Trapping of an assembly intermediate of photosynthetic LH1 antenna beyond B820 subunit. Significance for the assembly of photosynthetic LH1 antenna

Most photosynthetic LH1 antennae undergo dissociation into B820 subunits, suggesting their universal character as structural modules. However, dissociation into subunits seems to occur reversibly only in the absence of carotenoids and the subunits were never found to bind carotenoids. The interactions of carotenoids with B820 have been studied in a newly developed reconstitution assay of the LH1 antenna from Rhodospirillum rubrum (Fiedor, L., Akahane, J., and Koyama, Y. (2004) Biochemistry 43, 16487-16496). These model studies show that B820 subunits strongly interact with carotenoids and spontaneously form stable LH1-like complexes with substoichiometric carotenoid content. This is the first experimental evidence that B820 may occur as a short-lived intermediate in the assembly of LH1 in vivo. The resulting complex of B820 subunits with carotenoid, termed iB873, is homogeneous, according to ion exchange chromatography and reproducible pigment composition. The iB873-bound carotenoid is as efficient in energy transfer to bacteriochlorophyll as the one in native antenna. To our knowledge, iB873 is the first complex binding functional carotenoid, with the spectral and biochemical properties intermediate between that of B820 and the fully assembled LH1.     

Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides: II. A study of PufX- membranes

In the bacterium R. sphaeroides, the polypeptide PufX is indispensable for photosynthetic growth. Its deletion is known to have important consequences on the organization of the photosynthetic apparatus. In the wild-type strain, complexes between the reaction center (RC) and the antenna (light-harvesting complex 1 (LH1)) are associated in dimers, and LH1 does not fully encircle the RC. In the absence of PufX, the complexes become monomeric, and the LH1 ring closes around the RC. We analyzed the functional consequences of PufX deletion. Some effects can be ascribed to the monomerization of the RC.LH1 complexes: the number of RCs that share a common antenna for excitation transfer or a common quinone pool become smaller. We examined the kinetic effects of the closed LH1 ring on quinone turnover: diffusion across LH1 entails a delay of approximately 1 ms, and the barrier appears to be located directly against the quinone-binding (secondary quinone acceptor (Q(B))) pocket. The diffusion of ubiquinol from the RC to the cytochrome bc1 complex is approximately 2-fold slower in the mutant, suggesting an increased distance between the two complexes. The properties of the Q(B) pocket (binding of inhibitors, stabilization of Q(B-), and rate of Q(B)-H2 formation) appear to be modified in the mutant. Another specificity of PufX- is the accumulation of closed centers in the Q(A-) (where Q(A) is the primary quinone acceptor) state as the secondary acceptor pool becomes reduced, which is probably the origin of photosynthetic incompetence. We suggest that this is related to the Q(B) pocket alterations. The malfunction of the reaction center is probably due to a faulty association with LH1 that is prevented in the PufX-containing structure.     

Band Structure and Local Dynamics of Excitons in Bacterial Light-harvesting Complexes Revealed by Spectrally Selective Spectroscopy

Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

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 LH1-reaction 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.     

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

The assembly and organisation of photosynthetic membranes in Rhodobacter sphaeroides.

Recent AFM data demonstrate that mature photosynthetic membranes of R. sphaeroides are composed of rows of dimeric RC-LH1-PufX complexes with some LH2 complexes 'sandwiched' between these rows of core complexes, and others in discrete LH2-only domains which might form the light-responsive complement of the LH2 antenna. The present work applies membrane fractionation, radiolabelling and LDS-PAGE techniques to investigate the response of R. sphaeroides to lowered light intensity. The kinetics underlying this adaptation to low light conditions were revealed by radiolabelling with the bacteriochlorophyll (bchl) biosynthetic precursor, delta-aminolevulinate, which allowed us to measure only the bchls synthesised after the light intensity shift. We show that (1) the increase in LH2 antenna size is mainly restricted to the mature ICM membrane fraction, and the antenna composition of the precursor upper pigmented band (UPB) membrane remains constant, (2) the precursor UPB membrane is enriched in bchl synthase, the terminal enzyme of the bchl biosynthetic pathway, and (3) the LH2 and the complexes of intermediate migration in LDS-PAGE exhibit completely different labelling kinetics. Thus, new photosynthetic complexes, mainly LH2, are synthesised and assembled at the membrane initiation UPB sites, where the LH2 rings pack between the rows of dimeric cores fostering new LH2-LH1 interactions. Mature membranes also assemble new LH2 rings, but in this case the 'sandwich' regions between the rows of core dimers are already fully occupied and the bulk antenna pool is the favoured location for these new LH2 complexes.     

Characterisation of the photosynthetic complexes from the marine gammaproteobacterium Congregibacter litoralis KT71

Possibly the most abundant group of anoxygenic phototrophs are marine photoheterotrophic Gammaproteobacteria belonging to the NOR5/OM60 clade. As little is known about their photosynthetic apparatus, the photosynthetic complexes from the marine phototrophic bacterium Congregibacter litoralis KT71 were purified and spectroscopically characterised. The intra-cytoplasmic membranes contain a smaller amount of photosynthetic complexes when compared with anaerobic purple bacteria. Moreover, the intra-cytoplasmic membranes contain only a minimum amount of peripheral LH2 complexes. The complexes are populated by bacteriochlorophyll a, spirilloxanthin and two novel ketocarotenoids, with biophysical and biochemical properties similar to previously characterised complexes from purple bacteria. The organization of the RC-LH1 complex has been further characterised using cryo-electron microscopy. The overall organisation is similar to the complex from the gammaproteobacterium Thermochromatium tepidum, with the type-II reaction centre surrounded by a slightly elliptical LH1 antenna ring composed of 16 αβ-subunits with no discernible gap or pore. The RC-LH1 and LH2 apoproteins are phylogenetically related to other halophilic species but LH2 also to some alphaproteobacterial species. It seems that the reduction of light-harvesting apparatus and acquisition of novel ketocarotenoids in Congregibacter litoralis KT71 represent specific adaptations for operating the anoxygenic photosynthesis under aerobic conditions at sea.     

Native electrospray mass spectrometry reveals the nature and stoichiometry of pigments in the FMO photosynthetic antenna protein

The nature and stoichiometry of pigments in the Fenna-Matthews-Olson (FMO) photosynthetic antenna protein complex were determined by native electrospray mass spectrometry. The FMO antenna complex was the first chlorophyll-containing protein that was crystallized. Previous results indicate that the FMO protein forms a trimer with seven bacteriochlorophyll a in each monomer. This model has long been a working basis to understand the molecular mechanism of energy transfer through pigment/pigment and pigment/protein coupling. Recent results have suggested, however, that an eighth bacteriochlorophyll is present in some subunits. In this report, a direct mass spectrometry measurement of the molecular weight of the intact FMO protein complex clearly indicates the existence of an eighth pigment, which is assigned as a bacteriochlorophyll a by mass analysis of the complex and HPLC analysis of the pigment. The eighth pigment is found to be easily lost during purification, which results in its partial occupancy in the mass spectra of the intact complex prepared by different procedures. The results are consistent with the recent X-ray structural models. The existence of the eighth bacteriochlorophyll a in this model antenna protein gives new insights into the functional role of the FMO protein and motivates the need for new theoretical and spectroscopic assignments of spectral features of the FMO protein.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

Excitation trap approach to analyze size and pigment-pigment coupling: reconstitution of LH1 antenna of Rhodobacter sphaeroides with Ni-substituted bacteriochlorophyll

Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time range) radiationless de-excitation path, while the principal ground-state absorption and coordination properties of the pigment are retained. A method has been developed for substituting the native bacteriochlorophyll a by Ni-bacteriochlorophyll a ([Ni]-BChl) in the light harvesting antenna of the core complex (LH1) from the purple bacterium, Rhodobacter (Rb.) sphaeroides, to investigate its unit size and excited state properties. The components of the complex have been extracted with an organic solvent from freeze-dried membranes of an LH1-only strain of Rb. sphaeroides and transferred into the micelles of n-octyl-beta-glucopyranoside (OG). Reconstitution was achieved by solubilization in 3.4% OG, followed by dilution, yielding a complex nearly identical to the native one, in terms of absorption, fluorescence, and circular dichroism spectra as well as energy transfer efficiency from carotenoid to bacteriochlorophyll. By adding increasing amounts of [Ni]-BChl to the reconstitution mixture, a series of LH1 complexes was obtained that contain increasing levels of this efficient excitation trap. In contrast to the nearly unchanged absorption, the presence of [Ni]-BChl in LH1 markedly affects the emission properties. Incorporation of only 3.2 and 20% [Ni]-BChl reduces the emission by 50% and nearly 100%, respectively. The subnanosecond fluorescence kinetics of the complexes were monoexponential, with the lifetime identical to that of the native complex, and its amplitude decreasing in parallel with the steady-state fluorescence yield. Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the presence of a single excitation trap per LH1 unit suffices for efficient emission quenching and that this unit contains 20 +/- 1 BChl molecules.     

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH >11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH >9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q(A)Q(B)(-) state is stabilized by about 40 meV at 6.511. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and (31)P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.     

Low-temperature single-molecule spectroscopy on photosynthetic pigment–protein complexes from purple bacteria

The primary reactions of purple bacterial photosynthesis take place within two well characterized pigment–protein complexes, the core Reaction Center–Light Harvesting 1 (RC–LH1) complex and the more peripheral Light Harvesting 2 (LH2) complex. These antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction-centers, where it is used to ‘power’ the photosynthetic redox reaction. This review provides an overview of how the character of the electronically excited states of these pigment–protein complexes are determined by quantum mechanics and how the respective spectral signatures can be observed by single-molecule spectroscopy.     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Carotenoid-induced cooperative formation of bacterial photosynthetic LH1 complex

A simple reconstitution technique has been developed and then applied to prepare a series of light-harvesting antenna 1 (LH1) complexes with a programmed carotenoid composition, not available from native photosynthetic membranes. The complexes were reconstituted with different C(40) carotenoids, having two structural parameters variable: the functional side groups and the number of conjugated C-C double bonds, systematically increasing from 9 to 13. The complexes, differing only in the type of carotenoid, bound to an otherwise identical bacteriochlorophyll-polypeptide matrix, can serve as a unique model system in which the relationship between the carotenoid character and the functioning of pigment-protein complexes can be investigated. The reconstituted LH1 complexes resemble the native antenna, isolated from wild-type Rhodospirillum rubrum, but their coloration is entirely determined by carotenoid. Along with the increase in its conjugation size, the carotenoid absorption transitions gradually shift to the red. Thus, the extension of the conjugation size of the antenna carotenoids provides a mechanism for the spectral tuning of light harvesting in the visible part of the spectrum. The carotenoids in the reconstitution system promote the LH1 formation and seem to bind and transfer the excitation energy specifically only to a species with characteristically red-shifted absorption and emission maxima, apparently, due to a cooperative effect. Monitoring the LH1 formation by steady-state absorption and fluorescence spectroscopies reveals that in the presence of carotenoids it proceeds without spectrally resolved intermediates, leading directly to B880. The effect of the carotenoid is enhanced when the pigment contains the hydroxy or methoxy side groups, implying that, in parallel to hydrophobic interactions and pi-pi stacking, other interactions are also involved in the formation and stabilization of LH1.     

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

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

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

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 approximately 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 LH1-reaction 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.     

Conformation of bacteriochlorophyll molecules in photosynthetic proteins from purple bacteria.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [N?veke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.     

A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm

A recently isolated species of the photosynthetic purple sulfur bacteria, provisionally called strain 970, was investigated with respect to its antenna function by means of various spectroscopic techniques, including fluorescence and pump-probe absorption difference spectroscopy. The bacterium contains bacteriochlorophyll a and an as yet unidentified carotenoid, perhaps 3,4,3',4'-tetrahydrospirilloxanthin. It has a single antenna complex of the LH1 type, with a Q(y) absorption band situated at the unusually long wavelength of 963 nm at room temperature and 990 nm at 6 K. In contrast to many other species, the reaction center showed two well-separated absorption bands of bacteriopheophytin at 6 K, located at 747 and 762 nm. The primary electron donor showed a bleaching band centered at 925 nm upon photooxidation. Thus, the energy gap between LH1 and the primary electron donor is quite large in this strain: 425 cm(-1). Nevertheless, trapping occurred with a time constant of 65 +/- 5 ps, similar to the rates observed in other purple bacteria. As in other species, no back-transfer from the reaction center to the antenna was observed. Our results show that strain 970 is a unique subject for the study of antenna and reaction center function and organization.