Selective Excitation of Carotenoids of the Allochromatium vinosum Light-Harvesting LH2 Complexes Leads to Oxidation of Bacteriochlorophyll.

The mechanism of bacteriochlorophyll photooxidation in light-harvesting complexes of a number of purple photosynthetic bacteria when the complexes are excited into the carotenoid absorption bands remains unclear for many years. Here, using narrow-band laser illumination we measured action spectrum of this process for the spectral ranges of carotenoid and bacteriochlorophyll. It is shown that bacteriochlorophyll excitation results in almost no photooxidation of these molecules, while carotenoid excitation leads to oxidation with quantum yield of about 0,0003. Low value of the yield enabled an assumption that the studied process is initiated by the triplet states of the main carotenoids of the complexes with the number of conjugated double-bond chain length of N = 11. Interaction of these states with oxygen facilitates formation, though with low efficiency, of the excited singlet oxygen, which oxidizes bacteriochlorophylls. The carotenoid triplet states are formed in the process of the earlier studied singlet-triplet fission. The obtained results point at the necessity of reconsidering the functions of carotenoids in the light-harvesting complexes of purple bacteria.

LH2 Complex from Sulfur Bacteria Allochromatium vinosum - Natural Singlet Oxygen Sensor.

It was established that in a heterogeneous model system, which consisted of two types of complexes: reaction center or core complex of photosystem 2 of higher plants and LH2 complex of the sulfur bacterium Alc. vinosum, BChl850 oxidation of the LH2 complex could be observed under illumination by the light at a wavelength of 662 nm, which is the red absorption band of Chl. It has been shown that this process induces release of singlet oxygen, which is generated in photosystem II complexes and then partially diffuses into LH2 complex, where it oxidizes BChl850. It was established by HPLC that this results in formation of a product of BChl oxidation, 3-acetylchlorophyll. The process of BChl850 oxidation is inhibited by singlet oxygen quenchers (Trolox and Na ascorbate). It is suggested that the LH2 complex from the sulfur bacterium Alc. vinosum could be used to detect generation of singlet oxygen by the chlorophyll containing samples.

Carotenoids Do Not Protect Bacteriochlorophylls in Isolated Light-Harvesting LH2 Complexes of Photosynthetic Bacteria from Destructive Interactions with Singlet Oxygen.

The effect of singlet oxygen on light-harvesting (LH) complexes has been studied for a number of sulfur (S+) and nonsulfur (S-) photosynthetic bacteria. The visible/near-IR absorption spectra of the standard LH2 complexes (B800-850) of Allochromatium (Alc.) vinosum (S+), Rhodobacter (Rba.) sphaeroides (S-), Rhodoblastus (Rbl.) acidophilus (S-), and Rhodopseudomonas (Rps.) palustris (S-), two types LH2/LH3 (B800-850 and B800-830) of Thiorhodospira (T.) sibirica (S+), and an unusual LH2 complex (B800-827) of Marichromatium (Mch.) purpuratum (S+) or the LH1 complex from Rhodospirillum (Rsp.) rubrum (S-) were measured in aqueous buffer suspensions in the presence of singlet oxygen generated by the illumination of the dye Rose Bengal (RB). The content of carotenoids in the samples was determined using HPLC analysis. The LH2 complex of Alc. vinosum and T. sibirica with a reduced content of carotenoids was obtained from cells grown in the presence of diphenylamine (DPA), and LH complexes were obtained from the carotenoidless mutant of Rba. sphaeroides R26.1 and Rps. rubrum G9. We found that LH2 complexes containing a complete set of carotenoids were quite resistant to the destructive action of singlet oxygen in the case of Rba. sphaeroides and Mch. purpuratum. Complexes of other bacteria were much less stable, which can be judged by a strong irreversible decrease in the bacteriochlorophyll (BChl) absorption bands (at 850 or 830 nm, respectively) for sulfur bacteria and absorption bands (at 850 and 800 nm) for nonsulfur bacteria. Simultaneously, we observe the appearance of the oxidized product 3-acetyl-chlorophyll (AcChl) absorbing near 700 nm. Moreover, a decrease in the amount of carotenoids enhanced the spectral stability to the action of singlet oxygen of the LH2 and LH3 complexes from sulfur bacteria and kept it at the same level as in the control samples for carotenoidless mutants of nonsulfur bacteria. These results are discussed in terms of the current hypothesis on the protective functions of carotenoids in bacterial photosynthesis. We suggest that the ability of carotenoids to quench singlet oxygen (well-established in vitro) is not well realized in photosynthetic bacteria. We compared the oxidation of BChl850 in LH2 complexes of sulfur bacteria under the action of singlet oxygen (in the presence of 50 µM RB) or blue light absorbed by carotenoids. These processes are very similar: {[BChl + (RB or carotenoid) + light] + O2} → AcChl. We speculate that carotenoids are capable of generating singlet oxygen when illuminated. The mechanism of this process is not yet clear.

Lack of Excitation Energy Transfer from the Bacteriochlorophyll Soret Band to Carotenoids in Photosynthetic Complexes of Purple Bacteria.

The excitation energy transfer (EET) from the bacteriochlorophyll (BChl) Soret band to the second excited state(s) (S2) of carotenoids in pigment-protein complexes of purple bacteria was investigated. The efficiency of EET was determined, based on fluorescence excitation and absorption spectra of chromatophores, peripheral light-harvesting complexes (LH2), core complexes (LH1-RC), and pigments in solution. Carotenoid-containing and carotenoid-less samples were compared: LH1-RC and LH2 from Allochromatium minutissimum, Ectothiorhodospira haloalkaliphila, and chromatophores from Rhodobacter sphaeroides and Rhodospirillum rubrum wild type and carotenoid-free strains R-26 and G9. BChl-to-carotenoid EET was absent, or its efficiency was less than the accuracy of the measurements of ∼5%. Quantum chemical calculations support the experimental results: The transition dipole moments of spatially close carotenoid/BChl pairs were found to be nearly orthogonal. The structural arrangements suggest that Soret EET may be lacking for the studied systems, however, EET from carotenoids to Qx appears to be possible.

Incorporation of spheroidene and spheroidenone into light-harvesting complexes from purple sulfur bacteria.

Spheroidene and spheroidenone from the non-sulfur bacterium Rhodobacter (Rba.) sphaeroides were incorporated into diphenylamine (DPA) LH1-RC and LH2 complexes from sulfur bacteria Allochromatium (Alc.) minutissimum and Ectothiorhodospira (Ect.) haloalkaliphila in which carotenoid (Car) biosynthesis was inhibited by ~95%. A series of biochemical characteristics of the modified LH2 complexes was studied (electrophoretic mobility, absorption and CD spectra, Car composition, Car-to-BChl energy transfer and thermal stability). It was found that the electrophoretic mobility of the complexes with incorporated Cars did not change compared to that of the control and DPA-complexes, indicating the absence of any significant change in the structure of LH complexes upon DPA-treatment and subsequent incorporation of Cars. The analysis of fluorescence excitation spectra of the spheroidene-incorporated LH2 complex (LH2:sph) and the spheroidenone-incorporated LH2 complex (LH2:sph-ne) showed that spheroidene and spheroidenone exhibited relatively low efficiencies of energy transfer to BChl, when incorporated into the LH2 DPA-complexes from Alc. minutissimum and Ect. haloalkaliphila, although, they showed high efficiencies, being in their natural state in the LH2 complexes from Rba. sphaeroides. A significant increase in thermostability observed for the LH2:sph and LH2:sph-ne complexes with respect to the LH2 DPA-complexes indicated that the two incorporated Cars stabilized the structure of the LH2 complexes.

Distribution of colored carotenoids between light-harvesting complexes in the process of recovering carotenoid biosynthesis in Ectothiorhodospira haloalkaliphila cells.

The processes of recovering colored-carotenoid (Car) biosynthesis in Car-less cells of the purple sulfur bacterium Ectothiorhodospira haloalkaliphila grown with diphenylamine (DPA-cells) have been studied. It has been found that (1) the rate of recovering colored-Car biosynthesis in the lag-phase is far ahead of the growth rate of the cells themselves; (2) several Cars (ζ-carotene, neurosporene etc.) act as intermediates in Car biosynthesis; (3) because filling the "empty" Car pockets in the LH1-RC complexes is faster than in LH2, available spirilloxanthin is preferentially incorporated into the nascent LH1-RC core particles; (4) as a consequence of the resulting lack of spirilloxanthin availability, the biosynthetic intermediates (anhydrorhodovibrin, rhodopin and lycopene) fill the empty nascent LH2 Car pockets. In the present report, we further discuss the process of colored Car incorporation into LH complexes during the recovery of Car biosynthesis in the DPA-cells of Ect.haloalkaliphila.

Chemically modified reaction centers of photosystem II: Exchange of pheophytin a with 7-deformyl-7-hydroxymethyl-pheophytin b.

The native pheophytin a (Pheo a) in isolated reaction centers of photosystem II (PSII RCs) has been chemically exchanged with extraneous 7-deformyl-7-hydroxymethyl-Pheo b (7(1)-OH-Pheo b) which differs from Pheo a by the C-7 substituent (hydroxymethyl instead of methyl). The two pigments have similar reduction potentials in vitro [M. Meyer, Dissertation, Universität München, 1997], while their absorption spectra show small but distinct differences in the visible region. The resulting 7(1)-OH-Pheo b-modified reaction center preparations were characterized by high-performance liquid chromatography, electronic absorption and light-induced Fourier transform infra red absorption difference spectroscopies, together with photoaccumulation of the reduced pheophytin electron acceptor and NaBH4-treatment. About 70% of the total Pheo a molecules are found to be replaced by 7(1)-OH-Pheo b molecules in modified preparations, indicating that both the photochemically active (PheoD1) and inactive (PheoD2) binding sites were subjected to pigment exchange. The 7(1)-OH-Pheo b molecule located at the PheoD1 site is able to functionally replace the native Pheo a, participating in primary charge separation as an electron acceptor. The Qx absorption band of this modified pheophytin molecule is localized at ~546nm; its Qy band is blue-shifted with respect to the absorption of other reaction center core pigments, being located at ~665nm. The Qy and Qx optical transitions of the 7(1)-OH-Pheo b molecule exchanged into the PheoD2 site are identified at 677 and 543.5nm, respectively. The photochemically active double-modified PSII RCs additionally containing 7-deformyl-7-hydroxymethyl-13(1)-deoxo-13(1)-hydroxy-Pheo b at the PheoD2 site were obtained by treatment of the 7(1)-OH-Pheo b-modified RCs with NaBH4.

The LH2 complexes are assembled in the cells of purple sulfur bacterium Ectothiorhodospira haloalkaliphila with inhibition of carotenoid biosynthesis.

The effect of the inhibitor of carotenoid (Car) biosynthesis, diphenylamine (DPA), on the cells of the purple sulfur bacterium Ectothiorhodospira (Ect.) haloalkaliphila has been studied. There occurs an inhibition of the biosynthesis of colored Cars (≥99 %) at 71 µM DPA. Considering "empty" Car pockets (Moskalenko and Makhneva 2012) the content of Cars in the DPA-treated samples is first calculated more correctly. The total content of the colored Cars in the sample at 71 µM DPA does not exceed 1 % of the wild type. In the DPA-treated cells (membranes) a complete set of pigment-protein complexes is retained. The LH2 complex at 71 µM DPA is isolated, which is identical to the LH2 complex of the wild type in near IR absorption spectra. This suggests that the principles for assembling this LH2 complex in vivo in the absence of colored Cars remain the same. These results are in full agreement with the data obtained earlier for Allochromatium (Alc.) minutissimum (Moskalenko and Makhneva 2012). They are as follows: (1) DPA almost entirely inhibits the biosynthesis of the colored Cars in Ect. haloalkaliphila cells. (2) In the DPA-treated samples non-colored Cars are detected at 53.25 µM DPA (as traces) and at 71 µM DPA. (3) DPA may affect both phytoene synthase (at ≤71 µM DPA) and phytoene desaturase (at ≥53.25 µM DPA). (4) The assembly of LH2 complex does occur without any colored Cars.

Transient absorption study of two-photon excitation mechanism in the LH2 complex from purple bacterium Rhodobacter sphaeroides.

The mechanism of two-photon excitation of a peripheral light-harvesting complex LH2 (B800-850) from purple bacterium Rhodobacter sphaeroides was explained on the basis of femtosecond transient absorption data. Fast bleaching of the B850 absorption band was measured under two-photon excitation by 1350 nm femtosecond pulses, showing fast subpicosecond arrival of excitation energy to B850 circular aggregates. Any spectral changes connected with the B800 absorption band of B800-BChl molecules were absent. A similar picture was observed under one-photon excitation of the LH2 complex by 675 nm femtosecond pulses. We believe these effects may be attributed to direct excitation of high-energy excitonic states of a B850 circular aggregate or its vibrational manifold in accordance with the model of Abe [Chem. Phys. 2001, 264, 355-363].

Two-photon excitation spectroscopy of carotenoid-containing and carotenoid-depleted LH2 complexes from purple bacteria.

We applied two-photon fluorescence excitation spectroscopy to LH2 complex from purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides . Bacteriochlorophyll fluorescence was measured under two-photon excitation of the samples within the 1200-1500 nm region. Spectra were obtained for both carotenoid-containing and -depleted complexes of each bacterium to allow their direct comparison. The depletion of carotenoids did not alter the two-photon excitation spectra of either bacteria. The spectra featured a wide excitation band around 1350 nm (2x675 nm, 14,800 cm(-1)) which strongly resembled two-photon fluorescence excitation spectra of similar complexes published by other authors. We consider obtained experimental data to be evidence of direct two-photon excitation of bacteriochlorophyll excitonic states in this spectral region.

Heterogeneity of carotenoid content and composition in LH2 of the purple sulphur bacterium Allochromatium minutissimum grown under carotenoid-biosynthesis inhibition.

The effects brought about by growing Allochromatium (Alc.) minutissimum in the presence of different concentrations of the carotenoid (Car) biosynthetic inhibitor diphenylamine (DPA) have been investigated. A decrease of Car content (from approximately 70% to >5%) in the membranes was accompanied by an increase of the percentage of (immature) Cars with reduced numbers of conjugated C=C bonds (from neurosporene to phytoene). Based on the obtained results and the analysis of literature data, the conclusion is reached that accumulation of phytoene during inhibition did not occur. Surprisingly, DPA inhibited phytoene synthase instead of phytoene desaturase as generally assumed. The distribution of Cars in peripheral antenna (LH2) complexes and their effect on the stability of LH2 has been investigated using absorption spectroscopy and HPLC analysis. Heterogeneity of Car composition and contents in the LH2 pool is revealed. The Car contents in LH2 varied widely from control levels to complete absence. According to common view, the assembly of LH2 occurs only in the presence of Cars. Here, we show that the LH2 can be assembled without any Cars. The presence of Cars, however, is important for structural stability of LH2 complexes.