Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.     

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800–B850 gap from 750 cm^\s-1 at 0.1 MPa to 900 cm^-1 at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800–850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800–B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C₉-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (-0.4 cm⁰¹/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm⁰¹/MPa). The shift rate for B850 at 4.2 K is-0.28 cm^–1/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.Abbreviations B800 BChl antenna band absorbing (at room temperature) at 800 nm (B850, B875, B1015 are defined similarly) - CD circular dichroism - FC factor Franck-Condon factor - FMO comple Fenna-Matthews-Olson complex - L-S theory Laird-Skinner theory - LH1 core light-harvesting complex of the BChl antenna complexes - LH2 peripheral light-harvesting complex of the BChl antenna complexes - NPHB non-photochemical hole burning - P960 absorption band of special pair of Rhodopseudomonas viridis absorbing at 960 nm (room temperature). P870 of Rhodobacter sphaeroides is defined similarly - QM/MM results quantum mechanical/molecular mechanical results - RC reaction center - ZPH zero phonon hole     

Purification and Characterization of the B808–866 Light-harvesting Complex from Green Filamentous Bacterium Chloroflexus aurantiacus

The integral membrane light-harvesting complex B808–866 from the thermophilic green filamentous bacterium Chloroflexus aurantiacus has been isolated and characterized. Reversed-phase HPLC analysis demonstrated that the number of bacteriochlorophyll (BChl) in the B808–866 antenna complex is 36 ± 2 per reaction center. The main carotenoid type is γ-carotene, and the molar ratio of BChl to carotenoid is 3:2. The steady-state absorption and fluorescence spectroscopy of the B808–866 complex are reminiscent of the well-studied LH2 peripheral antenna of purple bacteria, whereas the protein sequence and the circular dichroism spectrum of B808–866 is more similar to the LH1 inner core antenna. The efficiency of excitation transfer from carotenoid to BChl is about 25%. The above results combined with electron microscopy and dynamic light scattering analysis suggest that the B808–866 antenna is more like the LH1, whereas surrounds the reaction center but probably consists of 24 building blocks with a ring diameter of about 20 nm. The above results suggested that there are probably two reaction centers inside the ring of B808–866. The unique properties of this light-harvesting complex may provide insights on the protein–pigment interactions in bacterial photosynthesis.     

Functional consequences of the organization of the photosynthetic apparatus in Rhodobacter sphaeroides. I. Quinone domains and excitation transfer in chromatophores and reaction center.antenna complexes

The purpose of this study was to gain information on the functional consequences of the supramolecular organization of the photosynthetic apparatus in the bacterium Rhodobacter sphaeroides. Isolated complexes of the reaction center (RC) with its core antenna ring (light-harvesting complex 1 (LH1)) were studied in their dimeric (native) form or as monomers with respect to excitation transfer and distribution of the quinone pool. Similar issues were examined in chromatophore membranes. The relationship between the fluorescence yield and the amount of closed centers is indicative of a very efficient excitation transfer between the two monomers in isolated dimeric complexes. A similar dependence was observed in chromatophores, suggesting that excitation transfer in vivo from a closed RC.LH1 unit is also essentially directed to its partner in the dimer. The isolated complexes were found to retain 25-30% of the endogenous quinone acceptor pool, and the distribution of this pool among the complexes suggests a cooperative character for the association of quinones with the protein complexes. In chromatophores, the decrease in the amount of photoreducible quinones when inhibiting a fraction of the centers implies a confinement of the quinone pool over small domains, including one to six reaction centers. We suggest that the crowding of membrane proteins may not be the sole reason for quinone confinement and that a quinone-rich region is formed around the RC.LH1 complexes.     

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.     

Site inhomogeneity and exciton delocalization in the photosynthetic antenna

The influence of energy disorder on exciton states of molecular aggregates (the dimer and the circular aggregate) was analyzed. The dipole strength and inhomogeneous line shapes of exciton states were calculated by means of numerical diagonalization of Hamiltonian with diagonal energy disorder without intersite correlation. The disorder degree corresponding to destruction of coherent exciton states was estimated. The circular aggregates were treated as a model of light-harvesting antenna structures of photosynthetic bacteria. It was concluded that the site inhomogeneity typical for LH1 and LH2 complexes of purple bacteria cannot significantly influence the exciton delocalization over the whole antenna.Abbreviations BChl- bacteriochlorophyll - LH1 and LH2- core and peripheral light-harvesting complexes from purple bacteria - RC- reaction center     

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.     

Three-dimensional reconstruction of a membrane-bending complex: the RC-LH1-PufX core dimer of Rhodobacter sphaeroides

A three-dimensional model of the dimeric reaction center-light harvesting I-PufX (RC-LH1-PufX) complex from Rhodobacter sphaeroides, calculated from electron microscope single particle analysis of negatively stained complexes, shows that the two halves of the dimer molecule incline toward each other on the periplasmic side, creating a remarkable V-shaped structure. The distribution of negative stain is consistent with loose packing of the LH1 ring near the 14th LH1 alpha/beta pair, which could facilitate the migration of quinone and quinol molecules across the LH1 boundary. The three-dimensional model encloses a space near the reaction center Q(B) site and the 14th LH1 alpha/beta pair, which is approximately 20 angstroms in diameter, sufficient to sequester a quinone pool. Helical arrays of dimers were used to construct a three-dimensional membrane model, which matches the packing lattice deduced from electron microscope analysis of the tubular dimer-only membranes found in mutants of Rba. sphaeroides lacking the LH2 complex. The intrinsic curvature of the dimer explains the shape and approximately 70-nm diameter of these membrane tubules, and at least partially accounts for the spherical membrane invaginations found in wild-type Rba. sphaeroides. A model of dimer aggregation and membrane curvature in these spherical membrane invaginations is presented.     

Theory of fluorescence induction in photosystem II: derivation of analytical expressions in a model including exciton-radical-pair equilibrium and restricted energy transfer between photosynthetic units.

The theoretical relationships between the fluorescence and photochemical yields of PS II and the fraction of open reaction centers are examined in a general model endowed with the following features: i) a homogeneous, infinite PS II domain; ii) exciton-radical-pair equilibrium; and iii) different rates of exciton transfer between core and peripheral antenna beds. Simple analytical relations are derived for the yields and their time courses in induction experiments. The introduction of the exciton-radical-pair equilibrium, for both the open and closed states of the trap, is shown to be equivalent to an irreversible trapping scheme with modified parameters. Variation of the interunit transfer rate allows continuous modulation from the case of separated units to the pure lake model. Broadly used relations for estimating the relative amount of reaction centers from the complementary area of the fluorescence kinetics or the photochemical yield from fluorescence levels are examined in this framework. Their dependence on parameters controlling exciton decay is discussed, allowing assessment of their range of applicability. An experimental induction curve is analyzed, with a discussion of its decomposition into alpha and beta contributions. The sigmoidicity of the induction kinetics is characterized by a single parameter J related to Joliot's p, which is shown to depend on both the connectivity of the photosynthetic units and reaction center parameters. On the other hand, the relation between J and the extreme fluorescence levels (or the deviation from the linear Stern-Volmer dependence of 1/phi f on the fraction of open traps) is controlled only by antenna connectivity. Experimental data are consistent with a model of connected units for PS II alpha, intermediate between the pure lake model of unrestricted exciton transfer and the isolated units model.     

Protein structural deformation induced lifetime shortening of photosynthetic bacteria light-harvesting complex LH2 excited state

Photosynthetic bacterial light-harvesting antenna complex LH2 was immobilized on the surface of TiO(2) nanoparticles in the colloidal solution. The LH2/TiO(2) assembly was investigated by the time-resolved spectroscopic methods. The excited-state lifetimes for carotenoid-containing and carotenoidless LH2 have been measured, showing a decrease in the excited-state lifetime of B850 when LH2 was immobilized on TiO(2). The possibility that the decrease of the LH2 excited-state lifetime being caused by an interfacial electron transfer reaction between B850 and the TiO(2) nanoparticle was precluded experimentally. We proposed that the observed change in the photophysical properties of LH2 when assembled onto TiO(2) nanoparticles is arising from the interfacial-interaction-induced structural deformation of the LH2 complex deviating from an ellipse of less eccentric to a more eccentric ellipse, and the observed phenomenon can be accounted by an elliptical exciton model. Experiment by using photoinactive SiO(2) nanoparticle in place of TiO(2) and core complex LH1 instead of LH2 provide further evidence to the proposed mechanism.     

A perturbed two-level model for exciton trapping in small photosynthetic systems.

The study of exciton trapping in photosynthetic systems provides significant information about migration kinetics within the light harvesting antenna (LHA) and the reaction center (RC). We discuss two random walk models for systems with weakly coupled pigments, with a focus on the application to small systems (10-40 pigments/RC). Details of the exciton transfer to and from the RC are taken into consideration, as well as migration within the LHA and quenching in the RC. The first model is obtained by adapting earlier local trap models for application to small systems. The exciton lifetime is approximated by the sum of three contributions related to migration in the LHA, trapping by the RC, and quenching within the RC. The second model is more suitable for small systems and regards the finite rate of migration within the LHA as a perturbation of the simplified model, where the LHA and the RC are each represented by a single pigment level. In this approximation, the exciton lifetime is the sum of a migration component and a single nonlinear expression for the trapping and quenching of the excitons. Numerical simulations demonstrate that both models provide accurate estimates of the exciton lifetime in the intermediate range of 20-50 sites/RC. In combination, they cover the entire range of very small to very large photosynthetic systems. Although initially intended for regular LHA lattices, the models can also be applied to less regular systems. This becomes essential as more details of the structure of these systems become available. Analysis with these models indicates that the excited state decay in LH1 is limited by the average rate at which excitons transfer to the RC from neighboring sites in the LHA. By comparing this to the average rate of transfer within the LHA, various structural models that have been proposed for the LH1 core antenna are discussed.     

Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution

Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.     

Dynamics of energy transfer and trapping in the light-harvesting antenna of Rhodopseudomonas viridis.

By low intensity picosecond absorption spectroscopy it is shown that the exciton lifetime in the light-harvesting antenna of Rhodopseudomonas (Rps.) viridis membranes with photochemically active reaction centers at room temperature is 60 +/- 10 ps. This lifetime reflects the overall trapping rate of the excitation energy by the reaction center. With photochemically inactive reaction centers, in the presence of P+, the exciton lifetime increases to 150 +/- 15 ps. Prereducing the secondary electron acceptor QA does not prevent primary charge separation, but slows it down from 60 to 90 +/- 10 ps. Picosecond kinetics measured at 77 K with inactive reaction centers indicates that the light-harvesting antenna is spectrally homogeneous. Picosecond absorption anisotropy measurements show that energy transfer between identical Bchlb molecules occurs on the subpicosecond time scale. Using these experimental results as input to a random-walk model, results in strict requirements for the antenna-RC coupling. The model analysis prescribes fast trapping (approximately 1 ps) and an approximately 0.5 escape probability from the reaction center, which requires a more tightly coupled RC and antenna, as compared with the Bchla-containing bacteria Rhodospirillum (R.) rubrum and Rhodobacter (Rb.) sphaeroides.     

Antenna organization in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum

Structural aspects of the core antenna in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum were studied by means of fluorescence emission and singlet-singlet annihilation measurements. In both species the number of bacteriochlorophylls of the core antenna between which energy transfer can occur corresponds to one core-reaction center complex only. From measurements of variable fluorescence we conclude that in C. tepidum excitation energy can be transferred back from the core antenna (B920) to the peripheral B800–850 complex in spite of the relatively large energy gap, and on basis of annihilation measurements a model of separate core-reaction center units accompanied by their own peripheral antenna is suggested. C. vinosum contains besides a core antenna, B890, two peripheral antennae, B800–820 and B800–850. Energy transfer was found to occur from the core to B800–850, but not to B800–820, and it was concluded that in C. vinosum each core-reaction center complex has its own complement of B800–850. The results reported here are compared to those obtained earlier with various strains and species of purple non-sulfur bacteria.Abbreviations BChl- bacteriochlorophyll - B800–820 and B800–850- antenna complexes with Q_y-band absorption maxima near 800 nm and 820 or 850 nm, respectively - B890 and B920- antenna complexes with Q_y-band absorption maxima near 890 and 920 nm, respectively - LH1- light harvesting 1 or core antenna - LH2- light harvesting 2 or peripheral antenna     

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum

The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm(-1) higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55+/-5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.     

Dynamics of excitation energy transfer in the LH1 and LH2 light-harvesting complexes of photosynthetic bacteria.

Photosynthetic light harvesting is a unique life process that occurs with amazing efficiency. Since the discovery of the structure of the bacterial peripheral light-harvesting complex (LH2), this process has been studied using a variety of advanced laser spectroscopic methods. We are now in a position to discuss the physical origins of excitation energy transfer and trapping in the LH2 and LH1 antennae of photosynthetic purple bacteria. We demonstrate that the time evolution of the state created by the light is determined by the combined action of excitonic pigment-pitment interactions, energetic disorder, and coupling to nuclear motion in a pigment-protein complex. A quantitative fit of experimental data using Redfield theory allowed us to determine the pathways and time scales of exciton and vibrational relaxation and analyze separately different contributions to the measured transient absorption dynamics. Furthermore, these dynamics were observed to be strongly dependent on the excitation wavelength. A numerical fit of this dependence turns out to be extremely critical to a variation of the structure and disorder parameters and, therefore, can be used as a test for different antenna models (disordered ring, elliptical deformations, correlated disorder, etc.). The calculated equilibration dynamics in the exciton basis allow a visualization of the exciton motion using a density matrix picture in real space.     

On the effects of PufX on the absorption properties of the light-harvesting complexes of Rhodobacter sphaeroides

Some species of purple bacteria as, e.g., Rhodobacter sphaeroides contain the protein PufX. Concurrently, the light harvesting complexes 1 (LH1) form dimers of open rings. In mutants without PufX, the LH1s are closed rings and photosynthesis breaks down, because the ubiquinone exchange at the reaction center is blocked. However, the main purpose of the LH1 is light harvesting. We therefore investigate the effects that the PufX-induced dimerization has on the absorption properties of the core complexes. Calculations with a dipole model, which compare the photosynthetic efficiency of various configurations of monomeric and dimeric core complexes, show that the dimer can absorb photons directly into the reaction centers more efficiently, but that the performance of the more sophisticated dimeric LH1 antenna degrades faster with structural perturbations. The calculations predict an optimal orientation of the reaction centers relative to the LH1 dimer, which agrees well with the experimentally found configuration. Based on experimental observations indicating that the dimeric core complexes are indeed rather rigid, we hypothesize that in PufX⁺ species the association between the LH1 and the reaction centers is enhanced. This mechanical stabilization of the core complexes would lead to the observed quinone blockage, when PufX is missing.     

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.