism spectrum of the LH1 complex was characterized by low intensity and nonconservative spectral shape, implying a high symmetry of the large LH1 ring and interaction between the BChl a and carotenoid molecules. A dimeric feature of the BChl a molecules in LH1 was revealed by magnetic circular dichroism spectrum. Crystals of the core complex were obtained which diffracted X-rays to about 10 Å.     

The puhE gene of Rhodobacter capsulatus is needed for optimal transition from aerobic to photosynthetic growth and encodes a putative negative modulator of bacteriochlorophyll production

A conserved orf of previously unknown function (herein designated as puhE) is located 3' of the reaction centre H (puhA) gene in purple photosynthetic bacteria, in the order puhABCE in Rhodobacter capsulatus. Disruptions of R. capsulatus puhE resulted in a long lag in the growth of photosynthetic cultures inoculated with cells grown under high aeration, and increased the level of the peripheral antenna, light-harvesting complex 2 (LH2). The amount of the photosynthetic reaction centre (RC) and its core antenna, light-harvesting complex 1 (LH1), was reduced; however, there was no decrease in expression of a lacZ reporter fused to the puf (RC and LH1) promoter, in RC assembly in the absence of LH1, or in LH1 assembly in the absence of the RC. In strains that lack LH2, disruption of puhE increased the in vivo absorption at 780 nm, which we attribute to excess bacteriochlorophyll a (BChl) pigment production. This effect was seen in the presence and absence of PufQ, a protein that stimulates BChl biosynthesis. Expression of puhE from a plasmid reduced A(780) production in puhE mutants. We suggest that PuhE modulates BChl biosynthesis independently of PufQ, and that the presence of excess BChl in PuhE(-)LH2(+) strains results in excess LH2 assembly and also interferes with the adaptation of cells during the transition from aerobic respiratory to anaerobic photosynthetic growth.     

Oligomers of bacteriochlorophyll and bacteriopheophytin with spectroscopic properties resembling those found in photosynthetic bacteria

Oligomers of bacteriopheophytin (BPh) and bacteriochlorophyll (BChl) were formed in mixed aqueous-organic solvent systems, and in aqueous micelles of the detergent lauryldimethylamine oxide (LDAO). Conditions were found that gave relatively homogeneous samples of oligomers and that allowed quantitative comparisons of the spectroscopic properties of the monomeric and oligomeric pigments. The formation of certain types of oligomers is accompanied by a large bathochromic shift of the long-wavelength (Q_y) absorption band of the BChl or BPh, and by a substantial increase in its dipole strength (hyperchromism). The hyperchromism of the Q_y band occurs at the expense of the Soret band, which loses dipole strength. The Q_x band shifts slightly to shorter wavelengths and also loses dipole strength. The CD spectrum in the near-infra-red (Q_y) region becomes markedly nonconservative. (The net rotational strength in the Q_y region is positive.) This also occurs at the expense of the bands at shorter wavelengths, which gain a net negative rotational strength. The spectroscopic properties of the oligomers resemble those of some of the BChl-protein complexes found in photosynthetic bacteria. The oligomerization of BPh in LDAO micelles is linked to the formation of large, cylindrical micelles that contain on the order of 10⁵ LDAO molecules. However, the spectral changes probably occur on the formation of small oligomers of BPh; they begin to be seen when the micelles contain about 10 molecules of BPh. The BPh oligomers formed in LDAO micelles fluoresce at 865 nm, but the fluorescence yield is decreased about 40-fold, relative to that of monomeric BPh. The fluorescence yield is insensitive to the BPh/LDAO molar ratio, suggesting that the oligomers formed under these conditions are predominantly dimers. When the oligomers are excited with a short flash of light, they are converted with a low quantum yield into a metastable form. This transformation probably involves alterations in the geometry of the oligomer, but not full dissociation.     

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.     

Electrostatic charge controls the lowest LH1 Qy transition energy in the triply extremophilic purple phototrophic bacterium,Halorhodospira halochloris

Halorhodospira (Hlr.) halochloris is a unique phototrophic purple bacterium because it is a triple extremophile—the organism is thermophilic, alkalophilic, and halophilic. The most striking photosynthetic feature of Hlr. halochloris is that the bacteriochlorophyll (BChl) b-containing core light-harvesting (LH1) complex surrounding its reaction center (RC) exhibits its LH1 Q_y absorption maximum at 1016 nm, which is the lowest transition energy among phototrophic organisms. Here we report that this extraordinarily red-shifted LH1 Q_y band of Hlr. halochloris exhibits interconvertible spectral shifts depending on the electrostatic charge distribution around the BChl b molecules. The 1016 nm band of the Hlr. halochloris LH1-RC complex was blue-shifted to 958 nm upon desalting or pH decrease but returned to its original position when supplemented with salts or pH increase. Resonance Raman analysis demonstrated that these interconvertible spectral shifts are not associated with the strength of hydrogen-bonding interactions between BChl b and LH1 polypeptides. Furthermore, circular dichroism signals for the LH1 Q_y transition of Hlr. halochloris appeared with a positive sign (as in BChl b-containing Blastochloris species) and opposite those of BChl a-containing purple bacteria, possibly due to a combined effect of slight differences in the transition dipole moments between BChl a and BChl b and in the interactions between adjacent BChls in their assembled state. Based on these findings and LH1 amino acid sequences, it is proposed that Hlr. halochloris evolved its unique and tunable light-harvesting system with electrostatic charges in order to carry out photosynthesis and thrive in its punishing hypersaline and alkaline habitat.     

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of 相似文献   

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.     

Excitation energy pathways in the photosynthetic units of reaction center LM- and H-subunit deletion mutants of <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis>

Light-induced reaction dynamics of isolated photosynthetic membranes obtained from wild-type (WT) and reaction center (RC)-subunit deletion strains SPUHK1 (an H-subunit deletion mutant) and SKΔLM (an (L+M) deletion mutant) of the purple non-sulphur bacterium Rhodospirillum rubrum have been investigated by femtosecond transient absorption spectroscopy. Upon excitation of the spirilloxanthin (Spx) S₂ state at 546 nm, of the bacteriochlorophyll Soret band at 388 nm and probing spectral regions, which are characteristic for carotenoids, similar dynamics in the SPUHK1, SKΔLM and WT strains could be observed. The excitation of Spx S₂ is followed by the simultaneous population of the lower singlet excited states S₁ and S* which decay with lifetimes of 1.4 and 5 ps, respectively for the mutants, and 1.4 and 4 ps, respectively, for the wild-type. The excitation of the BChl Soret band is followed by relaxation into BChl lower excited states which compete with excitation energy transfer BChl-to-Spx. The deexcitation pathway BChl(Soret) → Spx(S₂) → Spx(S₁) occurs with the same transition rate for all investigated samples (WT, SPUHK1 and SKΔLM). The kinetic traces measured for the Spx S₁ → S_N transition display similar behaviour for all samples showing a positive signal which increases within the first 400 fs (i.e. the time needed for the excitation energy to reach the Spx S₁ excited state) and decays with a lifetime of about 1.5 ps. This suggests that the Spx excited state dynamics in the investigated complexes do not differ significantly. Moreover, a longer excited state lifetime of BChl for SPUHK1 in comparison to WT was observed, consistent with a photochemical quenching channel present in the presence of RC. For long delay times, photobleaching of the RC special pair and an electrochromic blue shift of the monomeric BChl a can be observed only for the WT but not for the mutants. The close similarity of the excited state decay processes of all strains indicates that the pigment geometry of the LH1 complex in native membranes is unaffected by the presence of an RC and allows us to draw a model representation of the WT, SKΔLM and SPUHK1 PSU complexes.     

Exciton delocalization in the B808-866 antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy.

A model of pigment organization in the B808-866 bacteriochlorophyll a antenna of the green photosynthetic bacterium Chloroflexus aurantiacus based on femtosecond pump-probe studies is proposed. The building block of the antenna was assumed to be structurally similar to that of the B800-850 light-harvesting 2 (LH2) antenna of purple bacteria and to have the form of two concentric rings of N strongly coupled BChl866 pigments and of N/2 weakly coupled BChl808 monomers, where N = 24 or 32. We have shown that the Qy transition dipoles of BChl808 and BChl866 molecules form the angles 43 degrees +/- 3 degrees and 8 degrees +/- 4 degrees, respectively, with the plane of the corresponding rings. Using the exciton model, we have obtained a quantitative fit of the pump-probe spectra of the B866 and B808 bands. The anomalously high bleaching value of the B866 band with respect to the B808 monomeric band provided the direct evidence for a high degree of exciton delocalization in the BChl866 ring antenna. The coherence length of the steady-state exciton wave packet corresponds to five or six BChl866 molecules at room temperature.     

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm²) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure—polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC?+?QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC?+?QD) complexes.

     

Adaptation of intracytoplasmic membranes to altered light intensity in Rhodobacter sphaeroides

The model photosynthetic bacterium Rhodobacter sphaeroides uses a network of bacteriochlorophyll (BChl)-protein complexes embedded in spherical intracytoplasmic membranes (ICM) to collect and utilise solar energy. We studied the effects of high- and low-light growth conditions, where BChl levels increased approximately four-fold from 1.6×10(6) to 6.5×10(6) molecules per cell. Most of this extra pigment is accommodated in the proliferating ICM system, which increases from approximately 274 to 1468 vesicles per cell. Thus, 16×10(6)nm(2) of specialised membrane surface area is made available for harvesting and utilising solar energy compared to 3×10(6)nm(2) under high-light conditions. Membrane mapping using atomic force microscopy revealed closely packed dimeric and monomeric reaction centre-light harvesting 1-PufX (RC-LH1-PufX) complexes in high-light ICM with room only for small clusters of LH2, whereas extensive LH2-only domains form during adaptation to low light, with the LH2/RC ratio increasing three-fold. The number of upper pigmented band (UPB) sites where membrane invagination is initiated hardly varied; 704 (5.8×10(5) BChls/cell) and 829 (4.9×10(5) BChls/cell) UPB sites per cell were estimated under high- and low-light conditions, respectively. Thus, the lower ICM content in high-light cells is a consequence of fewer ICM invaginations reaching maturity. Taking into account the relatively poor LH2-to-LH1 energy transfer in UPB membranes it is likely that high-light cells are relatively inefficient at energy trapping, but can grow well enough without the need to fully develop their photosynthetic membranes from the relatively inefficient UPB to highly efficient mature ICM.     

Substitution of isoleucine L177 by histidine in Rhodobacter sphaeroides reaction center results in the covalent binding of PA bacteriochlorophyll to the L subunit

In this work, we report the unique case of bacteriochlorophyll (BChl) - protein covalent attachment in a photosynthetic membrane complex caused by a single mutation. The isoleucine L177 was substituted by histidine in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Pigment analysis revealed that one BChl molecule was missing in the acetone-methanol extract of the I(L177)H RCs. SDS-PAGE demonstrated that this BChl molecule could not be extracted with organic solvents apparently because of its stable covalent attachment to the mutant RC L-subunit. Our data indicate that the attached bacteriochlorophyll is one of the special pair BChls, P(A). The chemical nature of this covalent interaction remains to be identified.     

Analysis of absorption spectra of purple bacterial reaction centers in the near infrared region by higher order derivative spectroscopy

Reaction centers (RCs) of purple bacteria are uniquely suited objects to study the mechanisms of the photosynthetic conversion of light energy into chemical energy. A recently introduced method of higher order derivative spectroscopy [I.K. Mikhailyuk, H. Lokstein, A.P. Razjivin, A method of spectral subband decomposition by simultaneous fitting the initial spectrum and a set of its derivatives, J. Biochem. Biophys. Methods 63 (2005) 10-23] was used to analyze the NIR absorption spectra of RC preparations from Rhodobacter (R.) sphaeroides strain 2R and Blastochloris (B.) viridis strain KH, containing bacteriochlorophyll (BChl) a and b, respectively. Q(y) bands of individual RC porphyrin components (BChls and bacteriopheophytins, BPheo) were identified. The results indicate that the upper exciton level P(y+) of the photo-active BChl dimer in RCs of R. sphaeroides has an absorption maximum of 810nm. The blue shift of a complex integral band at approximately 800nm upon oxidation of the RC is caused primarily by bleaching of P(y+), rather than by an electrochromic shift of the absorption band(s) of the monomeric BChls. Likewise, the disappearance of a band peaking at 842nm upon oxidation of RCs from B. viridis indicates that this band has to be assigned to P(y+). A blue shift of an absorption band at approximately 830nm upon oxidation of RCs of B. viridis is also essentially caused by the disappearance of P(y+), rather than by an electrochromic shift of the absorption bands of monomeric BChls. Absorption maxima of the monomeric BChls, B(B) and B(A) are at 802 and 797nm, respectively, in RCs of R. sphaeroides at room temperature. BPheo co-factors H(B) and H(A) peak at 748 and 758nm, respectively, at room temperature. For B. viridis RCs the spectral positions of H(B) and H(A) were found to be 796 and 816nm, respectively, at room temperature.     

Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii

The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2?ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210?ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.     

Probing binding site of bacteriochlorophyll a and carotenoid in the reconstituted LH1 complex from Rhodospirillum rubrum S1 by Stark spectroscopy

Stark spectroscopy is a powerful technique to investigate the electrostatic interactions between pigments as well as between the pigments and the proteins in photosynthetic pigment–protein complexes. In this study, Stark spectroscopy has been used to determine two nonlinear optical parameters (polarizability change Tr(Δα) and static dipole-moment change |Δμ| upon photoexcitation) of isolated and of reconstituted LH1 complexes from the purple photosynthetic bacterium, Rhodospirillum (Rs.) rubrum. The integral LH1 complex was prepared from Rs. rubrum S1, while the reconstituted complex was assembled by addition of purified carotenoid (all-trans-spirilloxanthin) to the monomeric subunit of LH1 from Rs. rubrum S1. The reconstituted LH1 complex has its Q_y absorption maximum at 878 nm. This is shifted to the blue by 3 nm in comparison to the isolated LH1 complex. The energy transfer efficiency from carotenoid to bacteriochlorophyll a (BChl a), which was determined by fluorescence excitation spectroscopy of the reconstituted LH1 complex, is increased to 40%, while the efficiency in the isolated LH1 complex is only 28%. Based on the differences in the values of Tr(Δα) and |Δμ|, between these two preparations, we can calculate the change in the electric field around the BChl a molecules in the two situations to be E _Δ ≈ 3.4 × 10⁵ [V/cm]. This change can explain the 3 nm wavelength shift of the Q_y absorption band in the reconstituted LH1 complex.