Purple Bacteria

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by 13 C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced 13 C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.

Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-tobacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cationbacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.

“ Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181: 17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c ) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm an...

A DNA sequence has been obtained for a 35.6-kb genomic segment from Heliobacillus mobilis that contains a major cluster of photosynthesis genes. A total of 30 ORFs were identified, 20 of which encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, reaction-center (RC) apoprotein, and cytochromes for cyclic electron transport. Donor side electron-transfer components to the RC include a putative RC-associated cytochrome c 553 and a unique four-large-subunit cytochrome bc complex consisting of Rieske Fe-S protein (encoded by petC), cytochrome b 6 (petB), subunit IV (petD), and a diheme cytochrome c (petX). Phylogenetic analysis of various photosynthesis gene products indicates a consistent grouping of oxygenic lineages that are distinct and descendent from anoxygenic lineages. In addition, H. mobilis was placed as the closest relative to cyanobacteria, which form a monophyletic origin to chloroplast-based photosynthetic lineages. The consensus of the photosynthesis gene trees also indicates that purple bacteria are the earliest emerging photosynthetic lineage. Our analysis also indicates that an ancient gene-duplication event giving rise to the paralogous bchI and bchD genes predates the divergence of all photosynthetic groups. In addition, our analysis of gene duplication of the photosystem I and photosystem II core polypeptides supports a ''heterologous fusion model'' for the origin and evolution of oxygenic photosynthesis.

The electronic excitation population and coherence dynamics in the chromophores of the photosynthetic light harvesting complex 2 (LH2) B850 ring from purple bacteria (Rhodopseudomonas acidophila) have been studied theoretically at both physiological and cryogenic temperatures. Similar to the well-studied Fenna-Matthews-Olson (FMO) protein, oscillations of the excitation population and coherence in the site basis are observed in LH2 by using a scaled hierarchical equation of motion (HEOM) approach. However, this oscillation time (300 fs) is much shorter compared to the FMO protein (650 fs) at cryogenic temperature. Both environment and high temperature are found to enhance the propagation speed of the exciton wave packet yet they shorten the coherence time and suppress the oscillation amplitude of coherence and the population. Our calculations show that a long-lived coherence between chromophore electronic excited states can exist in such a noisy biological environment.

The interaction of metal ions with isolated photosynthetic reaction centers (RCs) from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas Viridis has been investigated with transient optical and magnetic resonance techniques. In RCs from all species, the electrochromic response of the bacteriopheophytin cofactors associated with Q transfer is slowed in the presence of Cu 2+ . This slowing is similar to the metal ion effect observed for RCs from Rb. sphaeroides where Zn 2+ was bound to a specific site on the surface of the RC [Utschig et al. (1998) Biochemistry 37, 8278]. The coordination environments of the Cu 2+ sites were probed with electron paramagnetic resonance (EPR) spectroscopy, providing the first direct spectroscopic evidence for the existence of a second metal site in RCs from Rb. capsulatus and Rps. Viridis. In the dark, RCs with Cu 2+ bound to the surface exhibit axially symmetric EPR spectra. Electron spin echo envelope modulation (ESEEM) spectral results indicate multiple weakly hyperfine coupled 14 N nuclei in close proximity to Cu 2+ . These ESEEM spectra resemble those observed for Cu 2+ RCs from Rb. sphaeroides [Utschig et al. (2000) Biochemistry 39, 2961] and indicate that two or more histidines ligate the Cu 2+ at the surface site in each RC. Thus, RCs from Rb. sphaeroides, Rb. capsulatus, and Rps. Viridis each have a structurally analogous Cu 2+ binding site that is involved in modulating electrontransfer process. Inspection of the Rps. Viridis crystal structure reveals four potential histidine ligands from three different subunits (M16, H178, H72, and L211) located beneath the Q B binding pocket. The location of these histidines is surprisingly similar to the grouping of four histidine residues (H68, H126, H128, and L211) observed in the Rb. sphaeroides RC crystal structure. Further elucidation of these Cu 2+ sites will provide a means to investigate localized proton entry into the RCs of Rb. capsulatus and Rps. Viridis as well as locate a site of protein motions coupled with electron transfer.

There is considerable variability in the transition energies of individual antenna complexes from purple photosynthetic bacteria, raising the question of how rapid energy transfer between these complexes is accomplished. Here, based on steady state and time-resolved absorption and emission studies of peripheral LH2 antenna complexes isolated from the membrane of the purple bacterium Rhodobacter sphaeroides, we present evidence that the absorbing and emitting states of this complex are qualitatively different. The free exciton created by light absorption dynamically localizes within ~100 fs by coupling with nuclear vibrations to form a self-trapped exciton. The accompanying spatial deformation covers ~20% of the complex circumference at low temperature. This self-trapping, the first in its kind observed in biological systems, results in a broad fluorescence spectrum and considerably improves energy resonance between heterogeneous LH2 antenna complexes. Furthermore, the red shift of t...

The variability of the exciton spectra of bacteriochlorophyll molecules in light-harvesting (LH) complexes of photosynthetic bacteria ensures the excitation energy funneling trend toward the reaction center. The decisive shift of the energies is achieved due to exciton spectra formation caused by the resonance interaction between the pigments. The possibility to resolve the upper Davydov sub-band corresponding to the B850 ring and, thus, to estimate the exciton bandwidth by analyzing the temperature dependence of the steady-state absorption spectra of the LH2 complexes is demonstrated. For this purpose a selfmodeling curve resolution approach was applied for analysis of the temperature dependence of the absorption spectra of LH2 complexes from the photosynthetic bacteria Rhodobacter (Rba.) sphaeroides and Rhodoblastus (Rbl.) acidophilus. Estimations of the intradimer resonance interaction values as follows directly from obtained estimations of the exciton bandwidths at room temperature give 385 and 397 cm -1 for the LH2 complexes from the photosynthetic bacteria Rba. sphaeroides and Rhl. acidophilus, respectively. At 4 K the corresponding couplings are slightly higher (391 and 435 cm -1 , respectively). The retained exciton bandwidth at physiological conditions supports the decisive role of the exciton coherence determining light absorption in bacterial light-harvesting antenna complexes.

Singlet-singlet annihilation is used to study exciton delocalization in the light harvesting antenna complex LH2 (B800-B850) from the photosynthetic purple bacterium Rhodobacter sphaeroides. The characteristic femtosecond decay constants of the high intensity isotropic and the low intensity anisotropy kinetics of the B850 ring are related to the hopping time t h and the coherence length N coh of the exciton. Our analysis yields N coh 2.8 6 0.4 and t h 0.27 6 0.05 ps. This approach can be seen as an extension to the concept of the spectroscopic ruler.

The structure of the exciton manifold in the core bacteriochlorophyll antenna complexes from the purple photosynthetic bacterium Rhodobacter sphaeroides has been studied at 5 K using conventional and polarized fluorescence excitation spectroscopy supported with model simulations. Exciton bandwidths in the range of 2100-2200 cm À1 , depending on sample integrity, have been determined in correlation with the largest mean coupling energy between the nearest-neighbor bacteriochlorophyll molecules close to 600 cm À1 . These numbers are considerably bigger than those in any other native antenna complex known. Another noteworthy feature of these complexes is dominance of structural (off-diagonal) static disorder over the energetic (diagonal) disorder.

Spectral characteristics of the optically excited states in the ring-shaped quasi-one-dimensional aggregates comprising 18 and 32 tightly coupled bacteriochlorophyll a molecules have been investigated using selective spectroscopy methods and theoretical modelling of the data. Distinguished by the lowest electronic transition energies in the LH2 and LH1 antenna complexes these aggregates govern the functionally important ultrafast funneling of solar excitation energy in the photosynthetic membranes of purple bacteria. It was found by using a sophisticated differential fluorescence line narrowing method that exciton-phonon coupling in terms of the dimensionless Huang-Rhys factor is strong in these systems, justifying an excitonic polaron theoretical approach for the data analysis. Although we reached this qualitative conclusion already previously, in this work essential dependence of the exciton-phonon coupling strength and reorganization energy on excitation wavelength as well as on excitation light fluence has been established. We then show that these results corroborate with the properties of excitonic polarons in diagonally disordered ensembles of the aggregates. Furthermore, the weighted density of states of the phonon modes, which is an important characteristic of dynamical systems interacting with their surroundings, was derived. Its shape, being similar for all studied circular aggregates, deviates significantly from a reference profile describing local response of a protein to the Q y electronic transition in a single bacteriochlorophyll a molecule. Similarities of the data for regular and B800 deficient mutant LH2 complexes indicate that the B800 pigments have no direct influence on the electronic states of the B850 aggregate system. Consistent set of model parameters was determined, unambiguously implying that excitonic polarons, rather than bare excitons are proper lowest-energy optical excitations in the LH1 and LH2 antenna complexes.

High-Potential Iron-Sulfur Proteins (HiPIP) are small electron carriers, present only in species of photosynthetic purple bacteria having a RC-bound cytochrome. Their participation in the photo-induced cyclic electron transfer was recently established for Rubrivivax gelatinosus, Rhodocyclus tenuis and Rhodoferax fermentans (Schoepp et al. 1995; Hochkoeppler et al. 1996a, Menin et al. 1997b). To better understand the physiological role of HiPIP, we

Several biophysical properties (absorption, fluorescence, linear dichroism) are reported for the chromatophore membranes of the thermophilic purple sulfur bacterium, Chromatium tepidum. Like the mesophilic strain Chromatium vinosum, two types of fight-harvesting complex are present: one absorbs at 800 nm and 855 nm; the second complex is equivalent to the 11890 complex of C. vinosum, hut absorbs at 918 nm, i.e., 30 nm higher. This is the highest absorption band observed so far for a light-harvesting complex containing bacteriochiorophyll a. In spite of the small overlap between the fluorescence and absorption bands of the two light-harvesting complexes, specially at low temperature, an efficient energy transfer occurs from the high-energy (B800-855) to the low-energy (B920) complexes. The B800-855 complexes have been isolated from the whole membrane by lauryldimethylamine N-oxide treatment, whereas only a partial purification was achieved for the B920 complexes.

Purple sulfur phototrophic bacteria accumulate various storage materials, such as sulfur globules, glycogen or polyhydroxy alkanoates (PHAs) under appropriate conditions. The formation of these materials requires reducing power which might be recovered upon their breakdown. This work aims at the understanding of the metabolism of PHA and its link to the nitrogenase mediated in vivo H 2 evolution in Thiocapsa roseopersicina BBS. The strain could accumulate 30% of the dry cell weight in the form of PHAs. Analysis of the genome sequence revealed the loci involved in PHAs synthesis and degradation. Phylogenetic analysis indicated independent evolution of the anabolic and catabolic proteins. A mutant carrying deleted PHA biosynthesis genes has been created in a host containing nitrogenase but none of the hydrogenases. Determination of the H 2 evolving capacity of the mutant revealed significantly reduced H 2 production in PHA deficient cells. Addition of excess electron sources such as thiosulfate stimulated the H 2 production via multiple effects.

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H 2 generation was utilized to study the production of H 2 from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H 2 production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H 2 from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids.

The dsr genes and the hydSL operon are present as separate entities in phototrophic sulfur oxidizers of the genera Allochromatium, Marichromatium, Thiocapsa and Thiocystis and are organized similarly as in Allochromatium vinosum and Thiocapsa roseopersicina, respectively. The dsrA gene, encoding the K subunit of `reverse' siroheme sulfite reductase, is also present in two species of green sulfur bacteria pointing to an important and universal role of this enzyme and probably other proteins encoded in the dsr locus in the oxidation of stored sulfur by phototrophic bacteria. The hupSL genes are uniformly present in the members of the Chromatiaceae family tested. The two genes between hydS and hydL encode a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, resembling subunits of heterodisulfide reductase from methanogenic archaea. These genes are similar but not identical to dsrM and dsrK, indicating that the derived proteins have distinct functions, the former in hydrogen metabolism and the latter in oxidative sulfur metabolism.

The orientation of the principal axes of the primary electron donor triplet state measured in single crystals of photosynthetic reaction centers is compared to the x-ray structures of the bacteria Rhodobacter (Rb.) sphaeroides R-26 and Rhodopseudomonas (Rps.) viridis. The primary donor of Rps. viridis is significantly different from that of Rb. sphaeroides. The measured directions of the axes indicate that triplet excitation is almost completely localized on the L-subunit half of the dimer in Rps. viridis but is more symmetrically distributed (approximately 63% on the L half of the special pair and approximately 37% on the M half) on the dimeric donor in Rb. sphaeroides R-26. The large reduction of the zero field splitting parameters relative to monomeric bacteriochlorophyll triplet in vitro suggests significant participation of asymmetrical charge transfer electronic configurations in the special pair triplet state of both organisms (approximately 23% in Rps. viridis and approximat...

The distance between the paramagnetic state of a native cofactor and a spin label is measured in the photosynthetic reaction centre from the bacterium Rhodobacter sphaeroides R26. A two-frequency pulsed electron paramagnetic resonance method [double-electronelectron spin resonance (DEER)] is used. A distance of 3.05 nm between the semiquinone anion state of the primary acceptor (Q A ) and the spin label at the native cysteine at position 156 in the H-subunit is found. Molecular-dynamics (MD) simulations are performed to interpret the distance. A 6 ns run comprising the entire RC protein yields a distance distribution that is close to the experimental one. The average distance found by the MD simulation is smaller than the distance obtained by DEER by at least 0.2 nm. To better represent the experiments performed at low temperature (60 K), a MD method to mimic the freezing-in of the room-temperature conformations is introduced. Both MD methods yield similar distances, but the second method has a trend towards a wider distance distribution.

The reaction center (RC)-bound primary acceptor quinone QA of the photosynthetic bacterium Rhodobacter sphaeroides R26 functions as a one-electron gate. The radical anion Q~-is proposed to have an asymmetric electron distribution, induced by the protein environment. We replace the native ubiquinone-10 (UQ10) with specifically 13C-labelled UQ10, and use Q-band (35 GI-Iz) EPR spectroscopy to investigate this phenomenon in closer detail. The direct observation of the ~3C-hyperfine splitting of the gz-COmponent of UQ I 0~,-in the RC and in frozen isopropanol shows that the electron spin distribution is symmetric in the isopropanol glass, and asymmetric in the RC. Our results allow qualitative assessment of the spin and charge distribution for Q~-in the RC. The carbonyl oxygen of the semiquinone anion nearest to the S = 2 Fe2+-ion and QB is shown to acquire the highest (negative) charge density.

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F V /F M , whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F V /F M of ~0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.

In this short communication we present the stoichiometric ratio of bacteriochlorophyll (Bchl), bacteriopheophytin (Bph) and carotenoids in a few photosynthetic purple bacteria complexes (whose 2D or 3D structures are well known) determined using the spectrum-reconstruction method (SRCM). An important conclusion of our pigment stoichiometric analysis is the evidence for the absence of the second carotenoid in LH2. In the process, we also highlight the useful application of SRCM in determining the molar extinction coefficients of carotenoids present in LH1, LH2 or RC for which these values are not known due to isolation problems and/or stability.

High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc 1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c 2 . For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity. To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species. Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c 2 . We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c 2 . We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures. There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree. The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.

It has already been established that the quaternary structure of the main light-harvesting complex (LH2) from the photosynthetic bacterium Rhodopseudomonas palustris is a nonameric 'ring' of PucAB heterodimers and under low-light culturing conditions an increased diversity of PucB synthesis occurs. In this work, single molecule fluorescence emission studies show that different classes of LH2 'rings' are present in "low-light" adapted cells and that an unknown chaperon process creates multiple sub-types of 'rings' with more conformational sub-states and configurations. This increase in spectral disorder significantly augments the cross-section for photon absorption and subsequent energy flow to the reaction centre trap when photon availability is a limiting factor. This work highlights yet another variant used by phototrophs to gather energy for cellular development.

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of C02). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.

A mildly thermophilic bacterium isolated from a New Mexican hot spring microbial mat is described. The organism is a budding bacterium and contained bacteriochlorophyll b as sole chlorophyll pigment. The organism grew up to 47 °C and grew optimally around 42°C. The new organism required only biotin and a reduced sulfur source (for biosynthetic purposes) as growth factors, and utilized a relatively restricted group of organic compounds as sole carbon sources. The hotspring anoxygenic phototroph also fixed N 2 up to 47°C and showed ammonia 'switch-off' of nitrogenase activity, typical of mesophilic phototrophic bacteria. The new organism, which we refer to as Rhodopseudomonas strain GI, is the first bacteriochlorophyll b-containing nonsulfur purple bacterium shown capable of growth above 45°C.

It is now well established that, for photosynthetic bacteria, the aerobic-to-microaerophilic transition activates the membrane-bound sensor kinase RegB, which subsequently phosphorylates the transcriptional activator RegA, thereby inducing elevated levels of intracellular photosynthetic membranes. The mechanism of RegB activation—in particular, the role of ubiquinone-10—is controversial at present. One problem here is that very limited quantitative in vivo data for the response of the ubiquinone redox state to different cultivation conditions exist. Here, we utilize Rhodospirillum rubrum to study the correlation of the quinone redox state to the expression level of photosynthetic membranes and determine an effective response function directly. Our results show that changes in the photosynthetic membrane levels between 50 and 95% of that maximally attainable are associated with only a twofold change in the ubiquinol/ubiquinone ratio and are not necessarily proportional to the total l...

Reaction centers (RC) from the species Erythrobacter (Eb.) litoralis, Erythromonas (Em.) ursincola and Sandaracinobacter (S.) sibiricus have been purified by LDAO treatment of light-harvesting-reaction center complexes and DEAE chromatography. The content and overall organisation of the RCs' chromophores, determined by linear dichroism (LD) and absorption spectroscopy, are similar to those isolated from anaerobic photosynthetic bacteria. The redox properties of the

Purple bacteria have thus far been considered to operate light-driven cyclic electron transfer chains containing ubiquinone (UQ) as liposoluble electron and proton carrier. We show that in the purple γ-proteobacterium Halorhodospira halophila , menaquinone-8 (MK-8) is the dominant quinone component and that it operates in the Q B -site of the photosynthetic reaction center (RC). The redox potentials of the photooxidized pigment in the RC and of the Rieske center of the bc 1 complex are significantly lower ( E m = +270 mV and +110 mV, respectively) than those determined in other purple bacteria but resemble those determined for species containing MK as pool quinone. These results demonstrate that the photosynthetic cycle in H. halophila is based on MK and not on UQ. This finding together with the unusual organization of genes coding for the bc 1 complex in H. halophila suggests a specific scenario for the evolutionary transition of bioenergetic chains from the low-potential menaquino...

Photosynthetic electron transfer has been examined in whole cells, isolated membranes and in partially purified reaction centers (RCs) of Roseicyclus mahoneyensis, strain ML6 and Porphyrobacter meromictius, strain ML31, two species of obligate aerobic anoxygenic phototrophic bacteria. Photochemical activity in strain ML31 was observed aerobically, but the photosynthetic apparatus was not functional under anaerobic conditions. In strain ML6 low levels of photochemistry were measured anaerobically, possibly due to incomplete reduction of the primary electron acceptor (Q A) prior to light excitation, however, electron transfer occurred optimally under low oxygen conditions. Photoinduced electron transfer involves a soluble cytochrome c in both strains, and an additional reaction center (RC)-bound cytochrome c in ML6. The redox properties of the primary electron donor (P) and Q A of ML31 are similar to those previously determined for other aerobic phototrophs, with midpoint redox potentials of ?463 mV and-25 mV, respectively. Strain ML6 showed a very narrow range of ambient redox potentials appropriate for photosynthesis, with midpoint redox potentials of ?415 mV for P and ?94 mV for Q A. Cytoplasm soluble and photosynthetic complex bound cytochromes were characterized in terms of apparent molecular mass. Fluorescence excitation spectra revealed that abundant carotenoids not intimately associated with the RC are not involved in photosynthetic energy conservation.

The vertical distribution of culturable anoxygenic phototrophic bacteria was investigated at five sites at or near the Juan de Fuca Ridge in the Pacific Ocean. Twelve similar strains of obligately aerobic phototrophic bacteria were isolated in pure culture, from depths ranging from 500 to 2,379 m below the surface. These strains appear morphologically, physiologically, biochemically, and phylogenetically similar to Citromicrobium bathyomarinum strain JF-1, a bacterium previously isolated from hydrothermal vent plume waters. Only one aerobic phototrophic strain was isolated from surface waters. This strain is morphologically and physiologically distinct from the strains isolated at deeper sampling locations, and phylogenetic analysis indicates that it is most closely related to the genus Erythrobacter. Phototrophs were cultivated from three water casts taken above vents but not from two casts taken away from active vent sites. No culturable anaerobic anoxygenic phototrophs were detected. The photosynthetic apparatus was investigated in strain JF-1 and contains light-harvesting I and reaction center complexes, which are functional under aerobic conditions. Keywords Aerobic phototrophic bacteria Á Anoxygenic photosynthesis Á Bacteriochlorophyll Á Erythrobacter Á Citromicrobium Á Juan de Fuca Ridge

Reaction centers (RCs)with one antenna complex attached (RC/LH~ complexes)were isolated from the alkaliphilic and moderately halophilic, phototrophic purple sulfur bacterium Ectothiorhodospira (E.) mobilis and analyzed with respect to quinone composition and charge recombination kinetics, using time-resolved (low-and room-temperature) spectroscopy and thin-layer chromatography. The RCs contain menaquinone as primary and ubiquinone as secondary quinone electron acceptor, QA and Q a, respectively. Q B is lost during isolation of the RC/LH I complexes. P+ QA charge recombination kinetics, which were shown to be pH independent, were only slightly temperature dependent, indicating that this recombination proceeds via the direct (electron tunnelling) route. At room temperature, its average lifetime was 34.5 ms. These decay kinetics were shown to be monophasic at room temperature and biphasic at low temperature. Addition of an excess of UQ 6 to RC/LH I complexes resulted in retardation of the P+ recovery, due to charge recombination of the state P+QAQa. Functional reconstitution of QB was also evident from flash-induced binary oscillations at 450 nm, which could be observed in RC/LH I complexes in the presence of an excess of UQ 6. This reconstitution of QB activity was, however, incomplete and decreased with increasing pH. The P+QAQB decay kinetics were pH independent; the average lifetime was about 6 s at room temperature. The apparent equilibrium constant g 2 between the states QAQB and QAQB, and consequently the free energy difference between these states, were relatively large and pH independent. K 2 was on average 167, whereas the mean value of the free energy difference between the two states was 130 meV. Finally, a scheme is presented for the kinetics and free energy changes of the electron transfer steps in isolated E. mobilis RCs.

Three photosynthetic membranes, called intra-cytoplasmic membranes (ICMs), from wild-type and the ∆pucBA mutant of the purple phototrophic bacterium Rps. palustris were investigated using optical spectroscopy. The ICMs contain identical light-harvesting complex 1-reaction centers (LH1-RC) but have various spectral forms of light-harvesting complex 2 (LH2). Spectroscopic studies involving steady-state absorption, fluorescence, and femtosecond time-resolved absorption at room temperature and at 77 K focused on inter-protein excitation energy transfer. The studies investigated how energy transfer is affected by altered spectral features of the LH2 complexes as those develop under growth at different light conditions. The study shows that LH1 → LH2 excitation energy transfer is strongly affected if the LH2 complex alters its spectroscopic signature. The LH1 → LH2 excitation energy transfer rate modeled with the Förster mechanism and kinetic simulations of transient absorption of the ICM...

The electronic excitation population and coherence dynamics in the chromophores of the photosynthetic light harvesting complex 2 (LH2) B850 ring from purple bacteria (Rhodopseudomonas acidophila) have been studied theoretically at both physiological and cryogenic temperatures. Similar to the well-studied Fenna-Matthews-Olson (FMO) protein, oscilla

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αβ-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αβpolypeptides that are hydrogen-bonded to BChl a. The Ca 2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of αand β-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.

Spectroscopic properties, including low-temperature absorbance, linear and circular dichroism and site-selection fluorescence of the antenna complexes from Rhodospirillum molischianum have been determined. The unique 'LHl-like' character of the amino acid sequence from LH2 of this bacterium is reflected in the circular dichroism of the B850 band of this complex. The wavelength dependence of the polarization of the LH2 complex shows an unusual shape that is attributed to the octameric state of this complex. The complete amino acid sequence for the LH1 a-polypeptide and most of the /3-polypeptides are presented. These conform to the general features of other LHI polypeptides. This result, in combination with spectroscopic data for LH1 imply that the organisation of the core in this bacterium is not much different from that in other purple non-sulphur bacteria.

Illumination of intact cells of Rhodobacter sphaeroides under anaerobic conditions has a dual effect on the redox state of the quinone pool. A large oxidation of the quinone pool is observed during the first seconds following the illumination. This oxidation is suppressed by the addition of an uncoupler in agreement with a lightinduced reverse electron transfer at the level of the complex I, present both in the non-invaginated part of the membrane and in the chromatophores. At longer dark times, this illumination increases the reducing power of the cells leading to a significant reduction of the others reaction centers (RCs). From the observation that a significant proportion of RCs could be reduced by the preillumination without affecting the numbers of charge separation for the RCs, we conclude that there is no rapid thermodynamic equilibrium between the quinones present in the noninvaginated part of the membrane and those localized in the chromatophores. Under anaerobic conditions where the chromatophores quinone pool is fully reduced, we deduce, on the basis of flash-induced fluorescence kinetics, that the reduced RCs are exclusively reoxidized by the quinone generated at the Q o site of the cyt bc 1 complex. The supramolecular association between a dimeric RC-LHI complex and one cyt bc 1 complex allows the confinement of a quinone between the RC-LHI directly associated to the cyt bc 1 complex. Keywords Rhodobacter sphaeroides Á Fluorescence Á Anaerobiosis Á Quinones Á Supercomplexes Á Complex I This article is dedicated to the memory of Pr. Roderick Clayton (23 October 2012) who introduced André Verméglio to the wonderful world of photosynthetic bacteria.

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by 13 C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced 13 C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.

Reaction centers from the photosynthetic bacterium Rhodopseudomonas viridis have been excited within the near-infrared absorption bands of the dimeric primary donor (P), of the “accessory” bacteriochlorophylls (B), and of the bacteriopheophytins (H) by using laser pulses of 150-fsec duration. The transfer of excitation energy between H, B, and P occurs in slightly less than 100 fsec and leads to the ultrafast formation of an excited state of P. This state is characterized by a broad absorption spectrum and exhibits stimulated emission. It decays in 2.8 ± 0.2 psec with the simultaneous oxidation of the primary donor and reduction of the bacteriopheophytin acceptor, which have been monitored at 545, 675, 815, 830, and 1310 nm. Although a transient bleaching relaxing in 400 ± 100 fsec is specifically observed upon excitation and observation in the 830-nm absorption band, we have found no indication that an accessory bacteriochlorophyll is involved as a resolvable intermediary acceptor ...

The reaction centre (RC) of green sulphur bacteria is a FeS-type RC, as are the RCs of Photosystems I (PS I) of oxygenic photosynthetic organisms and of heliobacteria. The core domains of both green sulphur bacterial and heliobacterial RCs are considered to be homodimeric, in contrast to those of purple bacteria, PS I and Photosystem II (PS II). This paper briefly describes the techniques of electron microscopy and image processing suited to investigate the structure of these proteins. Recent advances in the study of the structure of the green sulphur bacterial RC, primarily achieved by the application of scanning transmission electron microscopy, are reviewed.

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus arrrmriucus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be recc :tstituted by the addition of ubiquinone-IO. The equilibrium constant of electron transfer between primary (QA) and secondary (Qs) quinones was much higher than that in RC of purple bacteria. The Q. binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the Qs protonation was observed after the second gash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P'C&) formation. Since the C. auruntiuars RC lacks H-subunit, this subunit was not an obligatory component for electrogenic Qn protonation.

Electrogenic events in the photosynthetic reaction center complex (RC), accompanying single‐ and two‐electron reduction of the secondar quinone acceptor Qb, were investigated. In the presence of inhibitors of electron transfer via the bc1‐complex, the kinetics of formation of the transmembrane electric potential difference induced by two successive light flashes exhibit a few phases. Besides the fast phase A which is due to the charge separation between the bacteriochlorophyll dimer P and primary quinone acceptor qa, two slower atrazine‐sensitive phases, BI and BII, were observed. Phase BI is suggested to be due to proton transfer between the amino acid residues of the reaction center protein, and phase BII due to proton uptake during the second flash‐induced formation of ubiquinol. A possible model of electrogenesis in the acceptor moiety of the RC is discussed.

A three-dimensional model of the core proteins D1 and D2, including the cofactors, that form the Photosystem II reaction centre of pea (Pisum sativum), has been generated. This model was built with a rule-based computer modelling system using the information from the crystal structures of the photosynthetic reaction centres of Rhodopseudomonas viridis and Rhodobacter sphaeroides. An alignment of the primary sequences of twenty three D1, nine D2, eight bacterial L and eight bacterial M subunits predicts strong similarity between bacterial and higher plant reaction centres, especially in the transmembrane region where the cofactors responsible for electron transport are located. The sequence to be modelled was aligned to the bacterial structures using environment-dependent substitution tables to construct matrices, improving the alignment procedure. The ancestral relationship between the bacteria and higher plant sequences allowed both the L and M subunits to be used as structural templates as they were equally related to the higher plant polypeptides. The regions with the highest predicted structural homology were used as a framework for the construction of the structurally conserved regions. The structurally conserved region of the model shows strong similarity to the bacterial reaction centre in the transmembrane helices. The stromal and lumenal loops show greater sequence variation and are therefore predicted to be the structurally variable regions in the model. The key sidechain assignments and residues that may interact with cofactors are discussed.

The present work describes results obtained on hybrid systems formed in aqueous buffer solution by selfassembly of different CdSe quantum dots (QDs) surrounded by a ZnS shell and functionalized by covering the surface with anionic and cationic groups and various isolated pigment-protein complexes from the lightharvesting antennae of photosynthetic organisms (light-harvesting complexes 1 and 2 (LH1 and LH2, respectively) from purple bacteria, phycobiliproteins (PBPs) from cyanobacteria and the rod-shaped PBP from the cyanobacterium Acaryochloris marina). Excitation energy transfer (EET) from QDs to PBP rods was found to take place with varying and highly temperature-dependent efficiencies of up to 90%. Experiments performed at room temperature on hybrid systems with different QDs show that no straightforward correlation exists between the efficiency of EET and the parameter J / (R 12 6 ) given by the theory of Förster resonance energy transfer (FRET), where J is the overlap integral of the normalized QD emission and PBP absorption and R 12 the distance between the transition dipole moments of donor and acceptor. The results show that the hybrid systems cannot be described as randomly orientated aggregates consisting of QDs and photosynthetic pigment-protein complexes. Specific structural parameters are inferred to play an essential role. The mode of binding and coupling seems to change with the size of QDs and with temperature. Efficient EET and fluorescence enhancement of the acceptor was observed at particular stoichiometric ratios between QDs and trimeric phycoerythrin (PE). At higher concentrations of PE, a quenching of its fluorescence is observed in the presence of QDs. This effect is explained by the existence of additional quenching channels in aggregates formed within hybrid systems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

The light-harvesting complex 2 (LH2) of purple bacteria is one of the most studied photosynthetic antenna complexes. Its symmetric structure and ring-like bacteriochlorophyll arrangement make it an ideal system for theoreticians and spectroscopists. LH2 complexes from most bacterial species are thought to have eightfold or ninefold symmetry, but recently a sevenfold symmetric LH2 structure from the bacterium Mch. purpuratum was solved by Cryo-Electron microscopy. This LH2 also possesses unique near-infrared absorption and circular dichroism (CD) spectral properties. Here we use an atomistic strategy to elucidate the spectral properties of Mch. purpuratum LH2 and understand the differences with the most commonly studied LH2 from Rbl. acidophilus. Our strategy exploits a combination of molecular dynamics simulations, multiscale polarizable quantum mechanics/molecular mechanics calculations, and lineshape simulations. Our calculations reveal that the spectral properties of LH2 complexes are tuned by site energies and exciton couplings, which in turn depend on the structural fluctuations of the bacteriochlorophylls. Our strategy proves effective in reproducing the absorption and CD spectra of the two LH2 complexes, and in uncovering the origin of their differences. This work proves that it is possible to obtain insight into the spectral tuning strategies of purple bacteria by quantitatively simulating the spectral properties of their antenna complexes.

Photosynthetic light harvesting can occur with a remarkable near-unity quantum efficiency. The B800-850 complex, also known as light-harvesting complex 2 (LH2), is the primary light-harvesting complex in purple bacteria and has been extensively studied as a model system. The bacteriochlorophyll of the B800-850 complex are organized into two concentric rings, known as the B800 and B850 rings. However, depending on the species and growth conditions, the number of constituent subunits, the pigment geometry, and the absorption energies vary. While the dynamics of some B800-850 variants 1