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.     

Dynamics of energy transfer from lycopene to bacteriochlorophyll in genetically-modified LH2 complexes of Rhodobacter sphaeroides

LH2 complexes from Rb. sphaeroides were modified genetically so that lycopene, with 11 saturated double bonds, replaced the native carotenoids which contain 10 saturated double bonds. Tuning the S1 level of the carotenoid in LH2 in this way affected the dynamics of energy transfer within LH2, which were investigated using both steady-state and time-resolved techniques. The S1 energy of lycopene in n-hexane was determined to be approximately 12 500 +/- 150 cm(-1), by direct measurement of the S1-S2 transient absorption spectrum using a femtosecond IR-probing technique, thus placing an upper limit on the S1 energy of lycopene in the LH2 complex. Fluorescence emission and excitation spectra demonstrated that energy can be transferred from lycopene to the bacteriochlorophyll molecules within this LH2 complex. The energy-transfer dynamics within the mutant complex were compared to wild-type LH2 from Rb. sphaeroides containing the carotenoid spheroidene and from Rs. molischianum, in which lycopene is the native carotenoid. The results show that the overall efficiency for Crt --> B850 energy transfer is approximately 80% in lyco-LH2 and approximately 95% in WT-LH2 of Rb. sphaeroides. The difference in overall Crt --> BChl transfer efficiency of lyco-LH2 and WT-LH2 mainly relates to the low efficiency of the Crt S(1) --> BChl pathway for complexes containing lycopene, which was 20% in lyco-LH2. These results show that in an LH2 complex where the Crt S1 energy is sufficiently high to provide efficient spectral overlap with both B800 and B850 Q(y) states, energy transfer via the Crt S1 state occurs to both pigments. However, the introduction of lycopene into the Rb. sphaeroides LH2 complex lowers the S1 level of the carotenoid sufficiently to prevent efficient transfer of energy to the B800 Q(y) state, leaving only the Crt S1 --> B850 channel, strongly suggesting that Crt S1 --> BChl energy transfer is controlled by the relative Crt S1 and BChl Q(y) energies.     

Interaction of photosynthetic pigments with various organic solvents 2. Application of magnetic circular dichroism to bacteriochlorophyll a and light-harvesting complex 1

Magnetic circular dichroism (MCD) and absorption spectra of metal bacteriochlorin complexes have been measured on bacteriochlorophyll (BChl) a in various solvents and different forms of light-harvesting complexes 1 (LH1 complexes). In hydrophilic organic solvents, the MCD intensity of the Q(y)(0-0) transition of BChl a was sensitive to the wavelength of absorption maximum of Q(x)(0-0), and the ratio of MCD Q(y)(0-0) intensity to the dipole strength (B/D) was inversely proportional to the difference in energy between the Q(x)(0-0) and Q(y)(0-0). The similar correlation has been observed in metal chlorin derivatives as previously reported. The correlation depends on the coordination number of the Mg atom in BChl a and the molecules ligating to it. In a hydrophobic solvent such as carbon tetrachloride (CCl(4)), however, the correlation did not hold because of the existence of aggregates. Hence, the correlation between the values of B/D and the energy difference can be used to estimate the type and number of the molecules ligated to the Mg atom and to disclose the existence of aggregated pigments. We further apply the correlation to the LH 1 complex treated with n-octyl beta-D-glucopyranoside.     

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.     

Formation of a subunit form of the core light-harvesting complex from sulfur purple bacteria <Emphasis Type="Italic">Ectothiorhodospira haloalkaliphila</Emphasis> with different carotenoid composition

B820 subunits from a purple sulfur bacterium Ectothiorhodospira haloalkaliphila strain ATCC 51935^T were obtained by treatment of carotenoid free LH1-RC complexes of this bacterium with ß-octylglucopyranoside (ß-OG). The same complexes with 100% carotenoid content were unable to dißsociate to B820 subunits, but disintegrated to monomeric bacteriochlorophyll (BChl) regardless of their carotenoid composition. The degree of dissociation of the LH1-RC complexes with an intermediate content of carotenoids (the B820 formation) was directly dependent on the quantity of carotenoids in the samples. The resulting B820 subunits did not contain carotenoids. B820 subunits easily aggregated to form a complex with an absorption peak at 880 nm at decreased ß-OG concentration. Analysis of the spectra of the LH1-RC complexes isolated from the cells with different levels of carotenogenesis inhibition led to the conclusion of the heterogeneity of the samples with a predominance of them in (a) the fraction with 100% of carotenoids and (b) the fraction of carotenoid-free complexes.     

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 γ-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.     

Metal cations modulate the bacteriochlorophyll-protein interaction in the light-harvesting 1 core complex from Thermochromatium tepidum

The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium (Tch.) tepidum exhibits unusual Q(y) absorption by LH1 bacteriochlorophyll-a (BChl-a) molecules at 915nm, and the transition energy is finely modulated by the binding of metal cations to the LH1 polypeptides. Here, we demonstrate the metal-dependent interactions between BChl-a and the polypeptides within the intact LH1-RC complexes by near-infrared Raman spectroscopy. The wild-type LH1-RC (B915) exhibited Raman bands for the C3-acetyl and C13-keto CO stretching modes at 1637 and 1675cm(-1), respectively. The corresponding bands appeared at 1643 and 1673cm(-1) when Ca(2+) was biosynthetically replaced with Sr(2+) (B888) or at 1647 and 1669cm(-1) in the mesophilic counterpart, Allochromatium vinosum. These results indicate the significant difference in the BChl-polypeptide interactions between B915 and B888 and between B915 and the mesophilic counterpart. The removal of the original metal cations from B915 and B888 resulted in marked band shifts of the C3-acetyl/C13-carbonyl νCO modes to ~1645/~1670cm(-1), supporting a model in which the metal cations are involved in the fine-tuning of the hydrogen bonding between the BChl-a and LH1-polypeptides. Interestingly, the interaction modes were almost identical between the Ca(2+)-depleted B915 and Sr(2+)-depleted B888 and between B915 and Ca(2+)-substituted B888, despite the significant differences in their LH1 Q(y) peak positions and the denaturing temperatures, as revealed by differential scanning calorimetry. These results suggest that not only the BChl-polypeptide interactions but some structural origin may be involved in the unusual Q(y) red-shift and the enhanced thermal stability of the LH1-RC complexes from Tch. tepidum.     

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.     

The PufX protein of Rhodobacter capsulatus affects the properties of bacteriochlorophyll a and carotenoid pigments of light-harvesting complex 1

A pufX gene deletion in the purple bacterium Rhodobacter capsulatus causes a severe photosynthetic defect and increases core light-harvesting complex (LH1) protein and bacteriochlorophyll a (BChl) levels. It was suggested that PufX interrupts the LH1 alpha/beta ring around the reaction centre, allowing quinone/quinol exchange. However, naturally PufX(-) purple bacteria grow photosynthetically with an uninterrupted LH1. We discovered that substitutions of the Rhodobacter-specific LH1 alpha seryl-2 decrease carotenoid levels in PufX(-)R. capsulatus. An LH1 alphaS2F mutation improved the photosynthetic growth of a PufX(-) strain lacking the peripheral LH2 antenna, although LH1 BChl absorption remained above wild-type, suggesting that Rhodobacter-specific carotenoid binding is involved in the PufX(-) photosynthetic defect and LH1 expansion is not. Furthermore, PufX overexpression increased LH1-like BChl absorption without inhibiting photosynthetic growth. PufX(+) LH1 alphaS2-substituted mutant strains had wild-type carotenoid levels, indicating that PufX modulates LH1 carotenoid binding, inducing a conformational change that favours quinone/quinol exchange.     

Pigment organization of the B800–850 antenna complex of Rhodopseudomonas sphaeroides

The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer k_ET > 3 · 10¹² s^?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Q_y transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.     

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

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

Probing the structure of the core light-harvesting complex (LH1) of Rhodopseudomonas viridis by dissociation and reconstitution methodology

A subunit complex was formed from the core light-harvesting complex (LH1) of bacteriochlorophyll(BChl)-b-containing Rhodopseudomonas viridis. The addition of octyl glucoside to a carotenoid-depleted Rps. viridis membrane preparation resulted in a subunit complex absorbing at 895 nm, which could be quantitatively dissociated to free BChl b and then reassociated to the subunit. When carotenoid was added back, the subunit could be reassociated to LH1 with a 25% yield. Additionally, the Rps. viridis - and -polypeptides were isolated, purified, and then reconstituted with BChl b. They formed a subunit absorbing near 895 nm, similar to the subunit formed by titration of the carotenoid depleted membrane, but did not form an LH1-type complex at 1015 nm. The same results were obtained with the -polypeptide alone and BChl b. Isolated polypeptides were also tested for their interaction with BChl a. They formed subunit and LH1-type complexes similar to those formed using polypeptides isolated from BChl-a-containing bacteria but displayed 6–10 nm smaller red shifts in their long-wavelength absorption maxima. Thus, the larger red shift of BChl-b-containing Rps. viridis is not attributable solely to the protein structure. The -polypeptide of Rps. viridis differed from the other -polypeptides tested in that it could form an LH1-type complex with BChl a in the absence of the - and -polypeptides. It apparently contains the necessary information required to assemble into an LH1-type complex. When the -polypeptide was tested in reconstitution with BChl a and BChl b with the - and -polypeptides, it had no effect; its role remains undetermined.Abbreviations B820 the subunit form of the core light-harvesting complex in BChl-a-containing bacteria which has an absorption maximum at or near 820 nm - B875 the core light-harvesting complex of Rhodobacter sphaeroides which has an absorption maximum at 875 nm - B881 the core light-harvesting complex of wild-type Rhodospirillum rubrum which has an absorption maximum at 881 nm - B895 the subunit form of the core light-harvesting complex in Rps. viridis which has an absorption maximum near 888–895 nm - B1015 the core light-harvesting complex of Rps. viridis which has an absorption maximum at 1015 nm - CD circular dichroism - LH1 the core light-harvesting complex - OG n-octyl -d-glucopyranoside     

Effects of pH on the peripheral light-harvesting antenna complex for Rhodopseudomonas palustris

In this work steady-state absorption spectroscopy, circular dichroism spectroscopy and sub-micro-second time-resolved absorption spectroscopy were used to investigate the effect of pH on the struc-tures and functions of LH2 complex for Rhodopseudomonas palustris. The results revealed that: (1) B800 Bchla was gradually transformed to free pigments absorbing around 760 nm on the minutes timescale upon the induction of strong acidic pH, and subsequently there disappeared the CD signal for Qy band of B800 in the absence of B800. In addition, Carotenoids changed with the similar tendency to B850 BChl. (2) The introduction of strong basic pH gave rise to no significant changes for B800 Bchla, while B850 BChla experienced remarkable spectral blue-shift from 852 to 837 nm. Similar phe-nomenon was seen for the CD signal for Qy band of B850. Carotenoids displayed strong and pH-independent CD signals in the visible range. (3) In the case of both physiological and basic pH, broad and asymmetrical positive Tn←T1 transient absorption appeared following the pulsed photo-excitation of Car at 532 nm. By contrast, the featureless and weak positive signal was observed on the sub-microsecond timescale in the acidic pH environment. The aforementioned experimental results indicated that acidic pH-induced removal of B800 Bchla prevented the generation of the caro-tenoid triplet state (3Car*), which is known to be essential for the photo-protection function. Neverthe-less, carotenoids can still perform this important physiological role under the basic pH condition, where the spectral blue shift of B850 exerts little effect on the overall structure of the cyclic aggregate, therefore favoring the formation of carotenoid triplet state.     

Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.     

Formation of Bacteriochlorophyll Form B820 in Light Harvesting 2 Complexes from Purple Sulfur Bacteria Treated with Dioxane

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.     

Two-photon excitation spectrum of fluorescence of the light-harvesting complex B800–850 from Allochromatium minutissimum within 1200–1500 (600–750) nm spectral range is not carotenoid mediated

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ～ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S₀ → S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.     

Spirilloxanthin incorporation into the LH2 and LH1-RC pigment-protein complexes from a purple sulfur bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis>

Incorporation of spirilloxanthin into carotenoidless LH2 and LH1-RC complexes from a purple sulfur bacterium Allochromatium (Alc.) minutissimum was studied. Carotenoidless cells of Alc. minutissimum were obtained using diphenylamine, a carotenoid biosynthesis inhibitor. In the course of incorporation of the carotenoid mixture, the composition of which corresponded to that of Alc. minutissimum control photosynthetic membranes, no selective incorporation of spirilloxanthin into the LH1-RC complex was detected. It is assumed that in vivo carotenoids are not incorporated into the LH2 and LH1-RC complexes from a common pool. Pure spirilloxanthin destroys both the LH2 and LH1-RC complexes. Within the concentration range of spirilloxanthin in the incorporated mixture from 27% to 52%, it was found to be incorporated into the LH2 and LH1-RC complexes with the efficiency of 13% and 33%, respectively. The possible existence of different sites of assembly for the LH2 and LH1-RC complexes is discussed, as well as of two fractions of LH2 complexes, in one of which rhodopin may be integrated, and in the other (minor) one, spirilloxanthin.     

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.     

Excitation dynamics of two spectral forms of the core complexes from photosynthetic bacterium Thermochromatium tepidum

The intact core antenna-reaction center (LH1-RC) core complex of thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum is peculiar in its long-wavelength LH1-Q_y absorption (915 nm). We have attempted comparative studies on the excitation dynamics of bacteriochlorophyll (BChl) and carotenoid (Car) between the intact core complex and the EDTA-treated one with the Q_y absorption at 889 nm. For both spectral forms, the overall Car-to-BChl excitation energy transfer efficiency is determined to be ∼20%, which is considerably lower than the reported values, e.g., ∼35%, for other photosynthetic purple bacteria containing the same kind of Car (spirilloxanthin). The RC trapping time constants are found to be 50∼60 ps (170∼200 ps) for RC in open (closed) state irrespective to the spectral forms and the wavelengths of Q_y excitation. Despite the low-energy LH1-Q_y absorption, the RC trapping time are comparable to those reported for other photosynthetic bacteria with normal LH1-Q_y absorption at 880 nm. Selective excitation to Car results in distinct differences in the Q_y-bleaching dynamics between the two different spectral forms. This, together with the Car band-shift signals in response to Q_y excitation, reveals the presence of two major groups of BChls in the LH1 of Tch. tepidum with a spectral heterogeneity of ∼240 cm⁻¹, as well as an alteration in BChl-Car geometry in the 889-nm preparation with respect to the native one.     

固氮红细菌(Rhodobacter azotoformans) 423 nm特征吸收峰成因与定位

【背景】在不产氧光合细菌中,因420-425nm特征峰位于类胡萝卜素(Carotenoid,Car)吸收区域,通常被认为是由Car积累引起,但固氮红细菌R7菌株呈现的423 nm特征峰不具备Car三指峰特征。【目的】阐明R7菌株423 nm特征吸收峰形成的物质基础及胞内定位。【方法】采用吸收光谱、薄层层析、高效液相色谱、质谱、超速离心和离子交换层析等方法阐明423 nm吸收峰形成原因。【结果】谷氨酸钠明显促进R7菌株活细胞呈现423 nm特征峰,色素提取液中该峰蓝移至415 nm,但其生长、细菌叶绿素(Bacteriochlorophyll,BChl)和Car含量大幅度降低,而添加酵母提取物则反之。色素组成分析表明,在检测到的色素成分中,只有镁卟啉单甲基酯Ⅸ (Magnesium Protoporphyrin Ⅸ Monomethylester,MPE)呈现415 nm特征吸收峰。MPE可定位于光合膜上并呈现出423 nm特征峰。对色素蛋白复合体(Pigment Protein Complex,PPC)的研究显示,添加谷氨酸和酵母提取物的菌体细胞虽然都检测到3种PPC组分[2个外周捕光复合体(Peripheral Light Harvesting Complex 2,LH2)和1个光反应中心(Reaction Center,RC)],但源自谷氨酸菌体细胞的RC和1个LH2则呈现423 nm特征吸收峰,表明R7菌株可产生2种不同类型的LH2,且MPE可定位于一种LH2和RC。【结论】R7菌株所呈现的423 nm特征峰不是由Car积累所致,而是由MPE积累所形成,且能与LH2和RC结合定位于光合膜上。MPE是BChl合成的中间产物,其合成受严格调控,不容易获得。MPE代谢调控的深入研究可为光合作用光氧化损伤与保护机理增添新内容。